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  • J. exp. Biol. 170, 93-111 (1992) 9 3 Printed in Great Britain © The Company of Biologists Limited 1992

    ANALYSIS OF REPOLARIZATION OF PRESYNAPTIC MOTOR TERMINALS IN DROSOPHILA LARVAE USING POTASSIUM-

    CHANNEL-BLOCKING DRUGS AND MUTATIONS

    BY MICHEL GHO* AND BARRY GANETZKYt

    Laboratory of Genetics, 445 Henry Mall, University of Wisconsin-Madison, Madison WI53706, USA

    Accepted 3 June 1992

    Summary

    In Drosophila melanogaster muscles and neuronal cell bodies at least four different potassium currents have been identified whose activity shapes the electrical properties of these cells. Potassium currents also control repolarization of presynaptic terminals and, therefore, exert a major effect on transmitter release and synaptic plasticity. However, because of the small size of presynaptic terminals in Drosophila, it has not been possible to analyze the potassium currents they express. As a first approach to characterizing the ionic currents present at presynaptic motor terminals of Drosophila larvae, we recorded synaptic currents at the neuromuscular junction. From the alterations in evoked synaptic currents caused by various drugs and by mutations known to affect potassium currents in other tissues, we suggest that the repolarizing mechanism in presynaptic terminals consists of at least four distinct currents. One is affected by aminopyridines or Sh mutations, a second component is affected by the slo mutation, a third is sensitive to quinidine and one or more additional components are blocked by tetraethyl- ammonium. Depolarization depends on a presynaptic calcium current, which displays only slight voltage-dependent inactivation. Because the mechanism of repolarization exerts a major effect on synaptic activity, this analysis provides a framework for further genetic and molecular dissection of the basic processes involved in the regulation of transmitter release.

    Introduction

    The capability of combining genetic, electrophysiological and molecular tech- niques has made Drosophila melanogaster one of the best experimental systems for studying ion channels and membrane excitability. This multidisciplinary approach has permitted the electrophysiological and pharmacological characteriz-

    * Present address: Laboratoire de Neurobiologie Cellulaire et Moleculaire, CNRS, 91190 Gif-sur-Yvette, France.

    tTo whom reprint requests should be addressed.

    Key words: transmitter release, membrane repolarization, potassium currents, neurogenetics, Drosophila melanogaster.

  • 94 M. G H O AND B. GANETZKY

    ation of particular ion currents, the identification of the genes that specify these currents, and molecular analysis of the encoded proteins (for reviews see Wu and Ganetzky, 1988; Papazian etal. 1988). These currents, which determine the electrical properties of excitable cells, have been studied in vitro and in vivo in muscle cells, photoreceptor cells and neuronal cell bodies (Salkoff, 1985; Gho and Mallart, 1986; Sole and Aldrich, 1988; Hardie, 1991). However, little is known about the currents present in other functional regions of neurons in Drosophila. For example, at the presynaptic terminal, the mechanisms underlying depolariz- ation and repolanzation are of fundamental importance in regulating transmitter release but little is known about the potassium currents that contribute to repolarization. Furthermore, little is known about the mechanisms underlying the synaptic plasticity observed at the presynaptic terminal in Drosophila larvae (Zhong and Wu, 1991a). It is known that modification of potassium activity, via several different second messenger systems, has important consequences for synaptic modulation and plasticity (Kandel and Schwartz, 1982; Alkon, 1984). Direct study of the presynaptic terminals in Drosophila has been difficult because of their small size. Here, as a first approach to investigating the repolarization mechanism of larval presynaptic terminals, we investigate the effects of drugs and mutations known to block potassium currents on the time course of transmitter release at the neuromuscular junction.

    At least four distinct potassium currents have been described in Drosophila muscles and neurons (Salkoff, 1985; Wu and Haugland, 1985; Gho and Mallart, 1986; Wei and Salkoff, 1986; Sole and Aldrich, 1988; Saito and Wu, 1990). These include two fast, transient currents, IA and ICF. IA is voltage-dependent and similar to the molluscan A current (Connors and Stevens, 1971; Neher, 1971), whereas ICF is activated by calcium and is similar to other calcium-dependent currents previously described in muscles and neurons (Mounier and Vassort, 1975; Yamamoto and Washio, 1981; MacDermott and Weight, 1982). In addition, there are two slow, sustained potassium currents, IK and Ics- IK corresponds to the classical non-inactivating, voltage-dependent delayed rectifier first described in the squid axon (Hodgkin and Huxley, 1952). I c s is a calcium-dependent current similar to that originally described in molluscan neurons (Meech and Standen, 1975). These currents are selectively affected by several drugs and mutations in different genes. For instance, aminopyridines as well as Sh mutations block IA in muscles and some neurons (Salkoff and Wyman, 1981; Salkoff, 1983; Sole etal. 1987; Baker and Salkoff, 1990; Saito and Wu, 1991). In other neurons, IA is mediated by a distinct type of channel sensitive to aminopyridines but not affected by Sh mutations (Sole et al. 1987; Baker and Salkoff, 1990). ICF is eliminated by slo mutations (Elkins etal. 1986; Singh and Wu, 1989; Komatsu etal. 1990) and quinidine selectively affects IK (Singh and Wu, 1989; Hardie, 1991). In addition, calcium blockers or low calcium concentration in the external solution affect the calcium-activated currents IcF and ICs-

    Previous studies have inferred the presence of potassium currents in the presynaptic motor nerve terminal by the phenotypic effects of mutations on the

  • Presynaptic currents in Drosophila 95

    postsynaptic potential, referred to as an excitatory junctional potential (EJP). For example, the presence of presynaptic potassium channels encoded by the Sh locus has been indicated by the enlarged prolonged EJPs observed in Sh mutants, consistent with a failure of normal repolarization of the presynaptic terminal (Jan et al. 1977). Similarly, a potassium current specified by the slo locus, a structural gene for calcium-activated potassium channels (Atkinson et al. 1991), may also be present at motor terminals because abnormally prolonged postsynaptic potentials are also seen in slo mutants (B. Ganetzky, unpublished results). However, because slo is important for proper repolarization of muscles (Elkins and Ganetzky, 1988; Singh and Wu, 1990), it is not clear whether the effect of slo on the postsynaptic potential is pre- or postsynaptic. Thus, alteration of the EJP by a particular mutation or drug does not always provide sufficient evidence that the potassium channels affected by these agents are found in the presynaptic membrane and steps must be taken to eliminate the contribution of postsynaptic conductances to the duration or time course of the synaptic current. We have circumvented this problem in these experiments by recording the excitatory junctional current (EJC) while keeping the muscle held at a constant membrane potential under voltage-clamp conditions.

    At the neuromuscular junction, transmitter release is triggered by an influx of calcium ions into the presynaptic terminal (Katz and Miledi, 1965, 1969; Dodge and Rahamimoff, 1967; Llinds and Nicholson, 1975) and is not initiated until the repolarizing phase at the end of the presynaptic action potential (Llinas et al. 1981, 1982). Prior to this, the presynaptic terminal is depolarized to a value near the calcium equilibrium potential and there is no net calcium entry. The delay before release of neurotransmitter and the resultant synaptic current in the postsynaptic cell are therefore primarily dependent upon the kinetics of repolarization under the control of potassium channels (Katz and Miledi, 1967a,b; Benoit and Mambrini, 1970; Datyner and Gage, 1980; Mallart et al. 1991). By measuring the EJC in larval muscle cells, we were able to obtain quantitative measurements of the delay before transmitter release and the onset of this response. These measurements provide information about the time before onset of the repolarizing phase of the presynaptic action potential, which in turn depends upon activation of potassium currents. Using this experimental paradigm, we examined the effects on evoked synaptic currents of several different potassium-channel-blocking mu- tations and drugs. Our data suggest that at least four potassium currents contribute to the repolarization of motor nerve terminals. These results provide a foundation for further genetic and molecular analyses of the molecular mechanisms involved in the regulation of transmitter release in Drosophila.

    Materials and methods

    Animals

    Experiments were performed on body-wall muscle 6 (for nomenclature see Johansen et al. 1989) in the second and third abdominal segments of D. melano-

  • 96 M. GHO AND B. GANETZKY

    gaster third-instar larvae. The larval preparation was similar to that previously described by Jan and Jan (1976). Previous measurements have shown that the larval muscles are essentially isopotential (Wu and Haugland, 1985). Wild-type flies were of the Canton-S strain. The mutations used were ShKSJ33, slo' and eag1, which were all raised under standard laboratory conditions.

    Voltage-clamp recording

    Synaptic currents were recorded using a two-electrode voltage-clamp. By use of the voltage-clamp to measure the synaptic currents, we avoided the problem of non-linear

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