Gene name - pickpocket
Synonyms - Ppk1, dmdNaC1
Cytological map position -
Function - channel
Keywords - peripheral nervous system, locomotion, mechanosensory signal transduction of proprioceptive sensory information
Symbol - ppk
FlyBase ID: FBgn0020258
Genetic map position -
Classification - amiloride-sensitive sodium channel
Cellular location - surface transmembrane
|Recent literature||Koseki, N., Mori, S., Suzuki, S., Tonooka, Y., Kosugi, S., Miyakawa, H. and Morimoto, T. (2016). Individual differences in sensory responses influence decision making by Drosophila melanogaster larvae on exposure to contradictory cues. J Neurogenet: 1-39. PubMed ID: 27309770
Animals make decisions on behavioral choice by evaluating internal and external signals. Individuals often make decisions in different ways, but the underlying neural mechanisms are not well understood. This study describes a system for observing the behavior of individual Drosophila melanogaster larvae simultaneously presented with contradictory signals, in this case attractive (yeast paste) and aversive (NaCl) signals. Olfaction was used to detect the yeast paste, whereas the ENaC/Pickpocket channel was important for NaCl detection. Wild-type (Canton-S) larvae fall into two decision making groups: one group decided to approach the yeast paste by overcoming the aversive signal, whereas the other group decided to forgo the yeast paste because of the aversive signal. These findings indicate that different endogenous sensitivities to NaCl contribute to make differences between two groups and that diverse decision making steps occur in individual animals.
Coordination of rhythmic locomotion depends upon a precisely balanced interplay between central and peripheral control mechanisms. Although poorly understood, peripheral proprioceptive mechanosensory input is thought to provide information about body position for moment-to-moment modifications of central mechanisms mediating rhythmic motor output. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) family displaying limited expression in multiple dendritic (md) sensory neurons tiling the larval body wall and a small number of bipolar neurons in the upper brain (Adams, 1998). ppk1 null mutant larvae have normal external touch sensation and md neuron morphology but display striking alterations in crawling behavior. Loss of PPK1 function causes an increase in crawling speed and an unusual straight path with decreased stops and turns relative to wild-type. This enhanced locomotion results from sustained peristaltic contraction wave cycling at higher frequency with a significant decrease in pause period between contraction cycles. The mutant phenotype is rescued by a wild-type PPK1 transgene and duplicated by expressing a ppk1RNAi transgene or a dominant-negative PPK1 isoform. These results demonstrate that the Ppk1 channel plays an essential role in controlling rhythmic locomotion by providing mechanosensory signal transduction of proprioceptive sensory information (Ainsley, 2003).
The DEG/ENaC superfamily includes proteins involved in mechanotransduction, proprioception, neurotransmission, and fluid and electrolyte homeostasis. Members of this superfamily are united by similarities in their amino acid sequences and in some cases by their function. The first identified protein and the largest number of family members are from Caenorhabditis elegans; these include MEC-4, MEC-10, DEG-1, UNC-105, UNC-8, and DEL-1. Elegant genetic studies in that organism have suggested a role for DEG/ ENaC proteins in mechanotransduction. MEC-4 and MEC-10 are expressed in mechanosensory neurons and are required for normal sensitivity to touch. In addition, UNC-105 and UNC-8 are expressed in muscle and motor neurons, respectively, and are required for coordinated movement. Three lines of evidence suggest that C. elegans family members may be ion channels. (1) Other family members have been shown to be ion channels. (2) Residues in the transmembrane sequence of a related DEG/ ENaC channel, alphaENaC, can be substituted for residues in M2 of MEC-4 without loss of function in the worm and vice versa. (3) Specific mutations in several C. elegans family members cause a phenotype marked by neuronal swelling; this may suggest a loss of cell volume control, perhaps caused by unregulated opening of an ion channel. Nevertheless, these C. elegans, proteins have not been shown experimentally to be channels either in vivo or in heterologous expression systems (Adams, 1998 and references therein).
Subunits of the vertebrate epithelial Na+ channel (ENaC) subfamily form ion channels that mediate Na+ absorption across the apical membrane of epithelia. Studies of ENaC have served to define several functional properties of the family. (1) ENaC generates Na+ currents that are reversibly blocked by the diuretic amiloride. Although Na+ selectivity is not a general feature, all DEG/ENaC proteins shown to be ion channels are amiloride-sensitive and conduct cations. (2) ENaC functions as a multimeric complex of three subunits, alpha-, ß-, and γENaC. In Xenopus oocytes, expression of alphaENaC, but not ß- or γENaC, generates a small amiloride-sensitive Na+ current. However, when ß- and γENaC are coexpressed with alphaENaC, much larger Na+ currents are produced. This finding illustrates (3) -- some members can form ion channels when expressed alone, whereas others are ion channel subunits that do not function by themselves in oocytes. (4) Biochemical studies of alphaENaC reveal a membrane topology consisting of two transmembrane domains (M1 and M2), cytoplasmic NH2 and COOH termini, and a large extracellular region containing cysteine-rich domains. Other family members are thought to have a similar molecular organization (Adams, 1998 and references therein).
In addition to the C. elegans proteins, three other neuronal family members have been identified. FaNaCh is a neuronal channel activated by the neuropeptide FMRFamide. Brain Na+ channel 1 (BNC1, also named MDEG and BNaC1) is widely expressed in human brain. Although its physiologic function is unknown, its channel activity can be enhanced by mutation of a residue associated with neuronal swelling in C. elegans DEG/ENaC proteins. A channel very similar to BNC1, BNaC2 (also named ASIC), is expressed in brain and dorsal root ganglia, and is activated by extracellular protons. These observations demonstrate a fifth feature of the family; in contrast to ENaC, neuronal DEG/ENaC channels may open only in the presence of specific stimuli or an activating mutation (Adams, 1998 and references therein).
The Drosophila embryonic and larval peripheral nervous system (PNS) is composed of segmentally repeated neuronal clusters within the body wall (designated d, l, v', and v), each containing a defined set of sensory neurons responsible for the innervation of different sensory structures. Ciliated organs such as the adult external sensory bristles and the larval chordotonal organs, thought to sense external touch and cuticle deformation, are innervated by bipolar type I sensory neurons. Type II multiple dendritic (md) neurons within the PNS, also referred to as dendritic arborization (da) neurons, extend dendritic processes to uniformly tile the internal epithelial surface. md neurons have been proposed to play a proprioceptive mechanosensory function to coordinate muscle contractions for larval movement. However, recent reports have implicated md neurons as possible thermosensory and/or nociceptive neurons. Distinct differences in morphology between subsets of md neurons suggest that subtypes may serve different sensory functions (Ainsley, 2003).
pickpocket1 (ppk1), encoding a member of the degenerin/epithelial sodium channel(DEG/ENaC) family, is expressed in a single md neuron within each of the d, v', and v PNS clusters (Adams, 1998). DEG/ENaC proteins were first characterized based upon genetic studies in the nematode that sought touch-insensitive mutants (Lingueglia, 1995). These proteins have subsequently been implicated as central components of a heteromultimeric mechanotransduction channel (Goodman, 2003). DEG/ENaC family members may also function as peptide neurotransmitter receptors, as salt taste sensors, or pH sensors with a possible role in synaptic plasticity. Therefore, the role of PPK1 protein in Drosophila neurons cannot necessarily be predicted based upon protein structure alone (Ainsley, 2003 and references therein).
In a pattern identical to the endogenous ppk1 gene, a ppk1.9-GAL4 transgene was expressed in a single md neuron within each of the d, v', and v PNS clusters as well as in four bipolar neurons in each of the upper brain lobes. ppk1 is expressed in the class IV md neurons ddaC, v'ada, and vdaB, displaying a characteristically uniform extension of processes to tile the internal surface of the epithelium between segmental boundaries (see Grueber, 2000 for a portrayal of these neurons). Extensive dendritic branches approach but do not cross the anterior and posterior segmental boundaries and appear to never overlap. Each PNS neuronal cluster contains multiple md neurons, only one of which expresses PPK1. Each md neuron cell body produces a single axon extending toward the ventral nerve cord with extensive axonal branching at the midline of the ventral nerve cord. Expression of nuclear-localized lacZ controlled by ppk1.9GAL4 reveals no detectable ppk1 gene expression in neuronal cell bodies of the ventral nerve cord (Ainsley, 2003).
ppk1 is located at position 35B1 just upstream of the alcohol dehydrogenase (Adh) gene. The small genomic overlap (44 kb) between two existing deficiency breakpoints, Df(2L)b88h49 and Df(2L)A400, precisely removes ppk1 to create a null genotype in transheterozygous flies (Df/Df). ppk1 null mutant animals (Df/Df) are viable as larvae and adults with no morphological or behavioral abnormalities easily detected during routine culture (Ainsley, 2003).
No defects in md neuron morphology were detected in ppk1 mutant larvae carrying ppk1.9GAL4 and UAS-DsRed, suggesting that PPK1 is not required for basic developmental steps. ppk1 mutant larvae were also indistinguishable from wild-type when tested using previously published paradigms for external touch with an eyelash or single hair and for their response to four different odorants in traditional olfactory taxis assays. md neurons have been implicated as nociceptors based upon the loss-of-function phenotype for the painless gene encoding a TRP-like channel protein. This phenotype is manifested as a putative pain response displayed as larval twisting and rolling when a hot (>38°C) probe is placed nearby. ppk1 mutant larvae were indistinguishable from wild-type in their ability to respond to a hot probe, suggesting that ppk1 does not participate in the same signal transduction pathway defined by the painless mutation (Ainsley, 2003).
ppk1 mutants were examined for larval locomotion by testing for differences in larval wandering behavior. When placed on an agarose sheet in the absence of food, wild-type third instar larvae display a characteristic pattern of wandering behavior, in which short bursts of forward movement (3-10 forward contraction waves) are separated by stops and repeated side-to-side head probes followed normally by a change in direction. Larval contour trails were created using digital larval motion analysis and scored for total contour trail area as a useful and accurate indirect measure of speed and stops. In addition, larval centroid trails were used to more precisely quantitate a number of specific parameters describing larval locomotion pattern (Ainsley, 2003).
ppk1 mutants move with a decreased number of stops or turns, resulting in an extended straight or slightly arching contour trail and a total contour trail area nearly twice that of wild-type. This enhanced locomotion pattern can be returned to wild-type values by providing wild-type PPK1 from a UAS-PPK1 transgene driven by ppk1.9GAL4. As an independent means of disrupting PPK1 activity, a ppk1RNAi transgene was created and expressed in transgenic flies. ppk1RNAi larvae display the same increase in contour trail area in a dosage-dependent manner. The moderate mutant phenotype caused by a single copy of ppk1RNAi can be shifted to a level comparable to the Df/Df null genotype by adding an additional copy of the transposon or by reducing endogenous gene dosage using a ppk1 deficiency chromosome. The UAS-PPK1[E145X] transgene encoding a truncated form of PPK1 containing only the first transmembrane domain was used to express a dominant-negative PPK1 isoform. UAS-PPK1[E145X] expression duplicated the significant increase in total contour trail area previously observed in ppk1 null mutants (Ainsley, 2003).
Additional behavioral parameters were determined using computer-generated larval centroid trails. Maximum translocation from the starting centroid was drastically increased in ppk1 mutants to a value of 66.99 mm (± 5.9) relative to only 29.93 mm (± 3.6) in wild-type. Fraction of time spent stopped was decreased from 0.41 (± 0.046) in wild-type to only .097 (± 0.017) in ppk1 mutants. Changes in direction of forward motion (>20°) was found to be decreased to 2.92 turns (± 0.61) in ppk1 mutants relative to 6.85 turns (± 0.76) in wild-type. Each of these values was returned to wild-type levels by providing wild-type PPK1 controlled by ppk1.9GAL4. Crawling speed during a linear locomotion burst of 5 or more frames was increased to 55.76 mm/min (± 1.1) in ppk1 mutants compared to 41.97 mm/min (± 1.7) in wild-type, and the duration of linear locomotion bursts without stops, only 6.7 s (± 0.45) in wild-type, was increased to 41.4 s (± 4.1) in ppk1 mutants. This value was returned to 8.24 s (± 0.48) in rescued larvae (Ainsley, 2003).
Larval contraction wave cycles during sustained linear locomotion can be divided into two parts consisting of the actual progression of the telescoping peristaltic wave of contraction from the posterior to anterior ends of the larva, separated by a brief pause prior to initiation of the next wave of contraction. Using high-resolution digital video, wild-type larvae were demonstrated to complete a full contraction wave cycle every 1.94 s, but ppk1 mutants complete the cycle in only 1.11 s, resulting in an increase in linear speed. This is accomplished by more rapid progression of the contraction wave from posterior to anterior and by a significant decrease in the length of the pause period. Wild-type larvae pause an average of 1.06 s between contraction waves while ppk1 mutants pause only 0.43 s (Ainsley, 2003).
ppk1 mutants crawl with significantly fewer stops, side-to-side head probes, and changes in direction and also cycle peristaltic contraction waves at a higher rate, causing increased linear speed. These characteristics could be mechanistically separable or could result from disruption of the same PPK1-mediated function. Overall neural control of rhythmic locomotion such as swimming, walking, or crawling is a complex ensemble performance with contributions from both central neurons responsible for rhythmic motor output and peripheral mechanosensory neurons providing moment-to-moment proprioceptive input concerning relative body position. The presence of PPK1 in peripheral md neurons is consistent with a role in mechanosensory signal transduction of proprioceptive sensory information from individual larval segments. However, rhythmic motor output driving locomotion originates in neural networks known as central pattern generators (CPG) present in the spinal cord or the analogous ventral nerve cord. The absence of cellular PPK1 expression in the ventral nerve cord suggests that PPK1 does not participate directly in function of individual CPGs. Ample axonal projections of md neurons to the ventral nerve cord imply that PPK1 may function in the regulatory control of motor output from the nerve cord (Ainsley, 2003).
Regulation of CPG motor output also extends from higher brain centers. Mutations disrupting the central complex, a large median neuropil in the insect brain, result in impaired walking in adults and defective crawling behavior in larvae. These defects are invariably displayed as a decrease in locomotion and do not resemble the enhanced locomotion observed in ppk1 mutants. Limited expression of PPK1 in only a subset of neurons may indicate that it is involved in only a small component of the overall regulatory mechanism (Ainsley, 2003).
The exact physiological functions of PPK1 and other DEG/ENaC proteins are not well understood and could contribute to neuronal excitability in a wide variety of direct and indirect ways. This new genetic model of DEG/ENaC function in the Drosophila system should serve as a valuable tool for further studies. Using the unusual enhanced locomotion phenotype and the restricted ppk1 expression pattern, future work should be able to gather useful information concerning motor control and the specific function of DEG/ENaC proteins in both peripheral and central neurons (Ainsley, 2003).
The cDNAs encoding Ppk and Ripped pocket (Rpk) predict proteins of 69 kD (606 amino acids) and 65 kD (562 amino acids), respectively. Hydrophobicity analysis suggests that Ppk and Rpk, like other DEG/ENaC proteins, possess two transmembrane domains (M1 and M2). Both proteins contain all of the conserved sequences present in known DEG/ENaC proteins including conserved cysteines in the predicted large extracellular region. Ppk contains seven potential N-linked glycosylation sites within its predicted extracellular region, and Rpk possesses two. Among known DEG/ENaC proteins, Ppk and Rpk most closely resemble each other, with 38% amino acid identity and 60% similarity. The next closest relative is the human neuronal ion channel BNC1, which is 24% identical and 45% similar to Ppk and 22% identical and 46% similar to Rpk. Within their predicted extracellular regions, Ppk and Rpk possess a unique domain not found in other family members. This region is encoded largely by a single exon in the ppk gene. Phylogenetic analysis indicates that Ppk and Rpk may represent a new DEG/ENaC subfamily (Adams, 1998).
date revised: 15 May 2004
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