Presynaptic calcium-channel currents in normal and csp mutant Drosophila peptidergic terminals

Presynaptic calcium-channel currents in normal and cspmutant Drosophila peptidergic terminals

Miguel Morales,* Alberto FerruÂs and Manuel MartõÂnez-PadroÂnInstituto Cajal (CSIC) Ave. Doctor Arce 37, Madrid 28002, Spain

Keywords: calcium channels, cysteine string protein, Drosophila melanogaster, neuropeptide, release, synapse

Abstract

The study of regulated vesicle exocytosis, which underlies neurotransmitter and neuropeptide release, has bene®ted from aconvergence of several independent approaches. These include the use of genetically tractable organisms and model preparationsthat allow a direct characterization of presynaptic ionic currents. Aiming for a comprehensive analysis of release, we had alreadydeveloped a Drosophila preparation in which electrophysiological recordings from peptidergic terminals are feasible. Here, we reporton the characterization of the Ca2+-channel currents present in these terminals. With Ba2+ as the charge carrier, the presynapticmembrane expresses a current type with high-activation threshold and little inactivation. This current is blocked by verapamil anddiltiazem at micromolar concentrations, it is relatively insensitive to nifedipine and completely resistant to non-L-type Ca2+-channelantagonists. As a comparison, we also analysed the pharmacology of high-threshold Ba+2 currents on muscle ®bres. A high-activation threshold Ca2+-channel current is also present in muscle ®bres, albeit with a distinct pharmacological pro®le. Thus,peptidergic terminals and muscle ®bres exhibit different subtypes of voltage-gated Ca2+ channels. The putative role of cysteine stringprotein (CSP) as a neuronal Ca2+-channel modulator was tested by examining the peptidergic presynaptic current in csp nullmutants. We show that CSP is expressed in peptidergic boutons and abolished in the mutant. Direct recordings, under conditions thatinhibit calcium in¯ux into glutamatergic terminals, show that Ca2+-currents in peptidergic csp terminals are entirely normal. This resultindicates that CSP is not a generic Ca2+-channel modulator and it might perform different functions in fast versus slow forms ofrelease.

Introduction

Voltage-gated Ca2+ channels have been identi®ed as a ubiquitous

component of animal cells of virtually all phyla. They play a critical

role in depolarization±secretion coupling and, hence, their character-

ization is a requirement in the study of synaptic function. The

application of electrophysiological techniques to study presynaptic

currents has been hindered by the extremely small size of nerve

endings and, with some exceptions (e.g. LlinaÂs et al., 1981; Lemos &

Nowycky, 1989; Stanley & Goping, 1991), the nature of presynaptic

calcium channels has been inferred from the pharmacological

sensitivity of neurotransmitter release to selective antagonists. Based

on biophysical and pharmacological criteria, vertebrate calcium

channels have been classed into types T, N, L, P, Q and R. Thus, L-

type channels are highly sensitive to inorganic compounds, including

phenylalkylamines (PA), benzothiazepines (BTZ) and dihydropyr-

idines (DHP), while the peptide toxins w-Conotoxin GVIA and w-

Agatoxin IVA appear rather selective for channel types N and P,

respectively.

Transmitter release is triggered by a sudden increase in Ca2+

concentration restricted to the vicinity of the release machinery. Slow

exocytosis, as it occurs in neuropeptide release, has many features in

common with hormone release from endocrine cells. High-frequency

stimulation is usually required for optimal peptide release (Iverfeldt

et al., 1989), presumably due to the need for a sustained increase in

bulk calcium concentration at presynaptic terminals (Neher &

Zucker, 1993). It has been suggested that Ca2+ entry via strategically

located N- and/or P-type channels dominates the release of small

clear synaptic vesicles that contain `fast' neurotransmitters, whereas

L-type channels are often linked to the release of neuropeptide-

containing large dense-core vesicles (Dunlap et al., 1995). Larval

muscle ®bres in Drosophila are contacted by several types of synaptic

terminals (Atwood et al., 1993; Jia et al., 1993). Among these, type I

boutons contain small clear vesicles and mediate glutamatergic

release (Jan & Jan, 1976; Johansen et al., 1989), whereas peptidergic

type III boutons contain large-dense core vesicles and exhibit insulin-

like and proctolin immunoreactivity (Anderson et al., 1988; Gorczyca

et al., 1993).

As triggers of synaptic transmission, presynaptic calcium channels

interact with synaptic proteins of the docking-fusion machinery, such

as syntaxin and SNAP-25 (Wiser et al., 1996; Rettig et al., 1997), and

this interaction might depend on the channel type (Bezprozvanny

et al., 1995). Cysteine string proteins (CSP) are a major component of

all synaptic vesicle membranes (Mastrogiacomo et al., 1994; Pupier

et al., 1997). They have been proposed to modulate the activity of

presynaptic calcium channels (Gundersen & Umbach, 1992).

Drosophila csp mutants exhibit a temperature-dependent loss of

nerve-impulse evoked transmission, this defect being attributed to a

failure in depolarization-secretion coupling (Umbach et al., 1994).

Indirect evidence suggested that Ca2+ in¯ux into presynaptic

terminals could be impaired in csp mutants (Umbach & Gundersen,

Correspondence: Dr A. FerruÂs, as above. E-mail: [email protected]

*Present address: UCSD Department of Biology, 9500 Gillman Drive, LaJolla, CA 92093±0346, USA

Received 17 November 1998, accepted 13 January 1999

European Journal of Neuroscience, Vol. 11, pp. 1818±1826, 1999 Ó European Neuroscience Association

1997; Ranjan et al., 1998). This hypothesis has received support from

Ca2+ imaging experiments on csp mutant terminals (Umbach et al.,

1998). We have developed a preparation that allows patch-clamp

recordings from type III terminals in vivo (MartõÂnez-PadroÂn & FerruÂs,

1997; 1998). Here, we combine this technical achievement with the

genetic capabilities of Drosophila to characterize presynaptic Ca2+-

channel currents in these terminals, as well as to study their proposed

modulation by CSP. We show that this protein, readily expressed in

type III boutons, does not appear to be a generic Ca2+ channel

modulator.

Materials and methods

Mutants and experimental preparation

The ecdysone (ecd1) allele is a temperature sensitive recessive lethal

(Garner et al., 1977). When kept at 30 °C, ecd1 mutant larvae fail to

pupate and develop enlarged type III synaptic boutons. Although

electrophysiological recordings are feasible in wild type strains and

csp mutants, they are much easier in ecd1 mutants, and we have

systematically used this mutant to characterize presynaptic type III

Ca2+-currents, and as controls for comparison with csp mutants at

restrictive temperatures. Nevertheless, we have obtained a limited

number of recordings from the Oregon-R wild type strain in order to

ensure that ecd1 currents are very much like wild type. The

Drosophila cspR1 allele represents a deletion of the entire csp gene.

cspR1 is a temperature-sensitive paralytic mutant. About 4% of the

animals survive to adulthood at 25 °C, but none at 29 °C (Zinsmaier

et al., 1994); this temperature-sensitive paralysis is caused by a

reversible failure of evoked synaptic transmission (Umbach et al.,

1994; Zinsmaier et al., 1994).

The experimental preparation to record ionic currents from type III

synaptic boutons has been previously described (MartõÂnez-PadroÂn &

FerruÂs, 1997, 1998). Brie¯y, third instar larvae were pinned down

onto a Sylgard-coated experimental chamber, dissected open along

the dorsal midline in a solution containing (in mM): NaCl, 100; KCl,

5; MgCl2, 20; HEPES, 5; and Sucrose, 115 (pH 7.3), and digested for

8±10 min in the same solution with 200 U/mL collagenase (Type IA;

Sigma, St Louis, USA). Type III terminals were unambiguously

identi®ed using a 3 40 Nomarski water immersion objective (Nikon).

Unless otherwise indicated, all electrophysiological recordings were

performed at room temperature (21±23 °C) on type III synaptic

terminals innervating ventro-longitudinal muscle ®bre 12 (segments

A2±A6).

Electrophysiological recordings

The perforated patch-clamp technique (Horn & Marty, 1988) was

used to record whole-terminal currents as previously described

(MartõÂnez-PadroÂn & FerruÂs, 1997). Brie¯y, high resistance pipettes

(10±20 MW were pulled from thin-wall borosilicate glass (World

Precision Instruments). The pipette tip was ®lled with an internal

solution containing (in mM): CsCl, 140; MgCl2, 4; HEPES, 10;

EGTA, 5±10 (pH 7.3), and then back-®lled with the same solution to

which a ®nal concentration of 200 mg/mL Nystatin (Sigma) was

added. Whole-terminal currents were recorded with a patch-clamp

ampli®er (Axopatch 1D), ®ltered at 2 kHz, digitized and stored in a

computer for further analysis using PClamp Software (Axon

instruments, CA, USA). Upon seal formation and membrane

perforation, the pipette tip resistance increases 3±4 times, which

represents a voltage error of < 5 mV for a current magnitude of

100 pA (see MartõÂnez-PadroÂn & FerruÂs, 1997) and » 1 mV with 80%

series resistance compensation. Currents were leak-corrected by

subtracting the current produced by a pulse protocol of the same

magnitude but opposite polarity. Pooled data in the text are presented

as mean 6 SEM.

In most experiments (see below), the bath solution used to record

calcium-channel currents from synaptic terminals contained (in mM):

NaCl, 100; KCl, 5; BaCl2, 5; MgCl2, 20; HEPES, 5; Sucrose, 115, and

5mM TTX (pH 7.3). Because Ba2+ does not support muscle contrac-

tion, it yields more stable current recordings. Also, Ba2+ acts as a K+

channel blocker and helps to reduce contaminating K+ currents, which

are further blocked by the presence of Cs+ in the pipette solution.

Muscle calcium currents were recorded from muscle ®bre 6 (segments

A2±A3), using an Axoclamp-2 A ampli®er in the two-electrode

voltage-clamp mode. Short-shank micropipettes were pulled to a ®nal

resistance of 10±20 MW when ®lled with a 1 m KCl solution. The

external solution for muscle current recordings was the same as for

synaptic recordings, except that it included 100 mM quinidine and

10 mM 3,4-diaminopyridine to reduce contaminating K+ currents. In

those experiments addressing the function of CSP, the temperature of

the bath was controlled by a junction Peltier (HC10A controller) with a

HC10AD headstage (Dagan Corporation, Minneapolis, USA).

Space-clamp considerations

As type III terminals are connected by ®ne axonal processes, we were

concerned that current generated at poorly space-clamped adjacent

terminals could signi®cantly contaminate our recordings. In order to

assess the severity of this problem we performed simultaneous

recordings from two adjacent boutons. The results from one

successful experiment are shown (Fig. 1). The lower traces corre-

spond to Ba2+-inward currents recorded in one terminal (A) under

voltage-clamp conditions, in response to depolarizing voltage steps

from ±50 mV; while the upper traces represent the membrane

potential change recorded in an adjacent terminal (B) » 12 mm away.

These currents show a particularly poor clamp, and they are typical of

those recorded immediately after seal formation, but they have been

selected to illustrate the electrical relationship between the two

terminals. Voltage steps below ±20 mV caused a small, progressive

depolarization of terminal B, and no detectable inward current at

terminal A. However, voltage commands to ±20 mV and above,

trigger a much larger depolarization in B; its temporal course is

closely matched by the current pro®le at terminal A, and we attribute

it to Ba2+ in¯ux through Ca2+ channels in terminal B. Thus, Ba2+-

currents recorded at type III boutons under these conditions, are

signi®cantly contaminated by Ba2+ in¯ux at nearby terminals. As a

compromise solution, we sought to restrict activation of barium

currents to a single terminal by using focal application of 10 mM Ba2+

(or Ca2+), in extracellular solution, delivered from an unpolished

patch pipette located in close proximity to the bouton. Although

records using this procedure were often contaminated by current from

poorly clamped nearby boutons, as suggested by the sudden, all-or-

none activation of the current (Fig. 2A) and a progressive reduction in

latency to peak current with increasing depolarization, a number of

successful cases showed inward currents with progressive activation

and constant time to peak, suggestive of little or no contamination

(Fig. 2B). This latter set of data was used to derive the biophysical

parameters of the synaptic Ca2+-channel current.

Drugs

Verapamil hydrochloride and diltiazem hydrochloride (Roig Farma,

Barcelona, Spain) were dissolved in saline solution. Nifedipine

(Sigma) was dissolved in ethanol at a stock concentration of 10 mM.

Synthetic Funnel Web spider toxin was a gift from Dr R. LlinaÂs (New

York University, USA). All peptide toxins (Alomone Lab.,

Drosophila presynaptic calcium currents 1819

Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 1818±1826

Jerusalem, Israel) were dissolved in stock solution containing (in

mM): NaCl, 100; Tris, 10; EDTA, 1, and 0.1% BSA (pH 7.5), and

kept stored at ±20 °C. Bath solution exchanges were made by ¯ushing

the recording chamber with a solution volume 10 times larger at a

perfusion rate of 4 mL/min. The toxin peptides were added directly to

the recording chamber to obtain the desired ®nal concentration.

Immunolabelling

Third instar larvae were ®xed for 30 min in freshly made 4%

paraformaldehyde in Ca2+-free PBS containing 5 mM EGTA. Speci-

mens were incubated in mouse monoclonal anti-CSP antibody

(mAb49, 1 : 200, Buchner et al., 1988) overnight at 4 °C in PBT

(0.1% Triton in PBS), thoroughly washed in PBT and then incubated

in secondary antimouse antibody conjugated to Cy2 for 1 h at room

temperature. Preparations were mounted on PBS:glycerol (1 : 1) and

viewed under confocal microscopy.

Results

Calcium-channel currents in type III synaptic terminals

A typical family of Ba2+ currents, activated from a holding potential

of ±50 mV in 10 mV steps, where lack of space-clamp control

becomes evident, is depicted in Fig. 2A (left panel). In this particular

case, 10 mM Ba2+ solution was applied from an unpolished patch

pipette placed close to the bouton, but similar currents are obtained if

Ba2+ is present in the bath instead. Membrane steps below ±20 mV

did not elicit any inward current. In some cases, the current became

active in an all-or-none fashion at » ±15 to ±10 mV, whereas in other

cases some gradation in the activation could be observed, presumably

depending on the degree of coupling between boutons. On average,

the macroscopic current thus recorded reached a maximum peak at

» 0 mV, with little inactivation throughout the test pulses. Bath

application of 200 mM Cd2+caused a complete blockade of the

macroscopic inward current (not shown), indicating that it is, in all

likelihood, due to Ba2+ in¯ux through Ca2+ channels. With further

membrane depolarization, the current amplitude declines as the

electrochemical driving force for Ba2+ diminishes. A limited number

of Ca2+ current recordings (n = 3) was obtained by delivering Ca2+

(10 mM) from a nearby pipette (Fig. 2A, right traces). The currents

thus recorded, showed two important features when compared with

those recorded in Ba2+: (i) the current exhibited a more inactivating

pro®le; and (ii) current activation also occurred in an all-or-none

fashion, but at a more positive potential (0 mV) than with Ba2+

(Fig. 2C).

Using the procedure of focal delivery of divalent cations, and

restricting the voltage range to relatively lower values, we could

record a limited number of Ba2+ (n = 3) and Ca2+ (n = 1) currents

showing no contamination, as judged from their progressive

activation and constant time to peak (Fig. 2B). From a holding

potential of ±50 mV, and using Ba2+as the charge carrier, depolariz-

ing voltage steps above ±30 mV produced a relatively noninactivating

terminal inward current, which peaked at » 10 mV (Fig. 2B, left

traces). With extracellular Ca2+ (Fig. 2B, right traces), however, the

current showed considerable time-dependent inactivation. Also, the

threshold for current activation was 15±20 mV more depolarized than

in Ba2+. Membrane depolarization to the same potentials, before

delivery of the divalent cation from the pipette, produced only a small

outward leak current. This small current which was used for leak

subtraction, further indicates that the inward current is probably due

to the in¯ux of the cation used. The corresponding current±voltage

(I±V) relationships are shown in Fig. 2C.

We have explored the possibility that synaptic terminals could

contain a Ca2+ current component that would undergo steady-state

inactivation at the resting potential. Figure 2D shows superimposed

current traces activated by a test potential to 10 mV from a variety of

holding potentials (between ±100 mV and ±30 mV). Using this

voltage protocol, we observed no differences in either peak amplitude

or time course of the Ba2+ current evoked by the test pulse.

Notwithstanding the space-clamp problems, these results suggest that

no low-voltage activated component is present in type III terminals.

In conclusion, the single voltage-dependent Ca2+-channel current

detected exhibits a high activation threshold (above ±30 mV) and may

undergo Ca2+-dependent inactivation.

Pharmacology of synaptic and muscle Ca2+ currents

Accepting the fact that the Ca2+ channel current will be contaminated

by in¯ux at nearby terminals, these experiments were carried out

using 5 mM Ba2+ in the bath. Consequently, we aimed at a qualitative,

rather than a quantitative, pharmacological analysis. Presynaptic

Ba2+-currents, elicited from a holding potential of ±50 mV, were

reduced by verapamil in a concentration-dependent manner (Fig. 3A).

Bath application of 10 mM verapamil did not have any effect, whereas

50 and 100 mM reduced the current by 60% and 90%, respectively

(n = 3). These data indicate that 50% blockade would require » 30 mM

FIG. 1. Simultaneous recordings from two adjacent type III boutons. TerminalA (lower traces) was voltage-clamped at ±50 mV while terminal B (uppertraces) was recorded under current-clamp. Increasing depolarizing voltagecommands applied to terminal A in 10 mV steps are detected in terminal B,®rst as small depolarizations, which increase linearly, and then as much largerstepwise increases coincidental with the all-or-none activation of the inwardcurrents at terminal A. The time courses of terminal B membrane potentialsand terminal A currents are closely related. This is more obvious for the pulseto ±20 mV. In this particular case, the terminal B membrane potential at thebeginning of the pulse behaves very much the same as for morehyperpolarized pulses, re¯ecting electrotonic transmission from terminal A.However, later in the pulse, the membrane potential suddenly increases,presumably due to opening of terminal B Ca2+ channels, which is detected interminal A as a large, and also sudden, inward current.

1820 M. Morales et al.


verapamil. Diltiazem (Fig. 3B) also inhibits presynaptic currents in a

concentration range between 25 mM (29.25% 6 0.5, n = 3) and 200 mM

(87% 6 1.2, n = 3), with half-maximal effect at » 50 mM (55% 6 12,

n = 3). The inhibitory effect of DHPs on Ca2+ currents is voltage-

dependent (Lee & Tsien, 1983). For this reason, we assayed the effect

of nifedipine using a holding potential of ±30 mV. In these

conditions, 10 mM nifedipine had no effect on type III Ba2+ currents.

Increasing the concentration to 100 mM reduced the current amplitude

by 20% (n = 3, Fig. 3C), whereas the concentration needed for a 50%

blockade was near 200 mM (55%, n = 3). The highest concentration

tested (300 mM) only caused 80% inhibition of the total current. From

these results, we conclude that Ca2+ currents in type III terminals are

mediated by a population of channels which share some of the

pharmacological properties of L-type channels in vertebrates (see

Discussion section). Furthermore, vertebrate tissue-speci®c L-sub-

types are the preferential targets of certain snake-venom toxins. Thus,

calciseptine and calcicludine are potent neuronal L-type blockers but

are completely ineffective on L-channels from skeletal muscle (De

Weille et al., 1991; Schweitz et al., 1994). When tested on type III

terminals, neither calcicludine (10 mM) nor calciseptine (2 mM), even

at concentrations well beyond those reported for complete blockade

in vertebrates, had any effect on the Ba2+ current (n = 3 for each toxin,

data not shown).

In invertebrates, channels with mixed pharmacology are usually

found (Skeer et al., 1996), and thorough characterizations which

would help to assess the selectivity of calcium channel antagonists

further, is lacking. For these reasons we have analysed the effect of a

number of non-L-type calcium channel antagonists on the synaptic

current. These include N-, P- and Q-channel blockers (see Table 1)

and also PLTX, a potent toxin from the venom of the spider

Plectreurys tristes, which is speci®c for insect Ca2+ currents and

irreversibly blocks synaptic transmission in Drosophila (Branton

et al., 1987). None of these antagonists, however, had any effect on

the presynaptic Ba2+ current at peptidergic type III terminals.

A high-threshold calcium-channel current which is sensitive to 1,4-

dihydropyridines and diltiazem has been previously reported on larval

muscle ®bres (Gielow et al., 1995). For comparison, we have

reanalysed the pharmacological pro®le of muscle Ba2+ currents

under our experimental conditions. The pharmacology of the muscle

current showed noticeable differences from that of type III terminal

currents. In agreement with previous results, the muscle current

proved less sensitive to verapamil and diltiazem, exhibiting an IC50 of

178 mM and 262 mM, respectively (Fig. 3A and B), and it was almost

two orders of magnitude more sensitive to nifedipine (IC50 = 3.3 mM;

Fig. 3C). These results led us to conclude that Drosophila muscle

®bres express a Ca2+ channel type, reminiscent of vertebrate's L-type,

which is clearly different from the channel expressed in type III

synaptic terminals.

Presynaptic Ca2+-channel currents in normal versus cspmutants

Drosophila csp mutant larvae exhibit only moderate synaptic

depression at 22 °C but, when exposed to temperatures above

30 °C, they become paralysed and undergo a progressive decline

and ®nal failure of synaptic transmission (Umbach et al., 1994;

Zinsmaier et al., 1994; Heckmann et al., 1997). The interpretation has

been that, faced with a thermal challenge, the absence of CSP protein

reduces the dissociation threshold of a protein or protein complex

which is essential for evoked release, and this presumably

compromises presynaptic Ca2+ channels (Umbach et al., 1994;

FIG. 2. Whole-cell recordings from presynaptic type III terminals. Poorly clamped (A) and well-clamped (B) Ba2+ (left traces) and Ca2+ (right traces) inwardcurrents recorded in response to depolarizing pulses from a holding potential of ±50 mV (C) I±V relationship for the average peak Ba2+ (n = 3) and Ca2+ (n = 1)currents which displayed progressive activation. (D) To test for the presence of low-voltage activated currents (putative T channels), terminals were held for up to10 s at a holding potential between ±100 and ±30 mV, and then depolarized to 10 mV. No signi®cant differences in current amplitude or time course are observed.



Umbach & Gundersen, 1997). In this respect, Umbach et al. (1998),

using calcium imaging, have recently shown that nerve-stimulated

Ca2+ in¯ux into glutamatergic synaptic boutons is strongly reduced in

csp mutants at restrictive temperature. CSP protein, however, has

been reported to associate with both small clear and large dense-core

vesicle membranes (Mastrogiacomo et al., 1994; Pupier et al., 1997).

The protein is also abundant in non-neuronal secretory tissues

(Koham et al., 1995), suggesting that it may be present in all vesicles

targeted for exocytosis.

Given this widespread distribution, we were interested in

determining whether type III boutons express CSP protein and, in

that case, whether Ca2+ in¯ux into these terminals would also be

impaired in csp mutants. As a ®rst step, we applied immunolabelling

techniques to normal larvae, using a monoclonal antibody against

CSP. In toto preparations of wild type revealed an intense staining

restricted to synaptic endings at all neuromuscular junctions

(Fig. 4A). Close inspection of CSP immunoreactivity on muscle ®bre

12 (Fig. 4B) revealed that the protein is expressed in all types of

synaptic endings, which can be easily distinguished in ®xed tissue

because of their size, general shape and innervation pattern (Jia et al.,

1993). In particular, type III terminals appear as elliptical,

intermediate size varicosities connected by ®ne, long processes and,

very often, they span most of the muscle ®bre length (Fig. 4B).

Immunoreactivity against CSP was completely absent in cspR1

FIG. 3. Pharmacology of type III presynaptic and muscle Ba2+ currents. Effect of 100 mM verapamil (A), 200 mM diltiazem (B) and 10 mM nifedipine on type IIIterminal Ba2+currents (left). Percentage reduction of synaptic and muscle peak current as a function of drug concentration (right). The IC50 values for musclecurrent inhibition calculated as the slope of the double-reciprocal plot of the dose±response curve (not shown) were 178 mM for verapamil, 262 mM for diltiazem and3.3 mM for nifedipine. Each data point represents an average of at least 3 different cells. Ba2+ was at an extracellular concentration of 5 mM.

TABLE 1. Pharmacology of Ba2+ currents

Type III terminals: Muscle ®bres:concentrations for half- IC50maximal inhibition (mM) (mM)

Nifedipine » 200 3Verapamil » 30 178Diltiazem » 50 262Calcicludine No effect No effectCalciseptine No effect No effectN-type Blockers No effect Not testedP-type Blockers No effect Not testedQ-type Blockers No effect Not testedPLTX No effect No effect

N-type channel blockers used were: w-Conotoxin GVIA (1 mM);w-Conotoxin MVIIA (2 mM); and w-conotoxin SVIB (2 mM). The P-Typeblockers used were w-Agatoxin IVA (300 nM) and synthetic Funnel Webspider toxin (500 mM). The Q-type blocker was w-Conotoxin MVIIC (2 mM).PLTX was used at a concentration of 0.1 mM. At least three animals weretested per toxin.



mutant larvae (Fig. 4C), both in type III and other terminals, thereby

con®rming that cspR1 is a null mutation for the csp gene.

As temperature-induced paralysis is the characteristic feature of

csp mutants, we tested our laboratory stock for temperature-

dependent paralysis. All animals (n = 30) within a group of csp third

instar larvae exposed to 32 °C, became paralysed within 18 min (not

shown); in like manner, a similar group exposed to 34 °C, became

completely paralysed within 12 min. Control groups of ecd1 larvae

run in parallel in each case, were not affected by these procedures. In

addition, all csp larvae recovered within 1 h upon returning to room

temperature.

Proposals about the role of CSP, suggest that the thermal paralysis

results from a corresponding thermal dependence of a Ca2+ current in

the mutant. We examined type III synaptic Ba2+ currents in cspR1

mutants and compared them with those in ecd1 and wild type as

controls. Figure 5B represents average Ba2+ current peak-voltage

relationships for ecd1, Oregon-R and cspR1 larvae at room

temperature (22 °C). No major differences appear between the three

genotypes. The peak current amplitude measured at 0 mV was

97.3 6 5.4 pA (n = 19) for ecd1, 96.6 6 11.9 (n = 9) for Oregon-R and

93.2 6 7.9 (n = 5) for cspR1 larvae, suggesting that ecd1 Ba2+ currents

are entirely similar to wild type and that lack of CSP protein per se,

does not affect this current at the type III terminals.

Further, increasing the temperature from 22 °C to 32 °C had no

major effect on the average current peak amplitude of either ecd1

(98.6 6 22.2 pA at 0 mV, n = 5) or cspR1 larvae (108.7 6 13.4 pA at

0 mV, n = 9) (Fig. 5A and C). That is, temperature-dependent

paralysis of csp mutant larvae does not correlate with any signi®cant

reduction of type III terminal Ba2+ currents. Considering that type III

boutons do express CSP protein, our results suggest that CSP must

play an alternative role in these terminals.

Discussion

This study represents the ®rst attempt to characterize directly

presynaptic Ca2+-channel currents in peptidergic terminals of

Drosophila. Poor space-clamping posed a major problem which

prevented us from doing a precise quantitative study. We tried to

restrict Ca2+-channel activation to a single bouton by applying

divalent cations locally. The procedure yielded a modest rate of

success, and a large number of recordings was required to obtain a

few well-clamped currents, as judged by their progressive activation

and constant latency to peak.

Type III terminals express a high-voltage activated (HVA) Ca2+-

channel current with no evidence of a low-voltage activated (T-type)

component. With Ba2+ as charge carrier, the current shows little

inactivation during the test pulse. The I±V plot shows an activation

threshold at » ±20 mV and a maximal current of » +10 mV. The limited

data we have obtained with Ca2+ perfusion showed a clearly

inactivating current pro®le which suggests that these channels may

undergo Ca2+-dependent inactivation. Also, it would appear that Ba2+

causes a leftward shift of the I±V relationship as has been observed in

other preparations (e.g. Branchaw et al., 1997). Pharmacological tools

have been used in many studies to distinguish between HVA channel

types. Synaptic Ba2+ currents were not sensitive to drugs or to toxins

considered highly speci®c for vertebrates Ca2+-channel types. The

Drosophila synaptic current was blocked by PAs and BTZs only,

compounds which are considered less selective for L-type channels.

FIG. 4. CSP immunolabelling in larvalneuromuscular junctions. (A) Low-magni®cation confocal micrograph of ecd1

muscle ®bres showing high levels of CSPimmunoreactivity localized to presynapticendings (arrows). (B) High-magni®cationmicrograph showing the presence of CSPantigen in all types of synaptic boutons, aslabelled. Terminals of types Ib and II areclearly distinguishable because of their largeand small size, respectively. Elongated,medium size, type III boutons can be separatedfrom type Is because they are connected by®ne, long processes, and span most of muscle®bre 12. (C) CSP staining is completely absentin the null mutant cspR1. Scale bars, 50 mm (Aand C) and 10 mm (B).



As a further indication of the divergence between Drosophila and

vertebrate Ca2+ channels, there is evidence that the binding sites for

DHPs and PAs are likely to be dissociated in Drosophila and other

invertebrate neurons (SchaÈfer et al., 1994; Wicher & Penzlin, 1997).

Pelzer et al. (1989) found several Ca2+ channel types in reconstituted

Drosophila head membranes. One channel is highly sensitive to PAs,

but insensitive to micromolar concentrations of DHPs, whereas two

other channels proved DHP-sensitive but PA-insensitive. These

observations ®t well with radioligand binding studies of the

Drosophila head, showing that PA-binding sites lack allosterically

coupled DHP receptors (Pauron et al., 1987; Glossmann et al., 1991).

While vertebrate L-channels bind DHPs, PAs and BTZs on the same

subunit, in Drosophila a particular channel appears to have binding

sites for either PAs or DHPs, but not for both. In fact, the only putative

a1 subunit so far cloned from the Drosophila adult head contains a

conserved PA-binding domain, but not a canonical DHP-binding site,

as known in vertebrates (Zheng et al., 1995). The emergent picture is

that of a very large repertoire of Ca2+ channels among cell types and

species.

Direct studies of presynaptic calcium channels have been limited

by the availability of preparations with suf®ciently large nerve

endings. Bipolar neurons from gold®sh retina only express a slowly

inactivating L-current component (Heidelberger & Matthews, 1992).

In contrast, dissociated nerve terminals from rat neurohypophysis and

presynaptic calyces of chick ciliary ganglia both contain several

Ca2+-current components (Lemos & Nowycky, 1989; Yawo, 1990).

Drosophila type III synaptic Ca2+ currents are most similar to

currents described in somata and isolated terminals of crab

neurosecretory cells (Richmond et al., 1995, 1996). These peptidergic

neurons appear to express a single Ca2+ current type with a high-

activation threshold and calcium-dependent inactivation, which, as in

the ¯y, is rather insensitive to nifedipine w-Conotoxin GVIA and w-

Agatoxin IVA.

The presence of Ca2+ currents in other Drosophila neurons is not

characterized with enough resolution to allow conclusions on the

diversity of Ca2+ channels present in this insect. Nifedipine- and

diltiazem-resistant HVA Ca2+ currents with both sustained and

transient components have been reported in embryonic neurons in

culture (Byerly & Leung, 1988; Leung et al., 1989). PLTX

completely blocks both components, whereas the Hololena curta

spider venom HoTX appears speci®c for the sustained component.

Nanomolar concentrations of either toxin cause complete and

irreversible inhibition of synaptic transmission at the larval

neuromuscular junction (Bowers et al., 1987; Branton et al., 1987;

our own unpublished observations). This differential effect of PLTX

on type III terminals versus glutamatergic transmission indicates that

the channels linked to glutamate release from type I boutons might be

different from those present in peptidergic terminals. Drosophila

FIG. 5. Synaptic Ba2+ currents in type III terminals of csp null mutants. (A) Ba2+ currents recorded from ecd1 (left traces) and cspR1 (right traces) type III boutons atrestrictive temperature, in response to voltage depolarizing pulses from a holding potential of ±50 mV (B) Average Ba2+ peak I±V relationship for Oregon-R, ecd1

and cspR1, at a permissive temperature (22 °C). (C) Average Ba2+ peak I±V relationship for ecd1 and cspR1, at a restrictive temperature (32 °C). At this temperature,all mutant animals tested were paralysed (see text). Additional observations were carried out at temperatures up to 36 °C without signi®cant changes in the currentamplitudes and voltage dependence.



larval muscle ®bres also exhibit a voltage-dependent HVA Ba2+

current selectively blocked by L-type channel antagonists. This

current, however, proved almost two orders of magnitude more

sensitive to nifedipine, but less affected by PAs and BTZs (see also

Gielow et al., 1995), which agrees with the reported dissociation of

DHP and PA binding sites, as mentioned earlier.

The study of csp mutants is justi®ed because of the proposed role

of this protein as a presynaptic Ca2+ channel modulator. Thus,

coexpression in Xenopus oocytes of torpedo CSP cDNA positively

regulates the expression of N-type currents, and this expression is

selectively reduced by coinjection with a cloned antisense CSP

cRNA, which prompted the hypothesis that CSPs could act as

subunits or regulators of Ca2+ channels (Gundersen & Umbach,

1992). On the other hand, Drosophila null mutations for the csp gene

are semilethal during development and death is premature (Zinsmaier

et al., 1994). When exposed to high temperature, csp mutant larvae

exhibit a progressive decrement leading to ®nal blockade of nerve-

evoked, but not spontaneous, quantal release (Umbach et al., 1994).

Further characterization of the temperature-sensitive phenotype,

revealed that exocytosis can be promoted using methods that bypass

presynaptic Ca2+ channels, such as a-latrotoxin and Ca2+ ionophores,

but not by experimental procedures that rely on Ca2+ channel opening

(Umbach & Gundersen, 1997; Ranjan et al., 1998), suggesting that

calcium in¯ux might be impaired in csp mutants. A direct

demonstration of blocked Ca2+ entry in csp mutants at restrictive

temperature was recently provided by Umbach et al. (1998) using

imaging techniques. These authors, have also shown that calcium

in¯ux is blocked in vesicle-depleted shibiri synaptic terminals, in line

with the proposal that the interaction between CSP and Ca2+ channels

would take place during synaptic vesicle docking. Nonetheless,

evidence for a direct link between CSP and a Ca2+ channel is still

pending.

We have shown that type III boutons do express CSP protein, and

that this expression is abolished in cspR1 mutants. The electro-

physiological results, however, indicate that lack of CSP may not

affect type III terminal Ca2+ currents. Current peak amplitudes in csp

mutants measured at permissive (22 °C) versus restrictive (32 °C)

temperature were not signi®cantly different, and they were also

similar to those of control larvae at both temperatures. That is, Ba2+

currents at type III terminals stay the same in otherwise temperature-

sensitive-paralysed csp larvae. As Ca2+ in¯ux into csp type I

terminals is blocked at 32 °C (Umbach et al., 1998), these results

support the idea that absence of CSP has differential effects on

peptidergic versus glutamatergic release.

As glutamate (type I) and peptide (type III) release appear to

depend on different Ca2+ channel types, the interaction with CSP

might be channel-type speci®c. Alternatively, the putative CSP±Ca2+-

channel interaction might require the channels to be tightly coupled to

the docking complex (Sheng et al., 1996) which, under the present

hypothesis for CSP, is implied by the vesicular location of CSP. In

fact, one appealing aspect of this model is that only those channels in

close proximity to docked vesicles, and therefore in a prime position

to elicit effective exocytosis, would be allowed to open. However,

considering the widespread distribution of CSPs (see Buchner &

Gundersen, 1997) which includes non-neuronal tissues (Chamberlain

& Burgoyne, 1996), it is unlikely that presynaptic calcium channel

regulation is the only function of CSPs. In this respect, over-

expression of CSP enhances Ca2+-dependent and GTPgS-dependent

exocytosis from permeabilized PC12 cells, without affecting

depolarization-induced cytosolic Ca2+ signals (Chamberlain &

Burgoyne, 1998). An alternative hypothesis for CSP function

emphasizes the presence of a J-domain. These motives are known

to interact with heat-shock protein 70 (Hsp70) as chaperones to

regulate the folding of substrate proteins (Cyr et al., 1994). At the

synaptic terminal, Hsp70 is thought to participate in the uncoating of

clathrin-coated vesicles (Rothman & Schmid, 1986). In this context,

the function of CSP might be to contribute to rapid vesicle decoating

in conjunction with Hsp70 (Sudhof, 1995). In fact, CSP enhances

Hsc70 ATPase activity in a dose-dependent manner (Braun et al.,

1996). However, Ranjan et al. (1998) directly examined endocytosis

using the ¯uorescent probe FM1±43, and found no vesicle recycling

defect in Drosophila csp mutants. Taken together, the available data

on CSP indicate that there is a wider range of biological roles for this

protein than presented hereto.

Acknowledgements

We appreciate the critical comments on earlier versions of the manuscript ofDrs J. Lerma, W. BunÄo, A. Villarroel and O. Herreras. This research has beenfunded by grants DGICYT 93-0149 and 96-006 from the Spanish Ministry ofCulture

Abbreviations

CSP, cystein string protein; BTZ, benzothioazepines; DHP, dihydropyridines;Hsp70, Heat Shock Protein 70; HVA, high voltage activated; PA, phenylalk-ylamines; PBT, 0.1% Triton in PBS; PLTX, a potent toxin from the venom ofthe spider Plectreurys tristes.

References

Anderson, M.S., Halpern, M.E. & Keshishian, H. (1988) Identi®cation of theneuropeptide transmitter proctolin in Drosophila larvae: characterization of®ber-speci®c neuromuscular endings. J. Neurosci., 8, 242±255.

Atwood, H.L., Govind, C.K. & Wu, C.F. (1993) Differential ultrastructure ofsynaptic terminals on ventral longitudinal abdominal muscles in Drosophilalarvae. J. Neurobiol., 24, 1008±1024.

Bezprozvanny, I., Scheller, R.H. & Tsien, R.W. (1995) Functional impact ofsyntaxin on gating of N-type and Q-type calcium channels. Nature, 378,623±626.

Bowers, C.W., Phillips, H.S., Lee, P., Jan, Y.N. & Jan, L.Y. (1987)Identi®cation and puri®cation of an irreversible presynaptic neurotoxinfrom the venom of the spider Hololena curta. Proc. Natl Acad. Sci. USA,84, 3506±3510.

Branchaw, J.L., Banks, M.I. & Jackson, M.B. (1997) Ca2+- and voltage-dependent inactivation of Ca2+ channels in nerve terminals of theneurohypophysis. J. Neurosci., 17, 5772±5781.

Branton, W.D., Kolton, L., Jan, Y.N. & Jan, L.Y. (1987) Neurotoxins fromPlectreurys Spider venom are potent presynaptic blockers in Drosophila. J.Neurosci., 7, 4195±4200.

Braun, J.E.A., Wilbanks, S.M. & Scheller, R.H. (1996) The cysteine stringsecretory vesicle protein activates Hsc70 ATPase. J. Biol. Chem., 271,25989±25993.

Buchner, E., Bader, R., Buchner, S., Cox, J., Emson, P.C., Flory, E.,Heizmann, C.W., Hemm, S., Hofbauer, A. & Oertel, W.H. (1988) Cell-speci®c immuno-probes for the brain of normal and mutant Drosophilamelanogaster. I. Wildtype visual system. Cell Tissue Res., 253, 357±370.

Buchner, E. & Gundersen, C.B. (1997) The Dna-J like cysteine string proteinand exocytotic neurotransmitter release. Trends Neurosci., 20, 223±227.

Byerly, L. & Leung, H.T. (1988) Ionic currents of Drosophila neurons inembryonic cultures. J. Neurosci., 8, 4379±4393.

Chamberlain, L.H. & Burgoyne, R.D. (1996) Identi®cation of a novel cysteinestring protein variant and expression of cysteine string proteins in non-neuronal cells. J. Biol. Chem., 271, 7320±7323.

Chamberlain, L.H. & Burgoyne, R.D. (1998) Cysteine string protein functionsdirectly in regulated exocytosis. Mol. Biol. Cell, 9, 2259±2267.

Cyr, D.M., Langer, T. & Douglas, M.G. (1994) DnaJ-like proteins: molecularchaperones and speci®c regulators of Hsp70. Trends Biochem. Sci., 19, 176±181.

De Weille, J.R., Schweitz, H., Maes, P., Tartar, A. & Lazdunski, M. (1991)Calciseptine, a peptide isolated from black mamba venom, is a speci®cblocker of the L-type calcium channel. Proc. Natl Acad. Sci. USA, 88,2437±2440.



Dunlap, K., Luebke, J.I. & Turner, T.J. (1995) Exocytotic Ca2+ channels inmammalian central neurons. Trends Neurosci., 18, 89±98.

Garner, A., Kauvar, L. & Lepesant, J.A. (1977) Roles of ecdysone inDrosophila development. Proc. Natl Acad. Sci. USA, 74, 5099±5103.

Gielow, M.L., Gang-Guo, G. & Singh, S. (1995) Resolution andpharmacological analysis of the voltage dependent calcium channels ofDrosophila larval muscles. J. Neurosci., 15, 6085±6093.

Glossmann, H., Zech, C., Striessnig, J., Staudinger, R., Hall, L., Greenberg, R.& Armah, B.I. (1991) Very high af®nity interaction of DPI 201±106 andBDF 8784 enantiomers with the phenylalkylamine-sensitive Ca2+-channelin Drosophila head membranes. Br. J. Pharmacol., 102, 446±452.

Gorczyca, M.G., Augart, C. & Budnik, V. (1993) Insulin-like receptor andinsulin-like peptide are localized at neuromuscular junctions in Drosophila.J. Neurosci., 13, 3692±3704.

Gundersen, C.B. & Umbach, J.A. (1992) Suppression cloning of the cDNA fora candidate subunit of a presynaptic calcium channel. Neuron, 9, 527±537.

Heckmann, M., Adelsberger, H. & Dudel, J. (1997) Evoked transmitter releaseat neuromuscular junctions in wild type and cysteine string protein nullmutant larvae of Drosophila. Neurosci. Lett., 228, 167±170.

Heidelberger, R. & Matthews, G. (1992) Calcium in¯ux and calcium currentin single synaptic terminals of gold®sh retinal bipolar neurons. J. Physiol.(Lond.), 447, 235±256.

Horn, R. & Marty, A. (1988) Muscarinic activation of ionic currents measuredby a new whole-cell recording method. J. Gen. Physiol., 92, 145±159.

Iverfeldt, K., SerfoÈzoÈ, P., Diaz-Arnesto, L. & Bartfai, T. (1989) Differentialrelease of coexisting neurotransmitters: frequency dependence of the ef¯uxof substance P, thyrotropin releasing hormone and (3H (-serotonin fromtissue slices of rat ventral spinal cord. Acta Physiol. Scand., 137, 63±71.

Jan, L.Y. & Jan, Y.-N. (1976) Properties of the larval neuromuscular junctionin Drosophila melanogaster. J. Physiol. (Lond.), 262, 189±214.

Jia, X.-X., Gorczyca, M. & Budnik, V. (1993) Ultrastructure of neuromuscularjunctions in Drosophila: Comparison of wild type and mutants with increaseexcitability. J. Neurobiol., 24, 1025±1044.

Johansen, J., Halpern, M.E., Johansen, K.M. & Keshishian, H. (1989)Stereotypic morphology of glutamatergic synapses on identi®ed musclecells of Drosophila larvae. J. Neurosci., 9, 710±725.

Koham, S.A., Pescatori, M., Brecha, N.C., Mastrogiacomo, A., Umbach, J.A.& Gundersen, C.B. (1995) Cysteine string protein immunoreactivity in thenervous system and adrenal gland of rat. J. Neurosci., 15, 6230±6238.

Lee, K.S. & Tsien, R.W. (1983) Mechanism of calcium channel blockade byverapamil, D600, diltiazem and nitrendipine in single dialysed heart cells.Nature, 302, 790±794.

Lemos, J.R. & Nowycky, M.C. (1989) Two types of calcium channels coexistin peptide-releasing vertebrate nerve terminals. Neuron, 2, 1419±1426.

Leung, H.T., Branton, W.D., Phillips, H.S., Jan, L. & Byerly, L. (1989) Spidertoxins selectively block calcium currents in Drosophila. Neuron, 3, 767±772.

LlinaÂs, R., Steinberg, I.Z. & Walton, K. (1981) Presynaptic calcium currents insquid giant synapse. Biophys. J., 33, 289±322.

MartõÂnez-Padron, M. & FerruÂs, A. (1997) Presynaptic recordings fromDrosophila. Correlation of macroscopic and single-channel K+ currents. J.Neurosci., 17, 3412±3424.

MartõÂnez-PadroÂn, M. & FerruÂs, A. (1998) Patch-clamp recordings fromDrosophila: presynaptic terminals. In: de Pablo, F., FerruÂs, A. & Stern, C.D.(eds.), Cellular and Molecular Procedures in Developmental Biology:Current Topics in Developmental Biology, Vol. 36. New York: AcademicPress, pp. 303±312.

Mastrogiacomo, A., Parsons, S.M., Zampighi, G.A., Jenden, D.J., Umbach,J.A. & Gundersen, C.B. (1994) Cysteine string proteins: a potential linkbetween synaptic vesicles and presynaptic Ca2+ channels. Science, 263,981±982.

Neher, E. & Zucker, R.S. (1993) Multiple calcium dependent processes relatedto secretion in bovine chromaf®n cells. Neuron, 10, 21±30.

Pauron, D., Qar, J., Barhanin, J., Fournier, D., Cuany, A., Pralavorio, M.,Berge, J.B. & Lazdunski, M. (1987). Identi®cation and af®nity labeling ofvery high af®nity binding sites for the phenylalkylamine series of Ca2+

channel blockers in the Drosophila nervous system. Biochemistry, 26,6311±6315.

Pelzer, S., Barhanin, J., Pauron, D., Trautwein, W., Lazdunski, M. & Pelzer,D. (1989) Diversity and novel pharmacological properties of Ca2+ channelsin Drosophila brain membranes. EMBO J., 8, 2365±2371.

Pupier, S., Leveque, C., Marqueze, B., Kataoka, M., Takahashi, M. & Seagar,M.J. (1997) Cysteine string proteins associated with secretory granules ofthe rat neurohypophysis. J. Neurosci., 17, 2722±2727.

Ranjan, R., Bronk, P. & Zinsmaier, K.E. (1998) Cysteine string protein isrequired for calcium secretion coupling of evoked neurotransmission inDrosophila but not for vesicle recycling. J. Neurosci., 18, 956±964.

Rettig, J., Heinemann, C., Ashery, U., Sheng, Z.H., Yokoyama, C.T., Catterall,W.A. & Neher, E. (1997) Alteration of Ca2+ dependence of neurotransmitterrelease by disruption of Ca2+ channel/syntaxin interaction. J. Neurosci., 17,6647±6656.

Richmond, J.E., Penner, R., Keller, R. & Cooke, I.M. (1996) Characterizationof the Ca2+ current in isolated terminals of crustacean peptidergic neurons.J. Exp. Biol., 199, 2053±2059.

Richmond, J.E., Sher, E. & Cooke, I.M. (1995) Characterization of the Ca2+

current in freshly dissociated crustacean peptidergic neuronal somata. J.Neurophysiol., 73, 2357±2368.

Rothman, J.E. & Schmid, S.L. (1986) Enzymatic recycling of clathrin fromcoated vesicles. Cell, 46, 5±9.

SchaÈfer, S., Rosenboom, H. & Menzel, R. (1994) Ionic currents of Kenyoncells from the mushroom body of the honeybee. J. Neurosci., 14, 4600±4612.

Schweitz, H., Heurteaux, C., Bois, P., Moinier, D., Romey, G. & Lazdunsky,M. (1994) Calcicludine, a venom peptide of the Kunitz-type proteaseinhibitor family, is a potent blocker of high-threshold Ca2+ channels with ahigh af®nity for L-type channels in cerebellar granule neurons. Proc. NatlAcad. Sci. USA, 91, 878±882.

Sheng, Z.-H., Rettig, J., Cook, T. & Catterall, W.A. (1996) Calcium dependentinteraction of N-type calcium channels with the synaptic core complex.Nature, 379, 451±454.

Skeer, J.M., Norman, R.I. & Sattelle, D.B. (1996) Invertebrate voltage-dependent calcium channel subtypes. Biol. Rev., 71, 137±154.

Stanley, E.F. & Goping, G. (1991) Characterization of a calcium current in avertebrate cholinergic presynaptic nerve terminal. J. Neurosci., 11, 985±993.

Sudhof, T.C. (1995) The synaptic vesicle cycle: a cascade of protein±proteininteractions. Nature, 375, 645±653.

Umbach, J.A. & Gundersen, C.B. (1997) Evidence that cysteine string proteinsregulate an early step in the Ca2+-dependent secretion of neurotransmitter atDrosophila neuromuscular junctions. J. Neurosci., 17, 7203±7209.

Umbach, J.A., Minoru, S., Kidokoro, Y. & Gundersen, C.B. (1998) Attenuatedin¯ux of calcium ions at nerve endings of csp and shibire mutant drosophila.J. Neurosci., 18, 3233±3240.

Umbach, J.A., Zinsmaier, K.E., Eberle, K.K., Buchner, E., Benzer, S. &Gundersen, C.B. (1994) Presynaptic dysfunction in Drosophila csp mutants.Neuron, 13, 899±907.

Wicher, D. & Penzlin, H. (1997) Ca2+ currents in central insect neurons:electrophysiological and pharmacological properties. J. Neurophysiol., 77,186±199.

Wiser, O., Bennett, M.K. & Atlas, D. (1996) Functional interaction of syntaxinand SNAP-25 with voltage-sensitive L- and N-type Ca+2 channels. EMBOJ., 15, 4100±4110.

Yawo, H. (1990) Voltage-activated calcium currents in presynaptic nerveterminals of the chick ciliary ganglion. J. Physiol. (Lond.), 428, 199±213.

Zheng, W., Feng, G., Ren, D., Eberl, D.F., Hannan, F., Dubald, M. & Hall,L.M. (1995) Cloning and characterization of a calcium channel (1 subunitfrom Drosophila melanogaster with similarity to the rat brain type Disoform. J. Neurosci., 15, 1132±1143.

Zinsmaier, K.E., Eberle, K.K., Buchner, E., Walter, N. & Benzer, S. (1994)Paralysis and early death in cysteine string protein mutants of Drosophila.Science, 263, 977±980.



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Presynaptic calcium-channel currents in normal and csp mutant Drosophila peptidergic terminals