Potassium channels have been implicated in central roles in activity-dependent neural plasticity. The
giant fiber escape pathway of Drosophila has been established as a model for analyzing habituation and
its modification by memory mutations in an identified circuit. Several genes in Drosophila encoding K+
channel subunits have been characterized, permitting examination of the contributions of specific
channel subunits to simple conditioning in an identified circuit that is amenable to genetic analysis. Mutations altering each of four K+ channel subunits (Sh, slo, eag, and Hyperkinetic[HK]) have
distinct effects on habituation at least as strong as those of dunce and rutabaga, memory mutants with
defective cAMP metabolism. Habituation, spontaneous recovery, and dishabituation of the
electrically stimulated long-latency giant fiber pathway response have been shown in each mutant type.
Mutations of Sh (voltage-gated) and slo (Ca2+-gated) subunits enhance and slow habituation,
respectively. However, mutations of eag and Hk subunits, which confer K+-current modulation, have
even more extreme phenotypes, again enhancing and slowing habituation, respectively. In double
mutants, Sh mutations moderated the strong phenotypes of eag and Hk, suggesting that their
modulatory functions are best expressed in the presence of intact Sh subunits. Nonactivity-dependent
responses (refractory period and latency) at two stages of the circuit are altered only in some
mutants and do not account for modifications of habituation. Furthermore, failures of the long-latency
response during habituation, which normally occur in labile connections in the brain, can be induced in
the thoracic circuit stage in Hk mutants. This work indicates that different K+ channel subunits play
distinct roles in activity-dependent neural plasticity and thus can be incorporated along with second
messenger "memory" loci to enrich the genetic analysis of learning and memory (Engel, 1998).
Potassium channels control the repolarization of nerve terminals and thus play important roles in the control of synaptic transmission.
The effects of mutations in the slowpoke gene are described
on transmitter release at the neuromuscular junction in Drosophila. The slowpoke mutant exhibits
reduced transmitter release compared to normal. Similarly, the slowpoke mutation significantly suppresses the increased transmitter
release conferred either by a mutation in Shaker or by application of 4-aminopyridine, which blocks the Shaker-encoded potassium
channel at the Drosophila nerve terminal. Furthermore, the slowpoke mutation suppresses the striking increase in transmitter release that
occurs following application of 4-aminopyridine to the ether a go-go mutant. This suppression is most likely the result of a reduction of
Ca2+ influx into the nerve terminal in the slowpoke mutant. It is hypothesized that the effects of the slowpoke mutation are indirect,
perhaps resulting from increased Ca2+ channel inactivation, decreased Na+ or Ca2+ channel localization or gene expression, or by
increases in the expression or activity of potassium channels distinct from slowpoke (Warbington, 1996).
Drosophila provides an excellent model for delineating the role of ion channels in the origin and transmission of heartbeat. Tests in Drosophila are reported on a wide range of mutations and pharmacological agents known to interfere with K+, Ca2+, Na+, and Cl- ion
channels in well-characterized ways. K+ channels are central to heart function. Tetraethylammonium, which blocks all four K+
currents, slows the heart. Distinctions can be made among these currents. The mutation slowpoke and the agent charybdotoxin, both
of which affect a fast CaCa2+-gated K+ channel, virtually eliminate heartbeat. Shaker and ether-a-go-go, which encode subunits of K+
channels, have moderate, possibly regulatory effects. 'OPQ-type' Ca2+ channels are critical. omega-Conotoxin MVIIC, which blocks
these channels, virtually stops the heart. Amiloride, which may affect T-type Ca2+ channels, has no effect, nor do the L-type Ca2+
blockers verapamil and diltiazem. Temperature induced paralysis E, involved in the function of Na+ channels, the Na+ channel blockers
tetrodotoxin and amiloride, and the Cl- blockers mefanamic and niflumic acids have no effect. Na+ and Cl- channels thus appear
unnecessary for cardiac function (Johnson, 1998).
A mutation of Drosophila, slowpoke, specifically abolishes a Ca2+-dependent K+ current, IC,
from dorsal longitudinal flight muscles of adult flies. Other K+ currents remain normal, providing
evidence that IC is mediated by a molecularly distinguishable set of channels. The pharmacological
properties of IC are similar to those of Ca2+-dependent currents in some vertebrate cells. The muscle
action potential is significantly lengthened in slo flies, indicating that IC plays the major role in its
repolarization (Elkins, 1986).
Drosophila TRPA1 channel is required to avoid the naturally occurring insect repellent citronellal
Plants produce insect repellents, such as citronellal, which is the main component of citronellal oil. However, the molecular pathways through which insects sense botanical repellents are unknown. This study shows that Drosophila uses two pathways for direct avoidance of citronellal. The olfactory coreceptor OR83b contributes to citronellal repulsion and is essential for citronellal-evoked action potentials. Mutations affecting the Ca2+-permeable cation channel TRPA1 result in a comparable defect in avoiding citronellal vapor. The TRPA1-dependent aversion to citronellal relies on a G protein (Gq)/phospholipase C (PLC) signaling cascade rather than direct detection of citronellal by TRPA1. Loss of TRPA1, Gq, or PLC causes an increase in the frequency of citronellal-evoked action potentials in olfactory receptor neurons. Absence of the Ca2+-activated K+ channel (BK channel) Slowpoke results in a similar impairment in citronellal avoidance and an increase in the frequency of action potentials. These results suggest that TRPA1 is required for activation of a BK channel to modulate citronellal-evoked action potentials and for aversion to citronellal. In contrast to Drosophila TRPA1, Anopheles gambiae TRPA1 is directly and potently activated by citronellal, thereby raising the possibility that mosquito TRPA1 may be a target for developing improved repellents to reduce insect-borne diseases such as malaria (Kwon, 2010).
Two features of the citronellal responses were found to be abnormal in the trpA11 basiconic sensilla ab11a neurons. First, there was a higher citronellal-evoked action potential frequency than in wild-type. Second, there was a defect in deactivation in trpA11 ab11a neurons. The same two defective phenotypes were observed in ab11 neurons in the dGqα1 and norpAP24 mutants, although only the increase in the evoked responses was clearly different when testing significance by analysis of variance. These results support the conclusion that the dGqα1, norpAP24, and trpA11 mutations affect the citronellal response in an ORN in ab11 (Kwon, 2010).
The finding that there were increases in the frequency of citronellal-evoked action potentials was unexpected and raised a question as to the basis for these defects. TRPA1 is a Ca2+-permeable channel, and because reduced activity of Ca2+-activated K+ channels (BK channels) increases the frequency of action potential firing, it was of interest to see whether loss of TRPA1 caused reduced BK channel activity. If so, then a mutation in the gene (slowpoke, slo) encoding the fly BK channel might phenocopy the trpA1 phenotype. In support of this model, the slof05915 mutation caused an increase in the frequency of citronellal-evoked action potentials and impaired citronellal avoidance. Introduction of UAS-slo-RNAi in combination with either the trpA1-GAL4 or the Or83b-GAL4 resulted in a similar defect in citronellal avoidance (Kwon, 2010).
It is proposed that TRPA1 is required for activity of Slo, which in turn modulates citronellal-induced firing of action potentials. Slo might be required in many ORNs and be regulated by additional TRP channels. In support of this proposal, ab12 also responded to citronellal and displayed a higher frequency of action potentials in slof05915 but did not function through a Gqα/PLC/TRPA1 pathway. Knockout of a mammalian TRP channel, TRPC1, also disrupts the activity of a Ca2+-activated K+ channel (KCa) in salivary gland cells, and mutations affecting either TRPC1 or KCa result in similar defects in salivary gland secretion. Thus, a role for TRP channels in activating Ca2+-activated K+ channels might be a common but poorly appreciated general phenomenon that is evolutionarily conserved (Kwon, 2010).
The finding that loss of TRPA1 causes an increase rather than a decrease in citronellal-induced action potentials suggests that there might be a TRPA1 independent-pathway required for generating action potentials in response to citronellal. OR83b is a candidate for functioning in such a pathway, because mutation of Or83b interferes with the ability of the synthetic repellent DEET to inhibit the attraction to food odors. This study found that Or83b1 mutant flies, or Or83b1 in trans with a deficiency that uncovers the locus, exhibited an impairment in citronellal avoidance similar to that in trpA1 mutant flies. An Or83b1 defect in the DART assay was not specific to citronellal, because these flies were also impaired in the response to benzaldehyde. Tested were performed to see whether the frequency of citronellal-induced action potentials was altered in Or83b1 ab11 sensilla. In contrast to the trpA1 mutant phenotype, none of the mutant Or83b1 ab11 neurons responded to citronellal (Kwon, 2010).
These data indicate that there are dual pathways required for the response to citronellal. OR83b is necessary for producing citronellal-induced action potentials, and a Gq/PLC/TRPA1 pathway appears to function in the modulation of action potential frequency by activating BK channels. It is suggested that an abnormally high frequency of action potentials may lead to rapid depletion of the readily releasable pools of neurotransmitter, thereby muting the citronellal response. Interestingly, a loss-of-function mutation affecting a worm BK channel also results in a behavioral phenotype—increased resistance to ethanol. Although Drosophila TRPA1 functions downstream of a Gq/PLC signaling pathway, citronellal can also directly activate TRPA1, but with low potency. Nevertheless, because Anopheles gambiae TRPA1 is also expressed in the antenna and is activated directly by citronellal with high potency, it is suggested that mosquito TRPA1 represents a new potential target for in vitro screens for volatile activators that might serve as new types of insect repellents (Kwon, 2010).