InteractiveFly: GeneBrief

Na pump α subunit: Biological Overview | References

Gene name - Na pump α subunit

Synonyms -

Cytological map position - 93A4-93A4

Function - sodium/potassium transporter

Keywords - ion pump responsible for the cellular balance of sodium and potassium ions, change in neuronal excitability, auditory mechanosensation, epithelial junction function and tracheal tube-size control, septate junction function, neurodegeneration, CNS, muscles, malpighian tubules

Symbol - Atpα

FlyBase ID: FBgn0002921

Genetic map position - chr3R:16,774,552-16,802,113

Classification - ATPase-IIC_X-K: sodium or proton efflux -- potassium uptake antiporter, P-type ATPase, alpha subunit

Cellular location - surface transmembrane

NCBI link: EntrezGene
Atpα orthologs: Biolitmine

Recent literature
Talsma, A. D., Chaves, J. F., LaMonaca, A., Wieczorek, E. D. and Palladino, M. J. (2014).. Genome-wide screen for modifiers of Na+/K+ ATPase alleles identifies critical genetic loci. Mol Brain 7: 89. PubMed ID: 25476251
Mutations affecting the Na+/ K+ ATPase (a.k.a. the sodium-potassium pump) genes cause conditional locomotor phenotypes in flies and three distinct complex neurological diseases in humans. More than 50 mutations have been identified affecting the human ATP1A2 and ATP1A3 genes that are known to cause rapid-onset Dystonia Parkinsonism, familial hemiplegic migraine, alternating hemiplegia of childhood, and variants of familial hemiplegic migraine with neurological complications including seizures and various mood disorders. In flies, mutations affecting the Na pump α subunit gene have dramatic phenotypes including altered longevity, neural dysfunction, neurodegeneration, myodegeneration, and striking locomotor impairment. Locomotor defects can manifest as conditional bang-sensitive (BS) or temperature-sensitive (TS) paralysis: phenotypes well-suited for genetic screening. This study performed a genome-wide deficiency screen using three distinct missense alleles of ATPα and conditional locomotor function assays to identify novel modifier loci. A secondary screen confirmed allele-specificity of the interactions and many of the interactions were mapped to single genes and subsequently validated. Sixty-four modifier loci were identified, and classical mutations and RNAi were used to confirm 50 single gene interactions. The genes identified include those with known function, several with unknown function or that were otherwise uncharacterized, and many loci with no described association with locomotor or Na+/K+ ATPase function. In conclusion, this study has identified many critical regions and narrowed several of these to single genes. These data demonstrate there are many loci capable of modifying ATPα dysfunction, which may provide the basis for modifying migraine, locomotor and seizure dysfunction in animals.
Spong, K. E., Rodriguez, E. C. and Robertson, R. M. (2016). Spreading depolarization in the brain of Drosophila is induced by inhibition of the Na+/K+-ATPase and mitigated by a decrease in activity of protein kinase G. J Neurophysiol [Epub ahead of print] PubMed ID: 27358319
Spreading depolarization (SD) is characterized by a massive redistribution of ions accompanied by an arrest in electrical activity that slowly propagates through neural tissue. This study demonstrates the occurrence of SD in the brain of Drosophila providing a model system whereby cellular mechanisms can be dissected using molecular genetic approaches. Propagating waves of SD were reliably induced by disrupting the extracellular potassium concentration ([K+]o) either directly or by inhibition of the Na+/K+-ATPase with ouabain. The disturbance was monitored by recording the characteristic surges in [K+]o using K+-sensitive microelectrodes or by monitoring brain activity by measuring direct current (DC) potential. Using wild-type flies this study showed that young adults are more resistant to SD compared to older adults, evidenced by shorter bouts of SD activity and attenuated [K+]o disturbances. Furthermore, the susceptibility to SD was shown to differ between wild-type flies and w1118 mutants demonstrating that the ouabain-model is influenced by genetic strain. Lastly, flies with low levels of protein kinase G (PKG) had increased latencies to onset of both ouabain-induced SD and anoxic depolarization compared to flies with higher levels. These findings implicate the PKG pathway as a modulator of SD in the fly brain and given the conserved nature of the signalling pathway it could likely play a similar role during SD in the mammalian CNS.
D'Silva, N. M., Donini, A. and O'Donnell, M. J. (2017). The roles of V-type H+-ATPase and Na+/K+-ATPase in energizing K+ and H+ transport in larval Drosophila gut epithelia. J Insect Physiol [Epub ahead of print]. PubMed ID: 28188726
This study analyzed V- type H+-ATPase (VA) and Na+/K+-ATPase (NKA) along the caeca and midgut of third instar Drosophila larvae using immunohistochemistry and ATPase activity assays. Corresponding H+ and K+ fluxes were characterized using the Scanning Ion-Selective Electrode Technique (SIET), and the roles of transport ATPases in energizing ion transport across the larval gut were investigated by basal application of bafilomycin, a VA inhibitor, and ouabain, a NKA inhibitor. Addition of bafilomycin led to a decrease in H+ absorption along the caeca and midgut except at the copper cells and large flat cell zone of the middle midgut. H+ absorption was decreased by acetazolamide, consistent with carbonic anhydrase activity in all regions except at the large flat cell zone of the middle midgut. Bafilomycin or acetazolamide also led to decreased K+ absorption across the caeca and the anterior midgut. The data show the dependence of K+ transport on H+ gradients established by the VA in the latter regions, consistent with the presence of a Cation-Proton Antiporter (CPA2) identified in other insect epithelia. Addition of ouabain led to the increase of K+ absorption along the anterior midgut and the large flat cell zone of the middle midgut, suggesting a role for the NKA in these regions. This study shows the importance of both ATPases in driving ion transport across the gut of larval Drosophila.
Taverner, A. M., Yang, L., Barile, Z. J., Lin, B., Peng, J., Pinharanda, A. P., Rao, A. S., Roland, B. P., Talsma, A. D., Wei, D., Petschenka, G., Palladino, M. J. and Andolfatto, P. (2019). Adaptive substitutions underlying cardiac glycoside insensitivity in insects exhibit epistasis in vivo. Elife 8. PubMed ID: 31453806
Predicting how species will respond to selection pressures requires understanding the factors that constrain their evolution. This study used genome engineering of Drosophila to investigate constraints on the repeated evolution of unrelated herbivorous insects to toxic cardiac glycosides, which primarily occurs via a small subset of possible functionally-relevant substitutions to Na(+),K(+)-ATPase. Surprisingly, frequently observed adaptive substitutions were found at at two sites, 111 and 122, are lethal when homozygous and adult heterozygotes exhibit dominant neural dysfunction. A phylogenetically correlated substitution, A119S, was found that partially ameliorates the deleterious effects of substitutions at 111 and 122. Despite contributing little to cardiac glycoside-insensitivity in vitro, A119S, like substitutions at 111 and 122, substantially increases adult survivorship upon cardiac glycoside exposure. These results demonstrate the importance of epistasis in constraining adaptive paths. Moreover, by revealing distinct effects of substitutions in vitro and in vivo, the results underscore the importance of evaluating the fitness of adaptive substitutions and their interactions in whole organisms.
Cheslock, A., Andersen, M. K. and MacMillan, H. A. (2021). Thermal acclimation alters Na(+)/K(+)-ATPase activity in a tissue-specific manner in Drosophila melanogaster. Comp Biochem Physiol A Mol Integr Physiol: 110934. PubMed ID: 33684554
Insects, like the model species Drosophila melanogaster, lose neuromuscular function and enter a state of paralysis (chill coma) at a population- and species-specific low temperature threshold that is decreased by cold acclimation. Entry into this coma is related to a spreading depolarization in the central nervous system, while recovery involves restoration of electrochemical gradients across muscle cell membranes. The Na(+)/K(+)-ATPase helps maintain ion balance and membrane potential in both the brain and hemolymph (surrounding muscles), and changes in thermal tolerance traits have therefore been hypothesized to be closely linked to variation in the expression and/or activity of this pump in multiple tissues. This study tested this hypothesis by measuring activity and thermal sensitivity of the Na(+)/K(+)-ATPase at the tagma-specific level (head, thorax and abdomen) in warm- (25 °C) and cold-acclimated (15 °C) flies by Na(+)/K(+)-ATPase activity at 15, 20, and 25 °C. Differences in pump activity were related to differences in chill coma temperature, spreading depolarization temperature, and thermal dependence of muscle cell polarization. Differences in pump activity and thermal sensitivity induced by cold acclimation varied in a tissue-specific manner: While cold-acclimated flies had decreased thermal sensitivity of Na(+)/K(+)-ATPase that maintains activity at low temperatures in the thorax (mainly muscle), activity instead decreased in the heads (mainly brain). It is argued that these changes may assist in maintenance of K(+) homeostasis and membrane potential across muscle membranes and discuss how reduced Na(+)/K(+)-ATPase activity in the brain may counterintuitively help insects delay coma onset in the cold.
Andersen, M. K., Robertson, R. M. and MacMillan, H. A. (2022). Plasticity in Na+/K+-ATPase thermal kinetics drives variation in the temperature of cold-induced neural shutdown of adult Drosophila melanogaster. J Exp Biol 225(24). PubMed ID: 36477887
Most insects can acclimate to changes in their thermal environment and counteract temperature effects on neuromuscular function. At the critical thermal minimum, a spreading depolarization (SD) event silences central neurons, but the temperature at which this event occurs can be altered through acclimation. SD is triggered by an inability to maintain ion homeostasis in the extracellular space in the brain and is characterized by a rapid surge in extracellular K+ concentration, implicating ion pump and channel function. This study focused on the role of the Na+/K+-ATPase specifically in lowering the SD temperature in cold-acclimated Drosophila melanogaster. After first confirming cold acclimation altered SD onset, the dependency of the SD event on Na+/K+-ATPase activity was investigated by injecting the inhibitor ouabain into the head of the flies to induce SD over a range of temperatures. Latency to SD followed the pattern of a thermal performance curve, but cold acclimation resulted in a left-shift of the curve to an extent similar to its effect on the SD temperature. With Na+/K+-ATPase activity assays and immunoblots, it was found that cold-acclimated flies have ion pumps that are less sensitive to temperature, but do not differ in their overall abundance in the brain. Combined, these findings suggest a key role for plasticity in Na+/K+-ATPase thermal sensitivity in maintaining central nervous system function in the cold, and more broadly highlight that a single ion pump can be an important determinant of whether insects can respond to their environment to remain active at low temperatures.
Maor, G., Dubreuil, R. R. and Feany, M. B. (2023). alpha-synuclein promotes neuronal dysfunction and death by disrupting the binding of ankyrin to β-spectrin. J Neurosci. PubMed ID: 36653193
α-synuclein plays a key role in the pathogenesis of Parkinson's disease and related disorders, but critical interacting partners and molecular mechanisms mediating neurotoxicity are incompletely understood. This study shows that α-synuclein binds directly to β-spectrin. Using males and females in a Drosophila model of α-synuclein-related disorders this study demonstrated that β-spectrin is critical for α-synuclein neurotoxicity. Further, the ankyrin binding domain of β-spectrin is required for α-synuclein binding and neurotoxicity. A key plasma membrane target of ankyrin, Na(+)/K(+) ATPase, is mislocalized when human α-synuclein is expressed in Drosophila. Accordingly, membrane potential is depolarized in α-synuclein transgenic fly brains. The same pathway was examined in human neurons and it was found that Parkinson's disease patient-derived neurons with a triplication of the α-synuclein locus show disruption of the spectrin cytoskeleton, mislocalization of ankyrin and Na(+)/K(+) ATPase, and membrane potential depolarization. These findings define a specific molecular mechanism by which elevated levels of α-synuclein in Parkinson's disease and related α-synucleinopathies leads to neuronal dysfunction and death.
Maor, G., Dubreuil, R. R. and Feany, M. B. (2023). alpha-synuclein promotes neuronal dysfunction and death by disrupting the binding of ankyrin to ss-spectrin. bioRxiv. PubMed ID: 37333277
α-synuclein plays a key role in the pathogenesis of Parkinson's disease and related disorders, but critical interacting partners and molecular mechanisms mediating neurotoxicity are incompletely understood. This study shows that α-synuclein binds directly to β-spectrin. Using males and females in a Drosophila model of α-synuclein-related disorders this study demonstrated that β-spectrin is critical for α-synuclein neurotoxicity. Further, the ankyrin binding domain of β-spectrin is required for α-synuclein binding and neurotoxicity. A key plasma membrane target of ankyrin, Na (+) /K (+) ATPase, is mislocalized when human α-synuclein is expressed in Drosophila. Accordingly, membrane potential is depolarized in α-synuclein transgenic fly brains. The same pathway was examined in human neurons, and it was found that Parkinson's disease patient-derived neurons with a triplication of the α-synuclein locus show disruption of the spectrin cytoskeleton, mislocalization of ankyrin and Na (+) /K (+) ATPase, and membrane potential depolarization. These findings define a specific molecular mechanism by which elevated levels of α-synuclein in Parkinson's disease and related α-synucleinopathies leads to neuronal dysfunction and death.


The activity of Na+/K+-ATPase establishes transmembrane ion gradients and is essential to cell function and survival. Either dysregulation or deficiency of neuronal Na+/K+-ATPase has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and rapid-onset dystonia Parkinsonism. However, genetic evidence that directly links neuronal Na+/K+-ATPase deficiency to in vivo neurodegeneration has been lacking. This study used Drosophila photoreceptors to investigate the cell-autonomous effects of neuronal Na+/K+ ATPase. Loss of ATPα, an α subunit of Na+/K+-ATPase, in photoreceptors through UAS/Gal4-mediated RNAi eliminated the light-triggered depolarization of the photoreceptors, rendering the fly virtually blind in behavioral assays. Intracellular recordings indicated that ATPα knockdown photoreceptors were already depolarized in the dark, which was due to a loss of intracellular K+. Importantly, ATPα knockdown resulted in the degeneration of photoreceptors in older flies. This degeneration was independent of light and showed characteristics of apoptotic/hybrid cell death as observed via electron microscopy analysis. Loss of Nrv3, a Na+/K+-ATPase β subunit (see Nervana 1 and Nervana 2), partially reproduced the signaling and degenerative defects observed in ATPα knockdown flies. Thus, the loss of Na+/K+-ATPase not only eradicates visual function but also causes age-dependent degeneration in photoreceptors, confirming the link between neuronal Na+/K+ ATPase deficiency and in vivo neurodegeneration. This work also establishes Drosophila photoreceptors as a genetic model for studying the cell-autonomous mechanisms underlying neuronal Na+/K+ ATPase deficiency-mediated neurodegeneration (Luan, 2014).

The Na+/K+-ATPase transports Na+ and K+ against their concentration gradients across the cell membrane to maintain a low Na+ and high K+ concentration within the cells. These ion gradients determine the resting membrane potential and form the basis of the excitability of neurons. The Na+ gradient also provides the driving force for various secondary active transporters that import glucose, amino acids, and other nutrients into the cell. Additionally, the ion concentrations maintained by Na+/K+-ATPase are important for regulating cellular volume and preventing cells such as neurons from swelling and lysing (Luan, 2014).

Considering the importance of Na+/K+-ATPase in basic cellular functions, it is not surprising that either dysregulation or deficiency of neuronal Na+/K+-ATPase was observed in many neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD) and rapid-onset dystonia Parkinsonism (RDP). Thus, disrupting normal Na+/K+-ATPase activity in neurons has been proposed to contribute to the pathogenesis of neurodegeneration. Nevertheless, the link between the disrupting neuronal Na+/K+-ATPase activity and neuronal dysfunction/degeneration has yet to be clarified (Luan, 2014).

Na+/K+-ATPase is composed of at least two subunits: a large catalytic α subunit and a regulatory, single-transmembrane-domain β subunit. Mammals have three α-subunit and two β-subunit genes and may express six structurally distinct Na+/K+-ATPase isoforms. In the brain, although the α3 and β2 subunits are expressed predominantly in neurons, the α2 and β1 subunits are found primarily in glia, and the α1 subunit is ubiquitously expressed. The Na+/K+-ATPase in glia is required to maintain a low K+ level in the neuronal environment and thus has a large impact on neuronal function and survival. Na+/K+-ATPase inhibitors like ouabain act on all Na+/K+-ATPase isoforms and cannot differentiate between the cell-autonomous effects of the Na+/K+-ATPase in neurons from those derived from the neighboring glia. Thus, genetic approaches are needed to modulate the Na+/K+-ATPase level in neurons to investigate the function of neuronal Na+/K+-ATPase. Genetic studies on the impact of neuronal Na+/K+-ATPase deficiency in the past decade, which were mostly based on characterization of heterozygous mutant mice of the α3 subunit, have identified defects in the function of central brain neurons but have not provided direct evidence of neurodegeneration (Luan, 2014).

The Drosophila visual system expresses only one type of α subunit, ATPα, and three β subunits, Nrv1–3 (Ashmore, 2009; Baumann, 2010; Okamura, 2003; Palladino, 2003; Takeyasu, 2001). This study used Drosophila photoreceptors as a genetic model to study the cell-autonomous functions of neuronal Na+/K+-ATPase. Although ATPα mutants in Drosophila exhibit extensive neurodegeneration (Palladino, 2003), these mutants were not used because the degeneration is due to the loss of Na+/K+-ATPase not only in neurons but also in neighboring non-neuronal cells. Instead, using a UAS/Gal4-mediated RNAi approach, knocked down ATPα and Nrv1-3 were generated specifically in photoreceptors, and the impact of this knockdown was assessed on visual signaling and photoreceptor integrity in the fly (Luan, 2014).

Na+/K+-ATPase activity establishes and maintains the characteristic transmembrane gradients of Na+ and K+, which underlie essentially all vertebrate and invertebrate cellular physiology. Although the importance of Na+/K+-ATPase to the function and survival of both neurons and non-excitable cells has been demonstrated by decades of pharmacological studies, the involvement of neuronal Na+/K+ ATPase defects in neurodegeneration has yet to be demonstrated in vivo. This cell-specific RNAi study reveals for the first time that Na+/K+-ATPase is essential for normal neuronal function of Drosophila photoreceptors. More importantly, this work provides in vivo evidence that links neuronal Na+/K+-ATPase deficiency to age-dependent neurodegeneration (Luan, 2014).

Genetic studies on neuronal Na+/K+-ATPase function in the past decade, which were primarily based on the characterization of mice heterozygous for a mutation of the α3 subunit, have focused on central brain neurons (Clapcote, 2009, DeAndrade, 2011, Moseley, 2007: Shiina, 2010); however, the importance of functional Na+/K+-ATPase in sensory neurons remains largely unknown. This study shows that knockdown of ATPα in Drosophila photoreceptor neurons abolishes their response to light, resulting in complete blindness in the fly. These results confirm the importance of Na+/K+-ATPase in animal sensory functions. The light response of Drosophila photoreceptors is mediated by cation influx (mostly Na+) through light-stimulated TRP channels. Photoreceptors in ATPα knockdown flies were unresponsive to light for two reasons. First, without a sufficient K+ gradient across the cell membrane, the cell has no negative resting membrane potential and, thus, no electrical driving force for cation influx through TRP channels. Second, in the absence of Na+/K+-ATPase, photoreceptors may have already accumulated a high intracellular level of Na+ in the dark, which prevents extracellular Na+ flow into the cell through TRP channels during light stimulation. However, loss of the light response may not be attributed to morphological defects in the photoreceptor. First, temporally controlled knockdown of ATPα in the adult stage excludes the involvement of obvious developmental problems. Second, the light response of photoreceptors was abolished in 1-day-old ATPα knockdown flies despite the overall normal shape of the rhabdomeres. Based on these findings, it is concluded that loss of the light response is independent of degeneration in ATPα knockdown photoreceptors (Luan, 2014).

Drosophila photoreceptors have been used as a genetic model for retinal degeneration studies. Photoreceptor degeneration in many mutants is caused by defects in the regulation of the visual transduction cascade and is light-dependent. In ATPα knockdown photoreceptors, however, the visual cascade may not mediate or regulate degeneration because light deprivation does not change the severity of neurodegeneration in 10-day-old flies. Instead, the progressive degeneration in ATPα knockdown photoreceptors has demonstrated characteristics of apoptotic/necrosis hybrid cell death that are reminiscent of those observed in Na+/K+-ATPase-inhibited mammalian cells. When Na+/K+-ATPase is inhibited by ouabain, mammalian cells undergo hybrid cell death, which has been attributed to a loss of intracellular K+ ions. In vitro studies suggest that the depletion of intracellular K+ may induce apoptosis or act as a necessary cofactor to promote apoptosis. It is estimated that the intracellular levels of K+ in ATPα knockdown photoreceptors could be as low as 10 mM, which is comparable to the 50%-80% decrease in K+ observed in ouabain-treated mammalian cells. Thus, the low levels of intracellular K+ could have contributed to the degeneration of ATPα knockdown photoreceptors. Additionally, Ca2 + overload may also have a role in photoreceptor degeneration. The depolarization of the membrane potential in ATPα knockdown photoreceptors may activate voltage-gated Ca2 + channels and a reversed operation of the Na+-Ca2 + exchanger. Both activities will increase the intracellular Ca2 + concentration and could promote necrotic cell death. Finally, accumulation of Na+ inside ATPα knockdown photoreceptors impairs the driving force of nutrient import through the secondary membrane transporters, which may also play a role in cell degeneration. Hybrid cell death, an intermediate form of cell death falling along an apoptosis-necrosis continuum, can also be found in the neurodegeneration caused by excitotoxicity and ischemia. Therefore, further studies on the role Na+/K+-ATPase in hybrid cell death will elucidate the mechanism of the cell death bearing both apoptotic and necrotic features in different neuropathological conditions (Luan, 2014).

Until now, most in vivo studies on Na+/K+-ATPase-related neurodegeneration have relied on either pharmacological agents (Bignami, 1966 and Lees, 1994) or Drosophila ATPα mutants (Palladino, 2003). Those studies have suggested that both dysregulation and deficiency of Na+/K+-ATPase lead to extensive neurodegeneration. However, the degeneration observed in those studies could be partially derived from defects in non-neuronal tissues and cells in the brain. For example, Na+/K+-ATPase in the blood-brain-barrier participates in the maintenance of water and ion homeostasis in the central nervous system (CNS) (Harik, 1986; Keep, 1999), which is critical for neuronal function and survival. In the Drosophila auditory organ (Johnston's organ), Roy found that knocking down ATPα in scolopale cells, principal support cells that enclose neuronal dendrites, results in neuronal dysfunction and complete deafness (Roy, 2013). Additionally, a defect of Na+/K+-ATPase in astrocytes could be responsible for neonatal seizures and spongiform encephalopathy. In common neurodegenerative disorders such as AD, PD and RPD, however, Na+/K+-ATPase is only reduced in specific subgroups of neurons. To better mimic the neuropathological conditions of neurodegenerative diseases to study this degeneration mechanism, it is necessary to specifically downregulate Na+/K+-ATPase in particular neurons to avoid perturbations in other cells (Luan, 2014).

Because homozygous mutations in the mouse α3 subunit of Na+/K+-ATPase cause neonatal lethality, genetic studies on neuronal isoforms of Na+/K+-ATPase have so far been primarily based on the characterization of heterozygous α3 mutants. Those mouse studies, however, have not revealed direct evidence of neurodegeneration in the brain most likely due to the relatively moderate reduction of Na+/K+-ATPase activity in the heterozygous mutants. This in vivo model using Drosophila photoreceptors, which mimics the neuropathological conditions of those neurodegenerative disorders, could be a valuable tool for further investigating the mechanism of neuronal Na+/K+-ATPase deficiency-mediated neurodegeneration. In addition to the genetic tools for gene modulation, Drosophila utilizes simple assays such as ERG, phototaxis and optomotor responses, and the optical neutralization assay to evaluate both the function and morphology of photoreceptor neurons. The loss of Na+/K+-ATPase in photoreceptors does not change the environmental K+ levels, allowing the study the cell-autonomous effects of neuronal Na+/K+-ATPase deficiency. Taking advantage of this simple and convenient neuronal model may allow identification of the key players in Na+/K+-ATPase deficiency-mediated neurodegeneration, which will thereby guide in the design of new therapeutic strategies for neurodegenerative disorders (Luan, 2014).

This study has provided evidence that either dysregulation or deficiency of neuronal Na+/K+-ATPase causes abnormal depolarization of neurons by disrupting the intracellular ion balance instead of extracellular ion homeostasis, which leads to neuronal dysfunction and behavioral abnormality. Furthermore, disrupted neuronal Na+/K+-ATPase activity triggers progressive neurodegeneration. Therefore, this study suggests that early intervention against dysregulation or deficiency of neuronal Na+/K+-ATPase may alleviate the progression of neurodegenerative disorders (Luan, 2014).

Cell-type-specific roles of Na+/K+ ATPase subunits in Drosophila auditory mechanosensation

Ion homeostasis is a fundamental cellular process particularly important in excitable cell activities such as hearing. It relies on the Na(+)/K(+) ATPase (also referred to as the Na pump), which is composed of a catalytic α subunit and a β subunit required for its transport to the plasma membrane and for regulating its activity. This study shows that α and β subunits are expressed in Johnston's organ (JO), the Drosophila auditory organ. Expression of α subunits (ATPα and α-like) and β subunits (nrv1, nrv2, and nrv3) were knocked down individually in JO with UAS/Gal4-mediated RNAi. ATPα shows elevated expression in the ablumenal membrane of scolopale cells, which enwrap JO neuronal dendrites in endolymph-like compartments. Knocking down ATPα, but not α-like, in the entire JO or only in scolopale cells using specific drivers, resulted in complete deafness. Among β subunits, nrv2 is expressed in scolopale cells and nrv3 in JO neurons. Knocking down nrv2 in scolopale cells blocked Nrv2 expression, reduced ATPα expression in the scolopale cells, and caused almost complete deafness. Furthermore, knockdown of either nrv2 or ATPα specifically in scolopale cells causes abnormal, electron-dense material accumulation in the scolopale space. Similarly, nrv3 functions in JO but not in scolopale cells, suggesting neuron specificity that parallels nrv2 scolopale cell-specific support of the catalytic ATPα. These studies provide an amenable model to investigate generation of endolymph-like extracellular compartments (Roy, 2013).

Using the energy of ATP hydrolysis, the Na pump extrudes cytoplasmic Na+ (out) and extracellular K+ (in) in a 3:2 ratio and maintains the gradient of these cations across the membrane, thus controlling the electrolytic and fluid balance in the cells and organs throughout the body. Among its other functions, the Na pump helps maintain the resting potential of cells, regulates cellular volume, and facilitates transport of solutes in and out of cells. Ion homeostasis of most biological systems depends on the Na pump. In the auditory system, this pump has been linked to the maintenance of the inner ear osmotic balance (Bartolami, 2011). The scala media of the inner ear is filled with a K+-rich extracellular fluid known as endolymph, which is essential for preserving the sensory structures and supporting transduction. Maintaining the endolymph homeostasis is critical to sustain auditory functions. Loss of endolymphatic balance causes collapse of the endolymphatic compartment, leading to hearing loss in mammals. K+ channels and pumps, including the Na pump, ensure proper cycling and secretion of K+ ions in the stria vascularis cells of the cochlea. The Na pump has also been linked to age-related hearing loss and Ménière disease. A detailed functional analysis of this pump is therefore necessary to gain insight into the molecular physiology of hearing loss resulting from loss of auditory ionic homeostasis (Roy, 2013).

Although vertebrate and invertebrate auditory systems differ structurally, they evolved from the same primitive mechanosensors, and there are striking developmental genetic similarities between the two lineages. The fly auditory organ, Johnston's organ (JO), is a chordotonal organ (cho) housed in the second antennal segment (9-9292369817">9). The JO comprises an array of ~250 auditory units or scolopidia. Each scolopidium comprises two to three ciliated sensory neurons associated with several support cells. These bipolar neurons are monodendritic with a single distal cilium and a proximal axon. The scolopale cell, a principal support cell, encloses the neuronal dendrites in a fluid-filled lumen, the scolopale space. This fluid, the receptor lymph, resembles cochlear endolymph and, like the endolymph, is believed to be rich in K+ ions. The scolopale cells are structurally enforced with actin-based scolopale rods. Auditory mechanosensation involves the transduction of the mechanical sound stimulus through the rotation of distal antennal segments into a neuronal response in JO. Using electrophysiological techniques it is possible to record sound-evoked potentials (SEPs) from the auditory nerve. The JO also mediates gravity and wind detection, in addition to auditory mechanosensation (Roy, 2013).

This study used the auditory mechanosensory system of Drosophila melanogaster, with its molecular genetic and electrophysiological techniques, to understand the role of the Na pump in maintaining auditory ion homeostasis. It was hypothesized that the Na pump is important in maintaining the ion homeostasis of the auditory receptor lymph. This study shows that ATPα is the sole Na pump α subunit in JO and that it has elevated expression in scolopale cells. The β subunits show cell-type specific expression and functions in JO, with nrv2 being specific to scolopale cells and nrv3 specific to neurons. It was also shown that ATPα preferentially localizes to the scolopale cell ablumenal membrane. Such functional pump localization is consistent with a role in pumping K+ ions into the scolopale cell en route to the receptor lymph, a role that resembles its contribution to generating vertebrate inner ear endolymph (Roy, 2013).

This study has shown that the Na pump is localized preferentially to the scolopale cell ablumenal plasma membrane, from where it likely pumps K+ ions into the scolopale cell cytoplasm en route to the scolopale space. Several lines of evidence support this conclusion. First, it was observed that ATPα, the principal JO α subunit, has a strikingly high expression in the scolopale cell. This suggested that the ATPα gene has a scolopale cell-specific specialized role. Second, most ATPα protein localizes outside the scolopale rods, supporting an ablumenal plasma membrane localization. Third, ATPα knockdown in the scolopale cell resulted in deafness, loss of scolopale cell integrity, and morphological defects such as presence of distended cilia, implying ionic imbalance in the scolopale space. Taken together, the Na pump is likely to be involved in maintaining JO receptor lymph ion homeostasis. Other molecular players must work in conjunction with the Na pump to maintain the ion homeostasis of the system. However, their identification must await further study (Roy, 2013).

The Na pump may also have alternative functions that are not dependent on pump activity. The current results show subcellular localization of the ATPα-GFP fusion protein accumulating near the scolopale cell-cap cell junction and the scolopale cell-neuron inner dendritic segment junction. Septate junctions are known to be present between these cell types. Insect septate junctions form a transepithelial diffusion barrier that limits solute passage through the spaces between adjacent cells in an epithelium. The Na pump has a pump-independent cell junctional activity responsible for maintaining the epithelial barrier function in the Drosophila tracheal system, mediated by the Nrv2 β subunit (Paul, 2003; Paul, 2007). In the fly auditory system, failure to preserve junctional integrity of the scolopale cells because of a lack of functional pumps may also cause fluid retention inside the scolopale space, which could manifest as the morphological abnormalities seen when either ATPα or nrv2 were knocked down in the scolopale cell. However, a Lucifer Yellow dye exclusion assay, in animals in which ATPα has been knocked down with nompA-Gal4, argues against such a junctional role of the Na pump in scolopale cells, although one cannot absolutely rule out a junctional role of the pump because the Lucifer Yellow molecules may be too large to detect a mild junctional compromise, or RNAi may not completely inhibit the pump. Furthermore, septate junctions require numerous other components, so loss of only one component may not completely dismantle the septate junctions in these cells. Future genetic rescue experiments using constructs with inactivated pump function in an ATPα knockdown background would indicate whether the observed knockdown phenotype is a pump-independent function of the Na pump. However, because of the early requirement of the Na pump during development and cross-reactivity of the RNAi to both the endogenous and rescue construct gene copies, such experiments are currently not feasible (Roy, 2013).

The finding of organelles such as mitochondria in the scolopale space, often devoid of plasma membrane enclosure, raises the question of their origin. The most likely source of these is the scolopale cell itself. One possibility is that concomitantly compromised ion homeostasis and osmotic balance results in scolopale cell membrane rupture, releasing cellular contents. Torn membranes can rapidly reseal themselves through a Ca2+-dependent process. In Drosophila embryos, cells undergoing such a cell membrane tear form a membrane plug to reseal the gap in the lipid bilayer through a coordinated activity of the cell membrane and the cytoskeleton. A second possibility is that the extraneous material in the scolopale space results primarily from a developmental requirement of the Na pump. The cho cell lineage for each scolopidium comes from a single sense organ-precursor cell, specified in the imaginal disk epithelium. Lineage cell division occurs early, before massive changes in cell shape, with enormous subsequent elongation. Loss of ion homeostasis during this process may prevent the high developmental fidelity required for these cell shape changes. A third possible explanation may be partial apoptosis of the scolopale cell. Ultrastructurally, it is clear that the scolopale cell is still alive in the knockdown animals because the nuclei are not heteropyknotic and no cell shrinkage is seen; both of which are hallmarks of apoptotic cells. Nevertheless, ionic or osmotic imbalances in the cell may trigger a subset of apoptotic features such as blebbing of the plasma membrane. Such blebs into the scolopale space would initially be membrane-bound, but this membrane may be unstable and break down in the context of the scolopale space. The mitochondria found in the scolopale space also frequently display swelling or disrupted cristae, observations that are also consistent with apoptotic features in Drosophila (Roy, 2013).

Neuronal receptor currents resulting from auditory transduction likely cause depletion of ions in the receptor lymph; therefore, a mechanism must exist by which the receptor lymph is replenished with a constant supply of K+ ions. The receptor lymph filling the scolopale space in JO is likely to be highly enriched in K+ ions in analogy to the bristle receptors and campaniform sensilla in insects that have been shown to be rich in K+ ions. In addition, vertebrate endolymph is K+ enriched. The model of the Na pump in the fly auditory system is that it is present in the ablumenal plasma membrane of the scolopale cell where it actively transports K+ ions into the scolopale cell en route to the scolopale space to help maintain its K+-rich ionic composition. RNAi-mediated knockdown of Na pump subunits results in deafness and loss of morphological integrity. All of these findings are consistent with the model. However, to absolutely confirm the relevance of this model, additional experiments are required. First, it would be informative to measure the receptor lymph ionic concentration and directly demonstrate the high K+ concentration within the scolopale space. However, such experiments may prove to be technically challenging because of the small size of the scolopidium. It is also necessary to to identify other molecules that work in combination with the Na pump to maintain the receptor lymph for efficient auditory transduction (Roy, 2013).

In the vertebrate inner ear, the endocochlear potential is achieved by maintaining the ionic composition of the endolymph in the fluid compartment into which the stereocilia project. Deregulation of the ion concentration or fluid volume in the endolymph, mediated by cells in the stria vascularis and the lateral walls of the organ of Corti, is thought to underlie hearing disorders such as Alzheimer's disease, and may contribute to age-related hearing loss. Although the Na pump is thought to participate in enriching K+ in the endolymph and to contribute to fluid volume homeostasis in the endolymph, the precise mechanisms of Alzheimer disease and related diseases are not well understood. This article has taken advantage of rapidly advancing understanding of the Drosophila auditory system to systematically investigate the expression and functional roles of each Na pump subunit in the auditory organ. The cell-type specificity of Na pump subunits was defined as well as the functional and morphological consequences of cell type-specific loss of function of these subunits. It also sets the stage for future studies to elucidate the detailed pathways and mediators of ion transport and fluid regulation. The model of the Na pump subcellular localization in the ablumenal membrane of the scolopale cell provides a useful system to investigate endocochlear potential generation in endolymph-like extracellular compartments and its malfunctions in connection to inner ear disorders such as age-related hearing loss and Ménière disease (Roy, 2013).

Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals

Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, this study expressed the ouabain sensitive Na,K-ATPase α-subunit of Drosophila melanogaster together with the β-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC50 for the single mutations of N122H or T797A was increased roughly 250-fold relative to the wild-type, the double mutations of Q111V-N122H and Q111T-N122H proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on cardenolide exposed insects has resulted in very efficient substitutions that decrease cardenolide sensitivity extremely (Dalla, 2013).

Community-wide convergent evolution in insect adaptation to toxic cardenolides by substitutions in the Na,K-ATPase

The extent of convergent molecular evolution is largely unknown, yet is critical to understanding the genetics of adaptation. Target site insensitivity to cardenolides is a prime candidate for studying molecular convergence because herbivores in six orders of insects have specialized on these plant poisons, which gain their toxicity by blocking an essential transmembrane carrier, the sodium pump (Na,K-ATPase). This study investigated gene sequences of the Na,K-ATPase alpha-subunit in 18 insects feeding on cardenolide-containing plants (spanning 15 genera and four orders) to screen for amino acid substitutions that might lower sensitivity to cardenolides. The replacement N122H that was previously shown to confer resistance in the monarch butterfly (Danaus plexippus) and Chrysochus leaf beetles was found in four additional species, Oncopeltus fasciatus and Lygaeus kalmii (Heteroptera, Lygaeidae), Labidomera clivicollis (Coleoptera, Chrysomelidae), and Liriomyza asclepiadis (Diptera, Agromyzidae). Thus, across 300 Myr of insect divergence, specialization on cardenolide-containing plants resulted in molecular convergence for an adaptation likely involved in coevolution. The screen revealed a number of other substitutions connected to cardenolide binding in mammals. It was confirmed that some of the particular substitutions provide resistance to cardenolides by introducing five distinct constructs of the Drosophila melanogaster gene into susceptible eucaryotic cells under an ouabain selection regime. These functional assays demonstrate that combined substitutions of Q(111) and N(122) are synergistic, with greater than twofold higher resistance than either substitution alone and >12-fold resistance over the wild type. Thus, even across deep phylogenetic branches, evolutionary degrees of freedom seem to be limited by physiological constraints, such that the same molecular substitutions confer adaptation (Dobler, 2012).

Spike integration and cellular memory in a rhythmic network from Na+/K+ pump current dynamics

The output of a neural circuit results from an interaction between the intrinsic properties of neurons in the circuit and the features of the synaptic connections between them. The plasticity of intrinsic properties has been primarily attributed to modification of ion channel function and/or number. This study has found a mechanism for intrinsic plasticity in rhythmically active Drosophila neurons that was not based on changes in ion conductance. Larval motor neurons had a long-lasting, sodium-dependent afterhyperpolarization (AHP) following bursts of action potentials that was mediated by the electrogenic activity of Na(+)/K(+) ATPase. This AHP persisted for multiple seconds following volleys of action potentials and was able to function as a pattern-insensitive integrator of spike number that was independent of external calcium. This current also interacted with endogenous Shal K(+) conductances to modulate spike timing for multiple seconds following rhythmic activity, providing a cellular memory of network activity on a behaviorally relevant timescale (Pulver, 2010).

One of the most important tasks of a neuron is to keep track of its own activity. This is of obvious importance for neurons that are involved in memory processes, but it is also true for many other types of neurons that need to operate within a particular activity or input-output range. Many types of plasticity mechanisms have been described that allow cells to adjust synaptic weights and intrinsic properties to reflect their activity history and maintain optimal functionality. This study has demonstrated a new form of short-term cellular memory in Drosophila larval motor neurons that is mediated by spike-dependent activation of Na+/K+-ATPase. An AHP mediated by electrogenic activity of the Na+/K+ pump is proportional to the number of proceeding spikes, even when the pattern of activity is varied. This AHP effectively acts as a spike counter at behaviorally relevant spike rates. Furthermore, this study found that this AHP can release endogenous Ishal channels from inactivation during rhythmic firing, and that this modification persists for multiple seconds in the absence of rhythmic input, providing a memory trace of the rhythmically active state (Pulver, 2010).

Na+/K+ pumps are commonly portrayed as the necessary but unglamorous workhorses of neuronal membranes. By continually moving Na+ ions out and K+ ions into cells, Na+/K+ pumps generate an electrochemical gradient across the cellular membrane; this slow activity is crucial for generating the resting membrane potential in all neurons and setting basal excitability. Na+/K+ pumps can have other functions, however. Long lasting, Na+/K+ pump-mediated AHPs have been observed in a variety of neuronal types. In Drosophila they have been shown to be engaged by pharmacological manipulation of sodium channel inactivation kinetics. In various vertebrate preparations, pump-mediated AHPs regulate rhythmic bursting by suppressing excitability. Pump-mediated AHPs have also been shown to underlie changes in the efficacy of neuromodulatory synaptic input in leech sensory neurons. This same pump current has been shown to be intimately involved in sensory coding in these neurons due to its ability to allow adaptive scaling of input signals. The present study is the first to show that a persistent (many seconds long), dynamic change in neuronal excitability can be attributed to Na+/K+ pump function under normal physiological conditions in rhythmically active motor neurons (Pulver, 2010).

One striking feature of the AHP reported in this study is that it reflects overall previous spiking activity but remains relatively insensitive to the pattern of activity in which spikes are presented. This is not a feature of the long lasting AHPs which are mediated by ionic conductances. Spike counting has been shown to underlie memory formation in other systems. In weakly electric fish, a long-lasting shift in intrinsic excitability is responsible for a pulse integrating mechanism that is immune to frequency-dependent fluctuations. This process is critical to a form of long lasting sensorimotor adaptation in electric organ discharges (Pulver, 2010).

The role of spike counting in the mature larval locomotor circuit is less clear, but the ability of AHPs to act as spike integrators or 'spike counters' through a range of activity patterns has interesting implications for computational neuroscientists interested in homeostatic plasticity. In previous work, models of how neurons keep track of their own activity have been focused on sensors of intracellular Ca2+. Intracellular Ca2+ levels, however, are not always well correlated with spiking activity in neurons. Furthermore, Ca2+-sensing mechanisms that operate on time scales over 1 sec are sometimes difficult to justify in a model, given the fast time constant of Ca2+ decay after spiking (typically ~0.5 sec). Intracellular Na+ concentrations, by contrast, are more directly linked to spiking since they directly reflect the actions of voltage-gated Na+ channels. This work suggests that activity can modify intracellular Na+ levels over multi-second time scales. Such accumulation is likely to be most significant in small neurons or geometrically constrained subcellular compartments. These results suggest that activity sensors tuned to intracellular Na+ could be potentially useful as seconds - long time scale activity integrators in computational models of homeostatic plasticity. This has special relevance to rhythmic networks since many of these circuits operate with cycle periods of this magnitude (Pulver, 2010).

An additional interesting property of the hyperpolarization produced by the larval pump is that it can release endogenous Ishal channels from inactivation and thereby modify how a cell responds to the next depolarizing input. Previous studies in the larval motor circuit concluded that Ishal currents are largely inactivated at rest and do not affect spike timing in MN30-Ib cells. This conclusion, however, was based on measurements from silent cells. This is not the usual state of MN30-Ib cells; in a behaving animal, MN30-Ibs are rhythmically active. The importance of considering a network's endogenous activity in studies of synaptic plasticity has been demonstrated in other systems. The example shown in this study highlights the importance of considering the endogenous activity of a network when measuring intrinsic properties in neurons as well. The ability of the AHP to alter the intrinsic properties of motor neurons embedded in the firing locomotor circuit marks them as having been recently active and alters the timing of motor outputs (Pulver, 2010).

The extent to which activity-dependent intrinsic properties can lead to forms of cellular memory has been widely studied=. However, putting these phenomena in a functional context is often difficult. Genetic inhibition of Na+/K+ ATPase activity in motor neurons abolishes AHPs. This genetic manipulation clearly has an impact on network output: it slows forward peristalsis by reducing central pattern generator cycle period. One possibility is that normal AHPs facilitate proper segment-to-segment coordination by restricting the time-frame of rhythmic activity within a segment during a peristaltic wave. When this restriction is removed (as in the case when dnATPase is expressed in motor neurons), activity is 'slurred' over a longer time frame in each segment, potentially leading to longer overall peristaltic wave durations. Tight control of activity bursts has been shown to be an important factor in regulating cycle period in other segmentally coupled oscillating networks. At this time, there is not enough information about synaptic connectivity within the ventral ganglion to test this and other hypotheses using computational techniques, however, future work could address this question as circuit information becomes available (Pulver, 2010).

It is important to note that in addition to abolishing AHPs, expression of dnATPase (ATPaseα) also affects motor neuron response to current injection. As a result, some of the behavior effects seen could be caused by hyperexcitation. Unfortunately, currently available genetic tools do not allow manipulation of AHP amplitude and response to current injection independently. However, the dnATPase manipulation does not significantly affect resting membrane potential; in addition, the behavioral effects of acutely depolarizing motor neurons with the heat-activated ion channel dTRPA1 (i.e. full stop, no peristalsis) are different from those reported in this study. These observations suggest that the pump-mediated affects on CPG output observed in this study are not merely the result of massive motor neuron depolarization. Overall, the results suggest that the seconds-long time scale of AHPs could act to keep motor neuron properties primed for rhythmic action and provide a complement to the longer term plasticity processes that engage translational and transcriptional mechanisms 43 to tune intrinsic properties in this circuit (Pulver, 2010).

A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

The heterodimeric Na,K-ATPase has been implicated in vertebrate and invertebrate epithelial cell junctions, morphogenesis and oncogenesis, but the mechanisms involved are unclear. It has been shown that the Drosophila Na,K-ATPase is required for septate junction (SJ) formation and that of the three β-subunit loci, only Nrv2 isoforms support epithelial SJ barrier function and tracheal tube-size control. This study shows that Nrv1 is endogenously co-expressed with Nrv2 in the epidermis and tracheal system, but Nrv1 has a basolateral localization and appears to be excluded from the Nrv2-containing SJs. When the normally neuronal Nrv3 is expressed in epithelial cells, it does not associate with SJs. Thus, the β-subunit is a key determinant of Na,K-ATPase subcellular localization as well as function. However, localization of the Na,K-ATPase to SJs is not sufficient for junctional activity because although several Nrv2/Nrv3 chimeric β-subunits localize to SJs, only those containing the extracellular domain of Nrv2 have junctional activity. Junctional activity is also specific to different α-subunit isoforms, with only some isoforms from the major α-subunit locus being able to provide full barrier function and produce normal tracheal tubes. Importantly, mutations predicted to inactivate ATPα catalytic function do not compromise junctional activity, demonstrating that the Drosophila Na,K-ATPase has an ion-pump-independent role in junction formation and tracheal morphogenesis. These results define new functions for the intensively studied Na,K-ATPase. Strikingly, the rat α1 isoform has full junctional activity and can rescue Atpα-null mutants to viability, suggesting that the Na,K-ATPase has an evolutionarily conserved role in junction formation and function (Paul, 2007).

Cell junctions are multifunctional complexes that play many crucial roles in epithelial development by providing adhesion, diffusion barrier, polarity and signaling functions. These functions are generally evolutionarily conserved, but combinations of these functions in different junctions can be divergent. For example, although the adherens junctions (AJs) are very similar between vertebrates and Drosophila, the organization and function of junctions that create diffusion barriers show significant differences. In vertebrates, the barrier is provided by the claudin-based tight junctions, which are apical to the AJs and also contain apical polarity proteins such as Par-3, Crumbs and aPKC. In Drosophila, the barrier is provided by a claudin-containing junction termed the septate junction (SJ). However, SJs are basolateral and contain basal polarity proteins such as Scribble (also known as Scribbled), Discs Large and Lethal Giant Larvae, and thus have similarity to both vertebrate tight junctions and basolateral regions (Paul, 2007 and references therein).

The functions and composition of SJs are only partially defined, but in addition to barrier function, SJs are required for proper regulation of tracheal tube size in Drosophila (Paul, 2003). The tracheal system is a ramifying network of epithelial tubes that serves as a combined pulmonary/vascular system to deliver oxygen to the body. After the tracheal system forms, the tubes are enlarged by a process called 'tube expansion' that is coincident with SJs assembly. Without SJs, multicellular tubes such as the dorsal trunk (DT) become too long and can have diameter abnormalities, and some branches such as the ganglionic branches (GBs) lose staining of lumenal markers (Paul, 2003). These tracheal defects arise in SJ mutants because SJs mediate apical secretion of the lumenal matrix-associated protein Verm, which is required to control tracheal tube size. SJ-mediated secretion of Verm occurs via a specialized pathway, as SJs are not required for the secretion of other apical markers. The nature of this specialized pathway is unclear, but it is an important and assayable cellular function of the SJ that is distinct from its paracellular barrier function (Paul, 2007).

In screens for tracheal tube-size or barrier junction mutants, it has been found that the Na,K-ATPase localizes to and is required for SJ formation (Genova, 2003; Paul, 2003). This finding was unexpected because the Na,K-ATPase is expressed in essentially all animal cells and had not previously been reported to be part of a junctional complex. The Na,K-ATPase is a P-type ion transporter that is an α/β heterodimer. The α-subunit is a large, ~1000 amino acid (aa) ten-transmembrane protein that contains the Na+ and K+ antiporter function coupled to ATPase activity. The β-subunit is a small, 330 aa single-transmembrane protein that is thought to chaperone the α-subunit from the ER to the plasma membrane and to modulate ion transport (Paul, 2007 and references therein).

How could the ATPase function in SJ formation? One possibility is that Na,K-ATPase activity is required to keep intracellular Na+ concentration low to allow junction formation, which has been demonstrated for MDCK cells. Alternatively, there are multiple reports of ion-transport-independent roles for the Na,K-ATPase. For example, the human α3 Na,K-ATPase serves as a neural receptor for the agrin protein and, through mechanisms that are gradually being defined, the Na,K-ATPase appears to transduce a reactive oxygen-mediated signal initiated by ouabain. This report shows that the junctional activity of the Drosophila Na,K-ATPase is mediated by specific isoforms of the ATPα α-subunit and by the extracellular domain of the Nrv2 β-subunit, but is not affected by mutations predicted to block ion-pump activity. Furthermore, expression of the rat α1-subunit in Drosophila Atpα-null mutants can completely restore junctional function and rescue the mutants to viability. This result suggests that the Na,K-ATPase could have an evolutionarily conserved role in cell junction formation and is consistent with evidence that the Na,K-ATPase can promote cell junction formation and cell polarity in vertebrate systems (2004; Paul, 2007 and references therein).

The Na,K-ATPase has been intensively studied as an ion-transporter over the last 60 years, and although there is increasing evidence that it has roles beyond this, in most cases the details of these ion-transport-independent functions are unclear. In particular, in vertebrate epithelia there are multiple reports that implicate a role for Na,K-ATPase in cell adhesion and/or polarity. For example, evidence has been provided that the β1-subunit acts as a homophilic cell adhesion molecule, and it has been shown that expression of the β1-subunit and E-cadherin, but not of either alone, could cause viral-transformed unpolarized MDCK cells to form adherens and tight junctions, and to polarize. However, to date the vertebrate Na,K-ATPase has not been shown to be a component of known cell adhesion or polarity complexes. In Drosophila, the Na,K-ATPase is part of the SJ which also contains the basolateral polarity proteins Scrib, Dlg and Lgl (Genova, 2003; Paul, 2003). This study shows that the junctional activity of the Na,K-ATPase is mediated by the extracellular domain of the Nrv2 β-subunit and that the junctional activity does not require ion pumping by the α-subunit (Paul, 2007).

Using chimeric β-subunits composed of domains from the Nrv2 isoform that has junctional activity, and from the Nrv3 isoform that lacks junctional activity, it was discovered that only chimeras containing the Nrv2 extracellular domain could properly target the chimera to the SJ and provide junctional activity. Although the extracellular domains of Na,K-ATPase β-subunits have previously been shown to mediate α-subunit ion-transport activity and cell-cell adhesion interactions, this is the first demonstration that the extracellular domain of the β-subunit organizes a junctional complex rather than simply acting as a cell adhesion molecule. Although the extracellular domain could simply serve to localize the Na,K-ATPase to the SJ, evidence that the Nrv2 extracellular domain has additional roles in junctional activity is provided by the observation that the Nrv2IT/3E chimera localized to the SJ but did not provide junctional activity. Thus, the Nrv2 extracellular domain is likely to interact with other extracellular SJ components to help organize SJ complexes. Whether these are cis interactions that organize the other transmembrane SJ components such as Neurexin, Neuroglian, or LachesinF, or trans interactions that organize septa between cells, or both, remains to be determined (Paul, 2007).

One of the most surprising results from these studies is that the junctional and tube-size functions of the Na,K-ATPase apparently do not require ion pumping. This contrasts with the traditional view of the ATPase, as an ion pump required for ion homeostasis in many cellular functions and developmental events. For example, it has been shown that the low intracellular Na+ concentration maintained by the Na,K-ATPase is required for MDCK junction formation, and that ATPase-mediated ion transport was required for zebrafish neural tube inflation. Ion-transport by the α1β1 isoform is required for zebrafish heart morphogenesis. Heuronal α3 ATPase has been shown to bind to and act as a receptor for Agrin, but the signal is transduced via changes in ATPase ion transport activity. Thus, the apparent ion-pump-independent junctional activity of Nrv2 appears to be a novel activity for an Na,K-ATPase (Paul, 2007 and references therein).

Although ion pumping by the α-subunit is not required for SJ formation, the α-subunit nonetheless appears to have an important role in organizing SJs. All isoforms with the Long N-terminus fully support junctional assembly, whereas isoforms with the Short N-terminus have only partial activity at the phenotypic level and do not significantly support junctional assembly at the cellular level. The alternatively spliced sixth exon also appears to contain some junctional activity because different sixth exon isoforms vary widely in their ability to provide tube-size and barrier function when the Long N-terminus is absent (Paul, 2007).

A model for the role of the ATPase in SJ assembly that is consistent with the combined results of the α- and β-subunit data is that the extracellular domain of the β-subunit interacts with multiple extracellular SJ components to assemble an extracellular complex, whereas the α-subunit interacts with cytosolic proteins or intracellular portions of transmembrane proteins to promote junction formation, paracellular barrier formation and tracheal tube-size control. An example of a protein that could interact with the α-subunit to organize junctions is the cytoskeletal protein Ankyrin, which has been shown to bind two distinct sites on the rat α-subunit, sites which are conserved in the Drosophila ATPα (Paul, 2007).

The ability of the rat α1 isoform to rescue all junctional defects of Drosophila Atpα-null mutants is consistent with the 77% identity between the Drosophila ATPα and the rat α1 proteins, and supports the hypothesis that the Na,K-ATPase has a conserved role in cell junction formation. Why would the Na,K-ATPase have evolved and maintained a role in epithelial cell junction formation and/or polarity? Possibly, as metazoans first became multicellular, their epithelial cells would have needed to establish cell-cell junctions and asymmetrically distributed ion pumps (i.e., primitive cell polarity) to enable polarized ion transport. In the first epithelial cells, the asymmetric localization of the Na,K-ATPase may have been achieved by anchoring the pump to asymmetrically localized adhesion proteins. As cell junctional and polarity mechanisms evolved, the Na,K-ATPase could have transitioned from being associated with adhesion proteins only to serving an integral scaffolding role in a larger junctional complex. Although ATPase-mediated ion transport would still be required for ion homeostasis, the scaffolding function could be ion-transport-independent, consistent with the findings that Na,K-ATPase catalytic activity is required for Drosophila viability, but that ATPα-subunits predicted to be catalytically inactive fully support SJ formation (Paul, 2007).

The Na+/K+ ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal system

Although the correct architecture of epithelial tubes is crucial for the function of organs such as the lung, kidney and vascular system, little is known about the molecular mechanisms that control tube size. Mutations in the ATPα and nrv2 β subunits of the Na+/K+ ATPase cause Drosophila tracheal tubes to have increased lengths and expanded diameters. ATPα and nrv2 mutations also disrupt stable formation of septate junctions, structures with some functional and molecular similarities to vertebrate tight junctions. The Nrv2 β subunit isoforms have unique tube size and junctional functions because Nrv2, but not other Drosophila Na+/K+ ATPase β subunits, can rescue nrv2 mutant phenotypes. Mutations in known septate junctions genes cause the same tracheal tube-size defects as ATPα and nrv2 mutations, indicating that septate junctions have a previously unidentified role in epithelial tube-size control. Double mutant analyses suggest that tube-size control by septate junctions is mediated by at least two discernable pathways, although the paracellular diffusion barrier function does not appear to involved because tube-size control and diffusion barrier function are genetically separable. Together, these results demonstrate that specific isoforms of the Na+/K+ ATPase play a crucial role in septate junction function and that septate junctions have multiple distinct functions that regulate paracellular transport and epithelial tube size (Paul, 2003).

Previous showed that embryos homozygous for the l(2)k04223 strain had tracheal tube-size regulation defects. Using inverse PCR, it was determined that the transposable element in this strain was inserted in an intron in the nervana2 (nrv2) locus, which encodes two alternatively spliced isoforms of a Na+/K+ ATPase β subunit. Two lines of evidence demonstrate that the tracheal phenotype of l(2)k04223 homozygotes results from disruption of the nrv2 locus. First, an independent transposable element insertion in the nrv2 locus, l(2)k13315, causes the same tracheal phenotypes as l(2)k04223 and fails to complement l(2)k04223 for tracheal phenotypes and viability. Second, in an EMS non-complementation screen isolated two additional mutations that fail to complement l(2)k04223. Both of these mutants contain a single nonsense base change in the exons common to both nrv2 isoforms, and both have the same tracheal phenotypes as l(2)k04223. Together, these results show that a Na+/K+ ATPaseβ subunit is required for tracheal tube-size control (Paul, 2003).

To better define the role of nrv2 in epithelial morphogenesis, a putative nrv2 null allele, nrv2nwu3, was generated that deletes the first three common nrv2 exons which encode transmembrane and extracellular domains. A second putative null allele, nrv223B removes all nrv2 common exons. The phenotypes caused by nrv2nwu3 or nrv223B do not become more severe in trans to a chromosomal deficiency known to delete nrv2, providing genetic evidence that these are null alleles (Paul, 2003).

In nrv2-null embryos, beginning at the time of tracheal tube expansion, multicellular tracheal tubes become increasingly abnormal so that most tube lengths are significantly increased and all tube diameters are irregular with expansions and constrictions along their lengths. Defects are also present in regions of single cell tubes formed by autocellular junctions, particularly near the ends of the ganglionic branches where there are lumenal staining discontinuities (Paul, 2003).

Although the process of tracheal tube expansion is drastically disrupted in nrv2-null mutants, the earlier of phases of tracheal tube morphogenesis, including early tube-size regulation, are normal. Furthermore, overall embryonic morphogenesis of nrv2-null mutant embryos also appears to be grossly normal as evidenced by the correct outgrowth of all tracheal branches to their target tissues and the absence of major patterning or morphogeneic defects as assessed using DIC optics. One possible explanation for the apparent specific morphogenic requirement of nrv2 in tracheal tube expansion is that there is a maternal contribution of nrv2 that provides enough activity to support early morphogenic processes, but not enough to support tracheal tube expansion, which occurs late in embryonic development. However, in situ hybridization and microarray analysis did not reveal any significant maternal nrv2 transcript and embryos lacking both maternal and zygotic nrv2 are indistinguishable from nrv2 zygotic null embryos. Thus, nrv2 does not play a role in early epithelial formation or general embryonic morphogenesis, but instead appears to be specifically required for remodeling the length and diameter of tracheal tubes (Paul, 2003).

To test whether nrv2 functions as part of the Na+/K+ ATPase to control tracheal tube size, the embryonic phenotypes of mutations in the major Na+/K+ ATPase α subunit locus, ATPα was examined. The transposable element insertions ATPαl(3)1278, ATPαl(3)04694 and ATPαl(3)07008 caused tracheal defects similar or identical to nrv2-null mutations, including tube length increases, diameter expansions and ganglionic branch discontinuities. The ATPα-null mutations ATPαDTS1R1 and ATPαDTS1R2 (Palladino, 2003) also caused nrv2-like length and ganglionic branch defects, but caused only mild diameter defects. Although one would normally expect the ATPα-null mutations to cause more severe phenotypes than partial loss-of-function mutations, the hypomorphic ATPα mutations might cause strong nrv2-like phenotypes by producing inactive α subunits that could unproductively interact with Nrv2 and deplete the pool of Nrv2 available for productive interactions with other binding partners, such as α subunits expressed from the secondary Na+/K+ ATPase α subunit locus CG17923. However, despite some differences between the phenotypic effects of different ATPα mutations, the observation that both null and partial loss-of-function mutations cause nrv2-like tracheal tube-size defects demonstrates that the ATPα locus is required for tracheal tube-size control and suggests that the nrv2 β and ATPα α subunits function together in this process (Paul, 2003).

During the current investigations of the role of the Na+/K+ ATPase in tube-size control, Genova and Fehon reported that Na+/K+ ATPase mutants had salivary gland septate junction defects. Therefore this study tested whether tracheal septate junction barrier function was defective in Na+/K+ ATPase mutants using a dye exclusion assay, which tests the ability of an epithelium to exclude a 10 kDa dye. In wild-type embryos, tracheal septate junction barriers become functional and excluded dye starting at late stage 15. However, the tracheal and salivary gland epithelia in nrv2 and ATPα mutants do not acquire the ability to regulate paracellular transport and cannot prevent the dye from inappropriately diffusing into the tracheal and salivary gland lumens (Paul, 2003).

To understand the nature of the septate junction defects in Na+/K+ ATPase mutants, the subcellular distribution of septate junction and cell polarity components were determined in stage 16 nrv2 and ATPα null mutants. Three ectodermally derived epithelia -- trachea, epidermis and salivary glands -- were examined and similar defects were found in all three tissues. The effects were most clearly seen in the large columnar cells of the salivary gland in which the septate junction occupies only a small region of the lateral surface of the cell. By contrast, septate junctions occupy most of the lateral surface of tracheal cells, making visualization of mislocalized septate junction components more difficult. In both nrv2 and ATPα mutants, the septate junction components Coracle, Neurexin and Discs Large mislocalize along the lateral and sometimes the basal cell surfaces, rather than being tightly localized to the apicolateral septate junction. In some cases, the levels of these proteins appeared to also be reduced. The septate junction component Fasciclin III was undetectable in nrv2 and ATPα mutant salivary glands and strongly reduced in trachea (Paul, 2003).

Although every septate junction marker tested is severely affected by the Na+/K+ ATPase mutations, the effects appear to be specific for septate junctions since the localizations and levels of the adherens junction components E-cadherin (Shotgun) and β-catenin (Armadillo), and the apical determinant Discs Lost were unaffected in nrv2 and ATPα null mutants. Together, these data demonstrate that Na+/K+ ATPase mutations specifically disrupt septate junctions (Paul, 2003).

A simple explanation for the abnormal sizes of tracheal tubes in mutants having septate junction defects would be that ionic or hydrostatic disequilibria across the tracheal epithelium disrupts tracheal cell morphogenesis. If so, then all mutants with similar paracellular barrier defects should have equivalent tracheal morphologies. However, it was found that barrier mutants had tracheal morphologies ranging from near wild type in the case of cor14* to diameter- or length-specific defects in cystic and megatrachea. These results support the conclusion that septate junction control of tube size is not dependent on regulation of paracellular diffusion (Paul, 2003).

The mutant phenotypes and genetic interactions among tracheal tube-expansion and septate junction mutants suggest there are at least two genetic pathways by which septate junctions regulate tracheal tube size. For example, nrv2 and coracle appear to act in the same genetic pathway, since nrv2 and coracle null mutants have the same tracheal phenotypes as each other and as nrv2 coracle double null mutants. This genetic evidence is supported by observations that the localization of Coracle to septate junctions is disrupted in nrv2 and ATPα mutants. By contrast, although nrv2-null and varicose mutants have the same tracheal phenotypes, nrv2 and varicose are unlikely to act in the same linear genetic pathway because nrv2 varicose double mutants have more severe tracheal phenotypes than nrv2-null mutants. This result suggests that either varicose and nrv2 function in separate pathways to control tube size, or there is redundancy between the functions of these genes (Paul, 2003).

Additional genetic pathways may also be revealed by the differential effects of tube-expansion mutations on septate junction barrier integrity. For example, in contrast to nrv2 and other mutations, the existing convoluted mutations do not affect septate junction barrier integrity and therefore may define a size-control pathway that acts in parallel to septate junction pathways. Consistent with this proposal, the double mutant combination of a nrv2 null mutation and a convoluted mutation result in more severe tracheal morphology defects than either nrv2-null or convoluted mutations alone. Alternatively, genes such as convoluted may function in a branch of a septate junction pathway to link septate junctions to tube size control. Although these models are necessarily incomplete, they offer testable predictions about gene interactions and subcellular localizations of uncharacterized gene products that should help define tube-size control and paracellular barrier pathways at the molecular level (Paul, 2003).

A central issue raised by these findings is the nature of the molecular functions(s) of the Na+/K+ ATPase and septate junctions in tube-size control. Although the Na+/K+ ATPase has been studied intensively for more than 40 years for its function as an ion pump (Chow, 1995; Blanco, 1998), the data indicate that the tracheal tube-size function of the Na+/K+ ATPase is intimately associated with its role in septate junction function. Furthermore the paracellular barrier and tube-size control functions of the septate junction are separable (Paul, 2003).

In one class of model that accounts for these observations, the role of the Na+/K+ ATPase is to organize septate junctions, which control tube size by an undetermined mechanism. The many functions of vertebrate tight junctions provide possible examples of non-barrier mechanisms by which septate junctions could control tube size. In particular, tight junctions organize polarized apical secretion mediated by the exocyst, bind cytoskeletal components such as ankyrin and fodrin, contain potential signaling molecules such as the tyrosine kinases Src, Yes and protein kinase C, and have recently been shown to regulate the activity of a Y-box transcription factor. In addition, both septate and tight junctions complexes contain proteins that organize epithelial cell apical/basal domains (Paul, 2003).

Of the tube-size control models that do not invoke ion-transport functions of the Na+/K+ ATPase, models involving apicobasal domain organization are particularly attractive. Apical surface regulation is a common theme in tubulogenesis, and has been shown to play an important role in tube-size control in the Drosophila salivary gland. Several observations support the possibility that septate junctions control tracheal tube size through the apical cell surface. First, the differential regulation of tracheal apical and basal cell surfaces suggests that tracheal tube size control is mediated at the apical cell surface. Second, the increased tracheal tube lengths and diameters present in tube-expansion mutants necessitate an increased apical cell surface area. Given that the Dlg/Scrib/Lgl complex normally present in septate junctions has an early embryonic function to negatively regulate the extent of the apical membrane domain, this complex could also act later to negatively regulate tracheal apical surface area (Paul, 2003).

In an alternative class of models that are not exclusive of the above possibilities, the ion-pump activity of the Na+/K+ ATPase may directly or indirectly mediate tube-size control. For example, pharmacologically blocking Na+/K+ ATPase ion-transport activity leads to increased intracellular Ca2+ levels in some cell types, and Ca2+ signaling abnormalities may be the molecular defect that causes the enlarged tubules of polycystic kidney disease (PKD). Another example is that the low intracellular Na+ level maintained by the Na+/K+ ATPase is required for formation of tight junctions and stress fibers in Madin-Darby canine kidney (MDCK) cells, an epithelial cell line that can form tubules in response to hepatocyte growth. Septate junction formation might also require low intracellular Na+ levels. Finally, disruption of the cellular Na+/K+ electrochemical gradient could impact secondary active transport of other solutes that may be important for proper tube-size regulation (Paul, 2003).

Although the exact biochemical roles of the Na+/K+ ATPase and septate junctions in tube-size control are unclear, identification of these complexes as parts of a tube-size control mechanism is an important step towards further understanding these mechanisms at the molecular level (Paul, 2003).

The Na+/K+ ATPase has been implicated in vertebrate tube-size control by the abnormal subcellular localization of the Na+/K+ ATPase in the inappropriately expanded tubules in individuals with PKD and in several animal models of cystic kidney diseases. However, it has not yet been determined whether this mislocalization contributes to the progression of cystic diseases or whether it is merely a secondary effect of other cellular defects. The finding that the Na+/K+ ATPase is required for normal tube-size control in the Drosophila tracheal system suggests that the vertebrate Na+/K+ ATPase may play an important role in maintaining the normal size of kidney and other epithelial and endothelial tubes. Ultimately, a molecular understanding of the tube-size control mechanisms should allow development of new strategies for preventing and treating PKD and other diseases resulting from epithelial and endothelial tube defects (Paul, 2003 and references therein).

Neuroglian, Gliotactin, and the Na+/K+ ATPase are essential for septate junction function

One essential function of epithelia is to form a barrier between the apical and basolateral surfaces of the epithelium. In vertebrate epithelia, the tight junction is the primary barrier to paracellular flow across epithelia, whereas in invertebrate epithelia, the septate junction (SJ) provides this function. New proteins have been identified that are required for a functional paracellular barrier in Drosophila. In addition to the previously known components Coracle (Cora) and Neurexin (Nrx), four other proteins [Gliotactin, Neuroglian (Nrg), and both the alpha and ß subunits of the Na+/K+ ATPase] are required for formation of the paracellular barrier. In contrast to previous reports, it is demonstrated that the Na pump is not localized basolaterally in epithelial cells, but instead is concentrated at the SJ. Data from immunoprecipitation and somatic mosaic studies suggest that Cora, Nrx, Nrg, and the Na+/K+ ATPase form an interdependent complex. Furthermore, the observation that Nrg, a Drosophila homolog of vertebrate neurofascin, is an SJ component and is consistent with the notion that the invertebrate SJ is homologous to the vertebrate paranodal SJ. These findings have implications not only for invertebrate epithelia and barrier functions, but also for understanding of neuron-glial interactions in the mammalian nervous system (Genova, 2003).

The SJ has historically been thought of as an invertebrate-specific junction; however, recent studies of the vertebrate nervous system have identified a junction that is both molecularly and structurally homologous, the paranodal SJ (PSJ). The PSJ occurs between neurons and the glial cells that myelinate them, the oligodendrocytes and Schwann cells. Each glial cell wraps around and contacts the neuron multiple times in a spiral pattern to form the paranodal loops. The PSJ forms between the paranodal loops and the neuron and keeps the node of Ranvier distinct from the internodal region by providing a seal between the neuron and glial cell. This seal provides a barrier within the neuronal membrane that separates Na+ channels at the node of Ranvier from K+ channels under the glial cells, and a paracellular diffusion barrier between the neuron and the ensheathing glial cell. Consistent with these structural and functional similarities, the invertebrate epithelial SJ and the vertebrate PSJ also display similarities at the molecular level. Caspr (contactin-associated protein; also known as paranodin), a mammalian homolog of Nrx, is located on the neuronal face of the PSJ, where it interacts with protein 4.1, which is homologous to Drosophila Cor (Genova, 2003 and references therein).

To identify additional components of the Drosophila SJ, a collection of P element insertion mutations was screened for a phenotype attributable to a loss of the paracellular barrier. Two genes, Na pump alpha subunit (Atpalpha) and Nervana 2 (Nrv2), which encodes the ß subunit of the Na+/K+ ATPase) were identified as essential for the barrier function of the SJ. In addition, Neuroglian (Nrg), which is homologous to known components of the PSJ, and Gli, which is necessary for the blood-brain barrier, were tested and found to be necessary for the paracellular barrier. Direct immunostaining, epitope-tagged expression constructs, and GFP-tagged proteins indicate that Nrv2, ATPalpha, and Nrg localize to the SJ, and that they are interdependent for this localization. In keeping with this finding, the existence of a protein complex containing Cora, Nrx, Nrg, and Nrv is demonstrated. Taken together, these results suggest a novel complex involving the Na+/K+ ATPase that is necessary for establishing and maintaining the primary paracellular barrier in invertebrate epithelia, the SJs. Thus these studies provide new insights into the structure and function of SJs in both invertebrate epithelial cells and in the homologous PSJ of the vertebrate nervous system (Genova, 2003).

A novel approach has been used to identify components of the pleated SJ, which provides the barrier to paracellular diffusion in Drosophila epithelial cells. Three independent lines of evidence indicate that the proteins encoded by these genes are essential to the structure and function of epithelial SJs. (1) Mutations in all four identified loci, Nrg, Gli, Nrv2, and Atpalpha, disrupt the paracellular barrier of the salivary gland epithelium and alter the ultrastructure of epithelial SJs. (2) The proteins encoded by three of these genes localize to the region of the SJ as judged by antibody staining of fixed tissues and observation of GFP-tagged proteins expressed in living epithelial cells (reagents were unavailable for observations of the fourth protein, Gli). (3) Somatic mosaic studies and IP experiments indicate that these proteins form an interdependent complex at the SJ. This complex also includes two previously identified SJ components, Nrx, a transmembrane protein, and Cora, a membrane-associated cytoplasmic protein with a FERM domain (Genova, 2003).

One of the most intriguing results of this study is the identification of the Na+/K+ ATPase as a functional member of the SJ. Mutations in either the alpha subunit (ATPalpha) or ß subunit (NRV2) disrupt the paracellular barrier of the embryonic salivary gland and this functional loss corresponds to the structural loss of septae in the junction. Although the SJ is localized just basal to the adherens junction near the apical end of the cell, previous characterizations of the Na+ pump have described it as having a basolateral localization. The localization of the Na+/K+ ATPase was examined using immunofluorescence; both subunits are found highly concentrated at the SJ in imaginal epithelia. In embryonic epithelia, the results differed depending upon the fixation and staining method; methanol treatment resulted in staining that appeared basolateral whereas staining of embryos fixed without methanol was localized to the SJ. Observations of GFP-exon trap lines enabled the confirmation that both ATPase subunits localize to the SJ in live embryos and imaginal epithelia. These results are limited to the examination of ectodermally derived epithelia such as the embryonic epidermis, foregut, hindgut, and salivary glands. Interestingly, the midgut does not contain pleated SJs but rather smooth SJs, and so observed differences in subcellular localization of the ATPase may be cell type dependent (Genova, 2003).

The Nrv2 and Nrv1 genes encode ß subunits of the Na+/K+ ATPase that differ in their cytoplasmic tails. The P-element insertion [l(2)k13315] disrupts the Nrv2 gene product but appears to have no affect on the Nrv1 protein. In addition, both NRV2.1 and NRV2.2 are able to rescue the dye diffusion phenotype of l(2)k13315 whereas NRV1 is not. Together these results indicate that l(2)k13315 is a mutation in the Nrv2 locus, and that NRV2 normally functions in the SJ. Although both NRV2 and NRV1 were previously described as being nervous system specific, evidence from immunostaining and from a GFP gene trap inserted within the Nrv2 locus indicates that Nrv2 is highly expressed in epithelial cells. Because NRV1 expression is not affected by the l(2)k13315 mutation and l(2)k13315 homozygous mutant cells in the wing imaginal disc lack NRV staining, it is proposed that Nrv1 is nervous system specific and epidermal cells express only NRV2 (Genova, 2003).

The observation that an Nrv1 transgene cannot rescue the Nrv2 dye diffusion phenotype, even though it localizes to the SJ when ectopically expressed in epithelial cells, suggests that the proteins encoded by these genes, although quite similar in structure, are functionally distinct. Given the sequence diversity within the cytoplasmic tail, the observation that when expressed ectopically all three proteins localize to the SJ strongly suggests that this localization is mediated by the extracellular or transmembrane domain, rather than by the intracellular domain. This complex pattern of ß subunit expression and functional interactions suggests a surprising degree of functional regulation of the Na+/K+ ATPase in epithelial and neuronal cells (Genova, 2003).

The question still remains, What is the function of localizing the Na pump to such a specialized membrane domain, one of whose functions is to create a paracellular diffusion barrier? Several characteristics of the Na pump might be important in SJ function. Previous studies suggest that the Na+/K+ ATPase functions in cell adhesion, though whether its role is structural or regulatory is unclear. Other studies suggest that the Na pump could function as a scaffold on which proteins essential for the paracellular barrier are organized. For example, both subunits bind to a variety of proteins, from those involved in signal transduction to cytoskeletal elements. In addition, it is possible that the ion pumping activity of the Na pump actively participates in the formation or maintenance of the diffusion barrier. Studies in mammalian cells have demonstrated a requirement for the ATPase, and specifically the Na+ gradient it produces, in cell polarity, adhesion, and the formation of tight junctions. Because the tight junction is responsible for creating the paracellular barrier in vertebrate epithelial cells, the ATPase might perform a similar function in the paracellular barrier of the Drosophila SJ. Further experiments, using point mutations that specifically affect the pump function of the ATPase, could address these questions (Genova, 2003 and references therein).

Cora has been shown to bind to the cytoplasmic tail of Nrx in the SJ. Studies of the PSJ have shown that the mammalian homologs of Nrx and Nrg interact via their extracellular domains. Together, these observations suggest the existence of a multiprotein complex at the SJ in which Cora binds to Nrx, which in turn binds to Nrg. The finding that Nrx and Nrg coimmunoprecipitate when either anti-Cora or anti-Nrg antibodies are used to immunoprecipitate is consistent with this model. Because Drosophila epithelial cells express all three proteins, it is not possible to rigorously distinguish whether this interaction occurs within the same cell or between adjacent cells. However, the observation that wild-type cells are unable to efficiently assemble Cora and Nrx at the boundary with cor- cells suggests that intercellular interaction with the same complex on adjacent cells is required for SJ assembly. In addition, Nrv is found to coimmunoprecipitates with both Cora and Nrx. Nrg has not been detected in this complex, suggesting that the interaction between NRV2 and the Cora-Nrx complex occurs independently of Nrg, perhaps on the cytoplasmic side of the membrane. Although these results imply the possibility of an interaction between Cora and the cytoplasmic tail of NRV2, this seems unlikely in light of observations that NRV1, 2.1, and 2.2 all localize to the SJ, despite having different cytoplasmic tails. Thus, it is more likely that the interaction between Cora and the ATPase occurs either through Nrx or the alpha subunit (Genova, 2003).

Somatic mosaic analysis has demonstrated that this complex of Cora, Nrx, Nrv, ATPalpha, and Nrg can be disrupted without affecting overall polarity, or other components of the SJ. No component essential for the paracellular barrier has been identified that is unaffected in mutant cells, suggesting that the substrate upon which this complex assembles has yet to be found. Previous studies have demonstrated that Ankyrin binds both the cytoplasmic domain of Nrg and, as has been described in mammalian cells, the alpha subunit of NA+/K+ ATPase. In addition, Ankyrin colocalizes with Nrg at points of Nrg-induced S2 cell adhesion complexes. Thus, one candidate for a substrate upon which this complex assembles is Ankyrin, a well-known member of the membrane skeleton (Genova, 2003).

Other candidate proteins for this scaffold are Scribble and Dlg. Both of these proteins are required early in Drosophila development for the establishment of epithelial cell polarity and growth control. If either is absent from epithelial cells, then the apical junctional complexes do not properly form and epithelial integrity is lost. Thus, Scribble and Dlg may be among the first constituents of the SJ upon which the subsequently expressed SJ proteins assemble (Genova, 2003).

Previous studies have suggested that the SJ may function in intercellular signaling, particularly in the regulation of cell proliferation. For example, dlg, which encodes a PDZ repeat-containing, membrane-associated guanylate kinase protein, has tumor suppressor functions. Loss of function dlg mutations are characterized by disruption of apical-basal polarity and an overproliferation of the larval imaginal discs. However, it is not known whether this overproliferation is due to a direct involvement of Dlg in a signal transduction cascade or to the disruption of apical-basal polarity within epithelial cells that could result in a disruption of apical signaling complexes. In addition to dlg, cor mutations were first isolated as dominant suppressors of a gain of function allele of the EGF receptor, EgfrElp (also known as EgfrE3), suggesting that Cora may function to positively regulate EGFR pathway function. Interestingly, a recent study of Nrg function in the developing Drosophila nervous system has proposed that it positively regulates EGF receptor function during axon guidance. The role of Nrg in regulating EGFR function in epithelial cells has not been investigated, but preliminary results indicate that Nrg mutations also dominantly suppress the rough eye caused by EgfrElp. This result may suggest that Nrg (or the entire complex) must be localized to the SJ in epithelial cells to regulate Egfr function. Alternatively, it is possible that the SJ complex is necessary to maintain polarized localization of the Egfr to the apical membrane, though no effect of cor mutations on Egfr localization has been observed (Genova, 2003).

The recent discovery of molecular, structural, and functional similarities between the invertebrate epithelial SJ and the vertebrate PSJ in the nervous system gives added significance to the identification of new SJ components in Drosophila. In addition to Cora/protein 4.1 and Nrx/paranodin, the SJ and PSJ have been shown to share neurofascin-155 and a Drosophila homolog, Nrg. This level of molecular homology strongly suggests that these two SJs are structurally and functionally homologous as well. It is therefore somewhat surprising that published reports indicate that the Na pump is uniformly distributed along the axonal membrane rather than being restricted to the PSJ (Genova, 2003 and references therein).

One possible explanation is that only a subset of the several Na+/K+ ATPase isoforms found in the mammalian genome is localized to the PSJ, and that these isoforms have not yet been studied. Similarly, it is not known if the mammalian homologs of Drosophila Gli, the neuroligins, might localize to the PSJ, or if the Drosophila homolog of contactin, a protein that interacts with Nrx/paranodin, localizes to the SJ. Although it is possible that the invertebrate epithelial SJ and vertebrate PSJ are fundamentally different in some respects, this is unlikely given the remarkable degree of similarity between these two junctions. In any case, it is clear that the genetic and genomic tools available in Drosophila can provide important insights into both the SJ and its vertebrate counterpart, the PSJ (Genova, 2003).

Molecular characterization and expression of the (Na+ K+)-ATPase alpha-subunit in Drosophila melanogaster

The (Na+ + K+)-ATPase (sodium pump) is an ouabain-sensitive, electrogenic ion pump responsible for maintaining the balance of sodium and potassium ions in almost all animal cells. Robust, ouabain-sensitive rubidium uptake, indicative of the sodium pump, was found in tissue-cultured Drosophila cells, and both larvae and adults die when fed a diet containing ouabain. A monoclonal antibody to the avian sodium pump alpha-subunit was found to cross-react with the Drosophila sodium pump alpha-subunit. Immunofluorescence microscopy was used to obtain a semi-quantitative view of the expression of the sodium pump in Drosophila tissues: high levels of the sodium pump were detected in malpighian tubules, indirect flight muscles and tubular muscles, and throughout the nervous system. The cDNA encoding this sodium pump alpha-subunit in Drosophila melanogaster was cloned, sequenced and expressed in mouse L cells. At the amino acid level, its deduced sequence of 1038 residues (the first such sequence for an invertebrate) is approximately 80% similar to alpha-subunit sequences reported for vertebrates. Only one gene was found in Drosophila, located on the third chromosome at position 93B. A restriction site polymorphism has been found, and several mutations exist that may involve the alpha-subunit gene (Lebovitz, 1989).

The Drosophila Na,K-ATPase alpha-subunit gene: gene structure, promoter function and analysis of a cold-sensitive recessive-lethal mutation

The Drosophila Na,K-ATPase (or sodium pump) alpha-subunit gene was found to contain 10 exons and span approx. 25 kb. Two nearly adjacent transcriptional initiation sites were identified, and the 2085-nucleotide sequence upstream of the first transcriptional start was analysed for promoter activity in transfected Drosophila SL2 cells. This region was found to contain many cis-acting elements that influence promoter activity, including elements that confer 2- to 3-fold higher activity in SL2 cells cultured at 30 degrees C versus 22 degrees C. Temperature-sensitive transcriptional regulation of the Na,K-ATPase alpha-subunit in Drosophila is a plausible mechanistic candidate for the factor driving temperature-dependent up-regulation of the Na,K-ATPase alpha-subunit described here for fly strains homozygous for single P-element insertions in the alpha-subunit gene. Four new P-element insertion strains were identified in this study, each insertion site lying within the first intron of the Na,K-ATPase alpha-subunit gene. The insertion in strain 0462 resulted in cold-sensitive recessive lethality; flies homozygous for the 0462 mutation could be rescued by growth at 29-30 degrees C, a condition that partially corrected a deficiency in the level of Na,K-ATPase alpha-subunit. The high-temperature rescue of homozygous 0462 flies appeared to result primarily from improved Na,K-ATPase expression rather than an increase in the rate of ion transport per Na,K-ATPase molecule. These observations point to a role for sodium-pump activity in determining the range of temperature tolerance in Drosophila and demonstrate that relatively subtle changes in sodium-pump expression can have major consequences in whole organisms (Feng, 1997).

A mutation of the Drosophila sodium pump alpha subunit gene results in bang-sensitive paralysis

A bang-sensitive enhancer trap line was isolated in a behavioral screen. The flies show a weak bang-sensitive paralysis, recovering after about 7 s. The P element insert is localized at 93B1-2 on the salivary chromosomes, the site of the (Na+,K+)ATPase alpha subunit gene. Molecular characterization demonstrates that the transposon is inserted into the first intron of this gene. This insertion leads to normal-sized transcripts, but reduced levels of expression. This change is also reflected in lower amounts of a normal-sized alpha subunit protein. Mutant flies show a much greater sensitivity to ouabain, likewise indicating, on a functional level, a reduction in Na+ pump activity. Furthermore, the bang-sensitive behavior can also be mimicked by injecting sublethal doses of ouabain into wild-type flies. The molecular and functional evidence indicates that the insertion has produced a hypomorphic mutation of the (Na+,K+)ATPase alpha subunit gene, opening the way to future studies of the regulation of the Na+ pump (Schubiger, 1994).


Search PubMed for articles about Drosophila Na pump α subunit

Ashmore, L. J., Hrizo, S. L., Paul, S. M., Van Voorhies, W. A., Beitel, G. J. and Palladino, M. J. (2009). Novel mutations affecting the Na, K ATPase alpha model complex neurological diseases and implicate the sodium pump in increased longevity. Hum Genet 126: 431-447. PubMed ID: 19455355

Bartolami, S., Gaboyard, S., Quentin, J., Travo, C., Cavalier, M., Barhanin, J. and Chabbert, C. (2011). Critical roles of transitional cells and Na/K-ATPase in the formation of vestibular endolymph. J Neurosci 31: 16541-16549. PubMed ID: 22090480

Baumann, O., Salvaterra, P. M. and Takeyasu, K. (2010). Developmental changes in β-subunit composition of Na,K-ATPase in the Drosophila eye. Cell Tissue Res 340: 215-228. PubMed ID: 20336468

Bignami, A. and Palladini, G. (1966). Experimentally produced cerebral status spongiosus and continuous pseudorhythmic electroencephalographic discharges with a membrane-ATPase inhibitor in the rat. Nature 209: 413-414. PubMed ID: 5920254

Clapcote, S. J., Duffy, S., Xie, G., Kirshenbaum, G., Bechard, A. R., Rodacker Schack, V., Petersen, J., Sinai, L., Saab, B. J., Lerch, J. P., Minassian, B. A., Ackerley, C. A., Sled, J. G., Cortez, M. A., Henderson, J. T., Vilsen, B. and Roder, J. C. (2009). Mutation I810N in the alpha3 isoform of Na+,K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS. Proc Natl Acad Sci U S A 106: 14085-14090. PubMed ID: 19666602

Dalla, S., Swarts, H. G., Koenderink, J. B. and Dobler, S. (2013). Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals. Insect Biochem Mol Biol 43: 1109-1115. PubMed ID: 24121093

DeAndrade, M. P., Yokoi, F., van Groen, T., Lingrel, J. B. and Li, Y. (2011). Characterization of Atp1a3 mutant mice as a model of rapid-onset dystonia with parkinsonism. Behav Brain Res 216: 659-665. PubMed ID: 20850480

Dobler, S., Dalla, S., Wagschal, V. and Agrawal, A. A. (2012). Community-wide convergent evolution in insect adaptation to toxic cardenolides by substitutions in the Na,K-ATPase. Proc Natl Acad Sci U S A 109: 13040-13045. PubMed ID: 22826239

Feng, Y., Huynh, L., Takeyasu, K. and Fambrough, D. M. (1997). The Drosophila Na,K-ATPase alpha-subunit gene: gene structure, promoter function and analysis of a cold-sensitive recessive-lethal mutation. Genes Funct 1: 99-117. PubMed ID: 9680312

Genova, J. L. and Fehon, R. G. (2003). Neuroglian, Gliotactin, and the Na+/K+ ATPase are essential for septate junction function in Drosophila. J. Cell Biol. 161: 979-989. PubMed ID: 12782686

Harik, S. I. (1986). Blood--brain barrier sodium/potassium pump: modulation by central noradrenergic innervation. Proc Natl Acad Sci U S A 83: 4067-4070. PubMed ID: 3012548

Keep, R. F., Ulanski, L. J., 2nd, Xiang, J., Ennis, S. R. and Lorris Betz, A. (1999). Blood-brain barrier mechanisms involved in brain calcium and potassium homeostasis. Brain Res 815: 200-205. PubMed ID: 9878735

Lebovitz, R. M., Takeyasu, K. and Fambrough, D. M. (1989). Molecular characterization and expression of the (Na+ K+)-ATPase alpha-subunit in Drosophila melanogaster. EMBO J. 8: 193-202. PubMed ID: 2540956

Lees, G. J. and Leong, W. (1994). Brain lesions induced by specific and non-specific inhibitors of sodium-potassium ATPase. Brain Res 649: 225-233. PubMed ID: 7953637

Luan, Z., Reddig, K. and Li, H. S. (2014). Loss of Na/K-ATPase in Drosophila photoreceptors leads to blindness and age-dependent neurodegeneration. Exp Neurol 261C: 791-801. PubMed ID: 25205229

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Biological Overview

date revised: 23 June 2023

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