slowpoke

DEVELOPMENTAL BIOLOGY

Embryonic

slowpoke expression throughout development has been examined. It is expressed in muscle cells, neurons of the CNS and PNS, mushroom bodies, a limited number of cells in embryonic and larval midgut and in epithelial-derived tracheal cells. The promoter has been cloned and shown to direct expression in the same pattern as the endogenous gene in both neural and epithelial-derived cells. During pupariation and embryogenesis, slo is expressed in muscles many hours prior to the appearance of functional channels (Becker, 1995).

The entire developmental history of muscle membrane electrogenesis can be observed in the embryonic myotubes of Drosophila. The development of ionic currents and muscle properties was examined using whole-cell patch-clamp techniques throughout embryonic myogenesis. In the early stages of myogenesis, from myoblast fusion through to establishing epidermal insertions, the myotubes are electrically inert and are electrically and dye coupled to adjacent myotubes. Membrane electrogenesis begins in the mid-embryonic stages (early stage 16), when the myotubes abruptly uncouple, revealing the first of five prominent extrajunctional currents: a small, inward, voltage-gated calcium current (ICa). The uncoupling of the embryonic myotubes heralds the onset of extremely rapid electrogenesis; within several minutes both the fast, inactivating (IA; Shaker) and delayed, noninactivating (IK) outward potassium currents, the stretch-activated outward potassium current, and the junctional glutamate-gated inward current all appear and begin to develop in a current-specific manner. Very late in embryogenesis (late stage 17), the calcium-dependent, outward potassium currents [rapid, inactivating (ICF; slowpoke) then delayed, noninactivating (ICS)] develop, completing the complement of macroscopic currents in the mature larval muscle. Hence, the voltage-gated currents (ICa, IA, and IK, respectively) appear relatively early, and the calcium-dependent currents (ICF, ICS) appear only very late during myogenesis. This developmental progression of current maturation is reflected in dynamic changes in the voltage responses of the embryonic membrane, from wholly passive response to current injection in the early, coupled myotubes to regenerating, overshooting action potentials in the mature embryonic muscle. The earliest embryonic IA current has a midpoint of inactivation 40 mV more negative than the IA current in the mature embryo. As myogenesis proceeds, the inactivation curve develops a biphasic character, suggesting that a low-inactivation IA channel is present in early development and progressively replaced by the mature form as development proceeds. The current at all stages can be completely eliminated in Shaker mutants (ShKS133). These findings suggest that an embryonic form of the Shaker IA channel is present during early myogenesis. The prominent IA current present in early development is almost entirely inactivated at the physiological resting potential; the significance and mechanism of this developmental shift are unclear (Broadie, 1993).

The slowpoke gene of Drosophila encodes a pore-forming subunit of a BK-type Ca^Ca2+-activated K+ channel. The gene is expressed in neurons, muscles, tracheal cells and in the midgut. The P1 transgene gene contains the entire slowpoke transcriptional control region and drives the expression of a reporter protein comprised of slowpoke amino terminal sequences fused to beta-galactosidase. Midgut expression is limited to the copper cell and iron cell regions. The copper cell region is composed of two cell types: the copper cells and the interstitial cells. The P1 transgene is expressed in the interstitial cells but not the copper cells. Furthermore, it is shown that the reporter protein is apically localized in the interstitial cells. In these cells, the slowpoke Ca^Ca2+-activated K+ channel is thought to participate in the transport of ions between the hemolymph and the lumen of the gut. Subcellularly localized BK channels may be involved in the secretion of acid into the gut lumen. An analogous role for basolaterally localized BK channels has been proposed in the acid-secreting intercalating cells of the human kidney (Brenner, 1997).

Effects of Mutation or Deletion

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 Ca^Ca2+-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).

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