Ca2+ current, rather than enhancement of possible contaminating outward (K+, H+

Transcription

1 J. Phy8iol. (1987), 388, pp With 15 text-figures Printed in Great Britain SUPPRESSION OF CALCIUM CURRENT BY AN ENDOGENOUS NEUROPEPTIDE IN NEURONES OF APL YSIA CALIFORNICA BY V. BREZINA, THE LATE R. ECKERT AND C. ERXLEBEN From the *Department of Biology and Ahmanson Laboratory of Neurobiology, University of California, Los Angeles, CA 90024, and Friday Harbor Laboratories, Friday Harbor, WA, U.S.A. (Received 29 July 1986) SUMMARY 1. Actions of the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) and its derivative YGG-FMRFamide (Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2) on Ca2+ current were examined in identified, voltage-clamped neurones in the abdominal ganglion of Aplysia californica. 2. 'Puffed' application of either peptide at concentrations of 1-50,(M was followed by a transient partial suppression of pharmacologically isolated inward Ca2+ current elicited by a depolarizing step. At 20 C, suppression was maximal s following the brief puff of peptide, and lasted up to 90 s. Bath application of peptide had a steady suppressing effect, showing little if any desensitization. 3. Alternative sources of inward current suppression were ruled out, indicating that application of FMRFamide or YGG-FMRFamide produces a true decrease in Ca2+ current, rather than enhancement of possible contaminating outward (K+, H+ or Cl-) currents. 4. FMRFamide and YGG-FMRFamide were equally effective in suppressing Ca2+ current (apparent dissociation constant, K* 10/M). However, only 30-50% of the total Ca2+ current elicited by voltage steps to above +10 mv appeared to be susceptible to suppression by even saturating concentrations of peptide. This, as well as a reduced effect of the peptides on Ca2+ current which was observed at potentials below + 10 mv, may perhaps result from the presence of more than one class of Ca2+ channels, only one of which is sensitive to FMRFamide. 5. FMRFamide eliminated a constant fraction of Ca2+ current at all potentials above +10 mv, and had no direct effect on activation or inactivation of the remaining current. This behaviour is consistent with reduction in the number of functional Ca2+ channels by the peptide. 6. Suppression of Ca2+ current produced a concomitant depression of Ca2+dependent K+ current, which was shown previously to be insensitive to FMRFamide when activated by direct ionophoretic injection of Ca2+ into the cell. 7. The effect of FMRFamide on Ca2+ current was normal following interference with or activation of known second-messenger systems, those involving adenosine 3',5'-cyclic monophosphate (cyclic AMP), cyclic GMP, Ca2+, inositol trisphosphate and protein kinase C. * Address for reprint requests.

2 566 V. BREZINA, R. ECKERT AND C. ERXLEBEN 8. Suppression of Ca2+ current by FMRFamide appeared to be mediated by the same receptor as enhancement by the peptide of K+ current resembling IK(S) (K+ current suppressed by serotonin), an effect seen in most of the same cells. Both effects of FMRFamide were mimicked by injection of guanosine 5'-O-(3-thiotriphosphate) (GTP-y-S) into the cell, suggesting that the peptide may exert its effects by activating a guanosine 5'-triphosphate (GTP)-binding protein. INTRODUCTION Since the discovery of the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) in ganglion extracts of a clam (Price & Greenberg, 1977), FMRFamide-like peptides have been found in various animal phyla (Boer, Schot, Veenstra & Reichelt, 1980). In Aplysia californica, a gene encoding multiple copies of FMRFamide is expressed throughout the nervous system, and FMRFamide appears to be a principal peptide product of identified abdominal ganglion neurones (Schaefer, Picciotto, Kreiner, Kaldany, Taussig & Scheller, 1985). FMRFamide immunoreactivity is present in neuronal processes and varicosities, suggesting that the peptide may be released from them to function as a neurotransmitter or neurohormone (Weiss, Goldberg, Chohan, Stell, Drummond & Lukowiak, 1984; Schaefer et al. 1985); indeed, in clam ganglia FMRFamide is contained in neurosecretory granules (Nagle, 1981), from which it can be released by depolarization in a Ca2+-dependent manner (Nagle, 1982). In Aplysia, FMRFamide has been shown to influence synaptic activity and reflex behaviour (Voshart & Lukowiak, 1982), supporting the idea that FMRFamide-like peptides may act naturally as modulators of neural activity in this mollusc. In a previous paper (Brezina, Eckert & Erxleben, 1987) we presented evidence that FMRFamide and its derivative YGG-FMRFamide (Tyr-Gly-Gly-Phe-Met-Arg-Phe- NH2) influence two K+ currents in certain neurones in the abdominal ganglion of Aplysia. One of these, a Ca2+-insensitive current that resembles IK(S), the K+ current suppressed by serotonin in Aplysia sensory neurones (Siegelbaum, Camardo & Kandel, 1982), is activated by extracellular application of the peptides. The Ca2+-dependent K+ current, IK(Ca) (Meech & Standen, 1975), when elicited by direct injection of Ca2+ into the cell, is unaffected by FMRFamide or YGG-FMRFamide. In contrast, IK(Ca) elicited by depolarization is significantly depressed by the peptides, suggesting that they may exert their effect by suppressing the voltagedependent Ca2+ current. In the present paper we report results of voltage-clamp experiments that confirm this suggestion: we show that the Ca2+ current undergoes a transient decrease following a brief exposure to FMRFamide or YGG-FMRFamide. This effect on the Ca2+ current explains the suppression of Ca2+-dependent K+ current by these peptides, for reduced influx of Ca2+ leads to a reduction of IK(Ca) activated on depolarization. Further observations suggest that the peptides may exert their effects by activating a GTP-binding protein. Preliminary reports ofthis work have appeared (Brezina, Erxleben & Eckert, 1985; Erxleben, Brezina & Eckert, 1985; Brezina & Eckert, 1986).

3 SUPPRESSION OF Ca2+ CURRENT 567 METHODS Experiments were carried out primarily on the left upper quadrant (l.u.q.) neurones L2, L3, L4 and L6, and in a few cases on cells R2 and R15, in the abdominal ganglion of Aplysia californica (Frazier, Kandel, Kupfermann, Waziri & Coggeshall, 1967). Preparation of the tissue, methods of voltage clamping and other details of the methods have been described previously (Brezina et al. 1987). Compositions of artificial sea water (ASW) solutions are shown in Table 1. Experiments were carried out at C and C. TABLE 1. Principal solutions used. Artificial sea water (ASW) solutions contained 45 mm-mgcl2, 10 mm-glucose and the following concentrations (mm) of other compounds Solution name NaCl KCl CaC12 BaCl2 CoCl2 TEA Cl 4-AP Tris Cl* ASW TEA-4-AP ASW Ca2+-20 Co2+_ TEA-4-AP ASW Na+ ASW t 0Na+-4-AP ASW t 0Na+-50 K+-4-AP ASW t 0 Na+-TEA-4-AP ASW t 0 Na+-35 K+-TEA- 4-AP ASW t 0 Na+-O Ca2+-20 Co2+ ASW t 0 Na+-O Ca+-20 Co2+- TEA-4-AP ASW t 0 Na+-O Ca2+-20 Ba2+- TEA-4-AP ASW t * 25 mm-hepes buffer was sometimes used instead of 25 mm-tris. t Instead of Tris, N-methyl-D-glucamine was sometimes used to replace NaCl. The ph of all solutions was adjusted to 7-7 at 20 'C. Conventional depolarizing steps were used to identify peptide effects on voltage-dependent currents. At a fixed time (usually 20 s) following a puffed application (see below) of the peptide to the neurone (unless the peptide was bath-applied), the membrane potential was stepped from the holding to the test voltage. The procedure was then usually repeated using an equivalent hyperpolarization (or, for steps exceeding 60 mv, two or three hyperpolarizations of one-half or one-third amplitude); the corresponding currents were added to cancel symmetrical leakage and capacitive currents. Currents obtained following application of the peptide were compared with control currents obtained in the same manner but in the absence of the peptide. Voltage steps were given regularly at 30 or 60 s intervals. In recordings of Ca2+ currents, Na+-free solutions were generally used, and K+ currents were blocked with 200 mm-extracellular tetraethylammonium chloride (TEA Cl; Eastman Kodak, Rochester, NY, U.S.A.; or Sigma). This concentration of TEA reduces the voltage-dependent K+ current, IK(v), to below 10% of normal, and Iwo to below 1 % (Hermann / Gorman, 1981 b). As an additional measure, 5 mm-4-aminopyridine (4-AP, Sigma) was used to block IK(v) and the early transient K+ current, IK(A) (Connor & Stevens, 1971; Neher, 1971; Hermann & Gorman, 1981 a), and to some extent the proton current, IH (Byerly, Meech & Moody, 1984). A moderately low holding potential (-40 mv) was generally used to minimize activation Of IK(A) following depolarization. In most of the experiments, contamination by outward currents carried by K+ and H+ was further diminished by limiting depolarization to moderately positive potentials, generally no greater than +10 mv (Hermann & Gorman, 1981 a,b; Byerly et al. 1984). It has been shown that, under these conditions, current in Aplysia neurones is carried primarily by Ca2+, and that the decay of inward current is determined primarily by a Ca2+-dependent inactivation of Ca2+ conductance, with relatively little contribution by developing outward current (Chad, Eckert & Ewald, 1984).

4 568 V. BREZINA, R. ECKERT AND C. ERXLEBEN Ca21 tail current measurements were made at reduced temperature ( C) to slow deactivation kinetics, and thus enhance temporal resolution of the transient currents. Tail currents were recorded in Na+-free ASW containing outward current blockers, as described above, but containing in addition an elevated (35 mm) K+ concentration (Table 1). In this solution the K+ equilibrium potential, EK, is approximately equal to the holding potential (-40 mv) to which the membrane was repolarized for measurement of the tail currents, assuming that intracellular activity of K+ is close to the value of 142 mm reported by Kunze & Brown (1971; these experiments were done on l.u.q. cells). Often, several tail current traces were averaged to improve the signal-to-noise ratio. The peptides, FMRFamide, YGG-FMRFamide, FMRF (Phe-Met-Arg-Phe) and methionineenkephalin (Tyr-Gly-Gly-Phe-Met) (Peninsula Laboratories, Belmont, CA, U.S.A.), were made up from 1 mm stock solution by dilution with ASW identical to that present in the bath to a final concentration of 1-50 /M. Most of the experiments were performed by 'puffing' the peptide onto the cell surface from a pressure-ejection pipette as described previously (Brezina et al. 1987), since this allowed for alternate control traces during a series of measurements. In some experiments, however, the peptide was applied in the bath by steady superfusion of the ganglion. The experimental chamber was perfused continuously at the rate of 1 ml/min, and solution changes were made without significant perturbation of flow rate or temperature. Several experiments involved ionophoretic injection of the Ca2+ chelating agent EGTA (ethyleneglycol-bis-(,6-aminoethylether)n,n'-tetraacetic acid). The injection pipette contained 500 mm-egta (Sigma), and sA of current was passed until the block by EGTA of Ca2+ current inactivation (Chad et al. 1984) or, in the absence of K+ current blockers, of IK(ca) activation (Meech, 1978) was maximal (usually after several minutes of injection). In other experiments, ionophoretic injection of TEA+, Cs+ or adenosine 3',5'-cyclic monophosphate (cyclic AMP) was carried out using pipettes filled with 1 M-TEA Cl, 1 M-CsCl, or 200 mm-cyclic AMP (sodium salt, Sigma). Guanosine 3',5'-cyclic monophosphate (cyclic GMP; Sigma) was made up at 100 mm in solution containing 150 mm-kcl, 10 mm-hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid, Sigma), ph 7 4, and pressure injected. Finally, guanosine 5'-O-(2-thiodiphosphate) (GDP-,f-S; lithium salt, Sigma), guanosine 5'-O-(3-thiotriphosphate) (GTP-y-S; lithium salt, Sigma or Boehringer Mannheim), 5'-guanylylimidodiphosphate (GppNHp; sodium salt, Sigma), adenosine 5'-O-(3-thiotriphosphate)(ATP-y-S; lithium salt, Boehringer Mannheim), and D-myo-inositol trisphosphate (1P3; potassium salt, Sigma) were injected ionophoretically, from pipettes filled with 5 mm-gdp-f-s, 5 or 25 mm-gtp-y-s, 25 mm-gppnhp, 25 mm-atp-y-s or 1 mm-ip3, respectively, or made up at 2-5 mm in solution containing 150 mm-kcl, 10 mm-hepes, ph 7 4, and pressure injected. The phorbol esters 4,-phorbol 12-myristate 13-acetate (PMA, Sigma), 4/J-phorbol 12f, 13adibutyrate (PDB, Sigma) and 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA; LC Services, Woburn, MA, U.S.A.) were added to the bath solution, which in the case of PMA and PDB also contained 0 05 % (v/v) dimethylsulphoxide (DMSO) per 1 /LM-phorbol ester. Pertussis toxin (PTX; List Biological Laboratories, Campbell, CA, U.S.A.) was added to ASW to a final concentration of 1 jug/ml. Ganglia were incubated in the PTX suspension for up to 12 h at C; the response to application of peptide was measured before and after incubation. Other agents added to the bath solution in some experiments included serotonin hydrochloride (Sigma), naloxone hydrochloride (Sigma), sodium nitroprusside (Sigma), forskolin (Calbiochem) and RO (Hoffmann- LaRoche); in the case of naloxone, forskolin and RO , the bath solution also contained 0-25 % DMSO during these experiments. DMSO at this concentration had little or no effect on currents described in this paper. RESULTS FMRFamide and YGG-FMRFamide reduce Ca2+ current After introduction of Na+-free solution containing outward-current blockers (see Methods), the current recorded during a step depolarization from -40 to + 10 mv changed from a complex inward-outward waveform (see below) to a simpler, slowly decaying net inward current. Current measured in Aplysia neurones under these conditions with depolarizations not exceeding +10 mv is believed to be carried

5 SUPPRESSION OF Ca2+ CURRENT 569 A 2 25 ms ~ 0-2,A 3 30 na B C a 1601" 7 8e50 2K%g_ 140- ~~~~~ I- I FMRFamide ol-0 ' Time (min) Log [peptide] (M) Fig. 1. FMRFamide suppresses Ca2+ current (0 Na+-TEA-4-AP ASW). A, Ca2+ current elicited by 100 ms voltage step from -40 to + 10 mv (trace 1) is suppressed 20 s after 3 s puff of 50,uM-FMRFamide (trace 2). Trace 3 is the difference current obtained by subtracting trace 2 from trace 1 (see text). Inset, indicated parts of traces 1-3 have been further filtered and expanded vertically to show suppression of outward tail current by FMRFamide. All traces corrected for leakage current. Cell L3; 20 'C. B, plot of peak amplitude of (leakage-subtracted) Ca2+ currents elicited by regular voltage steps to + 10 mv. 10,sM-FMRFamide was perfused through the bath during the time shown. L.u.q. cell; 13 'C. C, dose-response relation for the effect of FMRFamide (@), YGG- FMRFamide (0) and FMRF (El) on Ca2+ current, showing the percentage of (leakagesubtracted) Ca2+ current, elicited by steps to +10 mv, remaining following 10s (FMRFamide and YGG-FMRFamide) or 2 s (FMRF) puff of peptide at concentrations between 10-8 and 10-3 M. FMRF had no effect at any concentration. The curve for FMRFamide and YGG-FMRFamide is the function (%ICa) = finsens +fsens/ (1 + [peptide]/k*), where finsens and fsens represent, respectively, the fractions of Ca2+ current insensitive (here 60%) and sensitive (40%) to the effect of peptide, and KrD the apparent dissociation constant, is equal to 10-5 M. Three l.u.q. cells; 13 'C. primarily by Ca2, with relatively little contamination by outward currents (Chad et al. 1984). In most cells, we found no evidence of contamination in experiments carried out with short (100 ms) voltage steps at low temperature (13 'C). Thus, the inward current elicited by these voltage steps was unaffected by prolonged injection of TEA+ or Cs+ into the cell (three cells), was completely blocked by superfusion of

6 570 V. BREZINA, R. ECKERT AND C. ERXLEBEN Ca2+-free solution containing 20 mm-co2+ leaving virtually no residual outward current (Fig. 2B; see below), and was not followed by an outward tail current. At higher temperature (20 C), however, a slow outward tail current was often seen following repolarization to -40 mv (compare Fig. 1 A, recorded at 20 C, and Fig. 3A, recorded at 13 C). The reversal potential of this tail current was, in the l.u.q. cells, between -60 and -65 mv in ASW containing 10 mm-k+, close to the reversal potential of Ca2+-dependent K+ current in these cells (Deitmer & Eckert, 1985; see also Meech & Standen, 1975), and it shifted with the extracellular K+ concentration (two cells). The tail current was therefore carried by K+; it may represent the Ca2+-dependent K+ current resistant to TEA and 4-AP which has recently been described in Aplysia neurones (Deitmer & Eckert, 1985). Appearance of the tail current on repolarization at 20 C indicates contamination of the Ca2+ current measured during the depolarizing step itself. To eliminate this problem, most of the experiments involving isolation of Ca2+ current were carried out at 13 'C. However, the magnitude of FMRFamide effect was about the same at both temperatures. FMRFamide or YGG-FMRFamide applied to certain cells from a pressure pipette caused a transient partial suppression of the inward Ca2+ current, at both 20 'C (Fig. 1 A) and 13 'C (Fig. 3A). At 20 'C, suppression was maximal s following the brief puff of peptide, and lasted usually up to 60 s (see Fig. 10), and in some cells up to 90 s; the time course of the response slowed considerably as the temperature was reduced to 13 'C (see below). At the higher temperature the peptides also depressed the outward tail current described above (Fig. 1 A, inset), consistent with its presumed Ca2+ dependence. Suppression of Ca2+ current by these peptides was regularly found in the l.u.q. neurones L2, L3, L4 and L6, and in cells R2 and R15, but not in cells L7 and L11. The degree of Ca2+ current suppression depended on several factors, such as the concentration of peptide in the pressure pipette, the proximity of the pipette to the cell surface, and variability in responsiveness from cell to cell. In most experiments 50 /tm-fmrfamide or YGG-FMRFamide was used in the pressure pipette; with this peptide concentration, suppression of Ca2+ current was occasionally as great as 50 %, but was generally closer to %. When the peptides were applied by superfusion in the bath, the concentration of peptide needed to produce a given degree of Ca2+ current suppression was often up to 5 times lower than with puffed application, presumably because peptide ejected with short puffs is diluted by the bath solution, particularly under continuous superfusion, before reaching its site of action. With bath application, the effect of FMRFamide was steady, showing little or no desensitization (Fig. 1 B; twelve cells). The dose-response relation for the effect of FMRFamide and YGG-FMRFamide on Ca2+ current was obtained (in four cells) by applying the peptides in puffs of long duration (10 s), thus combining the reversibility of puffing with the sensitivity of bath application. FMRFamide and YGG-FMRFamide appeared to be equally effective in suppressing Ca2+ current. Suppression was observed with concentrations greater than 0-2 sum. However, even at saturating concentrations above 200 /M only a fraction (usually %) of the total Ca2+ current was suppressed (Fig. 1 C). The concentration of FMRFamide or YGG-FMRFamide suppressing half of the peptidesuppressible current was 10 /LM. In contrast to FMRFamide and YGG-FMRFamide,

7 SUPPRESSION OF Ca2+ CURRENT FMRF (the non-amidated analogue of FMRFamide; three cells) and methionineenkephalin (five cells) were ineffective. The effect of FMRFamide was unchanged following superfusion of the opioid receptor blocker naloxone (up to 100#Um; two cells). 571 A B 3,4 Ca2+ 2 C2. A, 1Oms B, 25ms A, 1 ga B, 50 na Fig. 2. FMRFamide does not affect Na+ current or residual current remaining after elimination of Ca2+ current by replacement of bath Ca2+ with Co2+. Voltage steps from -40 to +10 mv. All records corrected for leakage current. A, mixed inward current recorded in TEA-4-AP ASW (trace 1) is reduced 20 s after 5 s puff of 50 /sm-fmrfamide (trace 2). Superfusion of 0 Ca2+-20 Co2+-TEA-4-AP ASW then eliminates Ca2+ current. The remaining Na+ current (trace 3) is unaffected by FMRFamide (trace 4). Beginning of traces superimposed. Cell R2; 20 'C. B, Ca2+ current recorded in 0 Na+-TEA-4-AP ASW (trace 1) is suppressed 20 s after 5 s puff of 50,M-FMRFamide (trace 2). The effect of FMRFamide is eliminated along with the Ca2+ current by superfusion of 0 Na+- 0 Ca2+-20 Co2+-TEA -4-AP ASW (traces 3,4). Cell L3; 13 'C. The suppression of inward current was not noticeably different when extracellular Ca2+ was replaced with Ba2+ (Fig. 3B; six cells). Since Ba2+ carries current through Ca2+ channels, this result is consistent with a decrease in Ca2+ conductance following application of FMRFamide. In contrast to the effect on Ca2+ current, the Na+ current, isolated in Ca2+-free ASW containing 200 mm-tea plus 5 mm-4-ap, was unaffected by FMRFamide (Fig. 2A; three cells). Possible alternative sources of inward-current suppression Accurate measurement of Ca2+ current is often complicated by special problems (Hagiwara & Byerly, 1981), such as the possible activation of simultaneous outward currents. The summing of such outward current with a Ca2+ current of constant amplitude would be expected to cause a reduction in net inward current that might be mistaken for a decrease of the Ca2+ current. As a result of such problems, some studies of Ca2+ current modulation have required reinterpretation (Klein & Kandel, 1978, 1980), or have given rise to controversy as to the identity of the modulated 3,4

8 572 V. BREZINA, R. ECKERT AND C. ERXLEBEN current (for example, Pellmar & Carpenter, 1980; Paupardin-Tritsch, Deterre & Gerschenfeld, 1981). In the Aplysia neurones examined here, careful separation of Ca2+ and outward currents is especially important since FMRFamide does, in fact, activate an outward current in these cells, namely the Ca2+-independent K+ current resembling IK(s). Although this current is blocked by the high concentrations of TEA and 4-AP used to isolate Ca2+ current (Brezina et al. 1987; see Discussion), we nevertheless carried out a number of experiments to rule out interference from this and other possible FMRFamide-sensitive currents. (i) FMRFamide still suppressed Ca2+ current in cells ionophoretically injected with TEA+ or Cs+ (three cells), agents that block the FMRFamide-activated IK(s)-like current (Brezina et al. 1987) and other K+ currents (for example, see Klein & Kandel, 1980; Deitmer & Eckert, 1985). (ii) The Ca2+ normally present in the bath was replaced with Co2+ to block the Ca2+ current (Hagiwara & Byerly, 1981). Under these conditions, and with voltage steps limited to potentials below + 10 mv, virtually no current remained after subtraction of leakage current, and FMRFamide had no effect (Fig. 2B; five cells). Thus, assuming that Co2+ blocks only Ca2+ channels, any sizable effect of FMRFamide on membrane current recorded in Ca2+-containing solution (Fig. 1) must originate either in the Ca2+ current itself or in a current that depends in some way on Ca2+ entry. (iii) The latter possibility, that FMRFamide modulates not the Ca2+ current, but another current that is Ca2+ dependent, was excluded by an experiment in which cells were injected with the Ca2+ chelating agent EGTA. This blocked two effects that Ca2, entering during a voltage step, is known to have, namely activation of IK(Ca) in the absence of K+ current blockers (Meech, 1978), and Ca2+-dependent inactivation of the Ca2+ conductance itself (Eckert & Chad, 1984). However, EGTA injection did not affect the suppression of Ca2+ current by FMRFamide (Fig. 3C; three cells). (iv) The time course of a control inward current was compared with that of the peptide-suppressed inward current and of the difference current (i.e. the current representing the net effect of FMRFamide, obtained by subtracting the suppressed current from the control current; even though the peptide produces an outward current shift, difference currents are shown, in Figs. 1 A and 3 A-C, as inward currents to facilitate comparison). All three currents had very similar time courses (Figs. 1 A and 3A,D); in particular, FMRFamide did not appear to affect the rate of activation of the current. When experimental conditions were varied (by injection of EGTA or replacement of bath Ca2+ with Ba2+) to change the shape of the control current, the shape of the FMRFamide-suppressed current and of the difference current changed correspondingly (Fig. 3B-D). Thus, while the amplitude of inward current varied considerably at different times during the 100 ms voltage step, the fraction of that current suppressed by FMRFamide remained constant, particularly in Ba2+_ containing solution and in EGTA-injected cells (Fig. 3E). In Ca2+-containing solution, however, the peptide-suppressed current decayed relatively more slowly than control current, resulting in a decrease in the fraction of current suppressed by FMRFamide towards the end of the voltage step (Fig. 3 E). This effect, and its absence in Ba2+-containing solution and in EGTA-injected cells, is consistent with the slower Ca2+-dependent inactivation of the Ca2+ conductance expected following reduction of Ca2+ current, and thus of intracellular accumulation of Ca2+, by FMRFamide (see below).

9 SUPPRESSION OF Ca2+ CURRENT 573 If the suppression of inward current by FMRFamide were the result of modulation by the peptide of a hidden current, and not of the Ca2+ current, the similarities in time course between the control current, carried primarily by Ca2+ (see above), and the difference current would require that two separate and independent currents (i.e. A B C Ca2+ Ba2+ * Ca2-EGTA 3> - t 3 3 r 2 2. A-C, 2;5m MA 1 D.E D... C Ba2~ E 20- ~ ~~B2+, C2+-EGTA a. Ba2+ 20 ms Ca Ca2-EGTA E1 Ba Ba2+, Ca2+-EGTA Time (ms) Fig. 3. FMRFamide suppresses Ca2+ conductance in Ca2+-containing or Ba2+_containing solution, and following injection of EGTA into the cell. Voltage steps from -40 to + 10 mv. All records corrected for leakage current. In A-C, trace 1 is the control current recorded before FMRFamide application, trace 2 is the current recorded 20 s after 1-3 s puff of 50 /SM-FMRFamidc, and trace 3 is the difference current obtained by subtracting trace 2 from trace 1 (see text). A, currents recorded in 0 Na+-TEA-4-AP ASW. L.u.q. cell; 13 'C. B, 0 Na+-0 Ca2+-20 Ba2+-TEA-4-AP ASW. Cell L2; 20 'C. C, 0 Na+-TEA-4- AP ASW, following ionophoretic injection of EGTA into the cell (see Methods). Cell L6; 20 'C. D, top panel shows control currents from A-C scaled to same size for comparison; bottom panel shows difference currents from A-C scaled to same size. E, fraction of inward (Ca2+ or Ba2+) current suppressed by FMRFamide (i.e. difference current expressed as percentage of control current) plotted as function of time into 100 ms voltage step, for each of A-C. the large Ca2+ current and the smaller hidden current) exhibit identical kinetics. Such congruity would appear to be rather improbable and fortuitous. The similarity in time course between the control current and the difference current argues in particular (together with the results of the EGTA-injection experiment described in (iii), above) against the possibility that the difference current might represent modulation by FMRFamide of a separate but Ca2+-dependent current. Modulation of such a current, activated by the rise in intracellular Ca2+ that develops during the flow of Ca2+ current, would be expected to result in a difference current related not to the instantaneous amplitude of the Ca2+ current, but rather to its integral, in the same

10 574 V. BREZINA, R. ECKERT AND C. ERXLEBEN Aa b 2f 1 ~~~~~~~~~~~ Aa, 7 ms Ab,1ms 0-2,pA B 0O C 0*5 - co *0- c 0._ a a A 20-0 cm CD el 0-, A~ *\' 0 0~~~ I/ _ V (mv) V (mv) Fig. 4. Effect of membrane potential on suppression of Ca21 current by FMRFamide (0 Na+- 35 K+-TEA-4-AP ASW; 13 C). All records and measurements corrected for leakage current. Aa, Ca21 current elicited by voltage steps to + 10 mv and Ca2+ tail current on repolarization to -40 mv (trace 1) are both reduced following superfusion of 10 /M-FMRFamide (trace 2). Ab shows horizontal expansion of tail currents from Aa. Cell L6, unusually responsive to FMRFamide. B and C, measurements of peak Ca2+ currents elicited by 20 ms voltage steps to the potentials indicated (B) and Ca2+ tail currents (measured 600 #us following end of the voltage step) on repolarization to -40 mv (C), with (0) and without (@) repeated puffed application of 50,LM-FMRFamide. Bottom shows percentage suppression of peak Ca2+ current (B) or Ca2+ tail current (C) as function of membrane potential. Two different l.u.q. cells. way in which, for example, activation of IK(ca) is related to Ca2+ current (Gorman & Thomas, 1980; Eckert & Ewald, 1982). (v) Ca2+ tail currents were recorded under conditions that minimize or eliminate interference from currents carried by K+ or H±. The extracellular concentration of K+ was adjusted (see Methods) so that the equilibrium potential for K+, EK, was close

11 SUPPRESSION OF Ca2+ CURRENT to -40 mv, the holding potential to which the membrane was repolarized for measurement of the tail currents. Any K+ channels remaining open following repolarization should therefore have carried little or no current. Similarly, assuming that intracellular concentrations of H+ in these cells are close to the values reported for Helix neurones (Thomas, 1974), EH should have been mv more positive than the holding potential of -40 mv. Enhancement by FMRFamide of current carried by H+ should therefore have appeared as an outward current shift during steps to depolarized potentials, but as an inward shift of the tail currents measured after repolarization to -40 mv. Fig. 4, however, shows that, under these conditions, the Ca2+ current during the depolarizing step itself and also the following Ca2+ tail current both invariably experienced an outward shift (i.e. were suppressed) after application of FMRFamide. The tail current exhibited approximately the same degree of suppression by the peptide that was experienced by the current recorded during the depolarizing step, for steps to potentials up to + 50 mv, at 13 C (Fig. 4B and C; six cells). For steps to higher potentials, where the degree of suppression of Ca2+ current during the step could not be measured since this current was clearly contaminated with outward current (see below), the effect of FMRFamide on the Ca2+ tail current remained undiminished (Fig. 4C; two cells). (vi) Measurement of Ca2+ tail currents under the conditions just described would not be expected to rule out modulation by FMRFamide of a current carried by Cl-, since the Cl- reversal potential, EC1 in these cells is generally more negative than the holding potential of -40 mv (Kunze & Brown, 1971); thus the current during the depolarizing step and the tail current would both be shifted outward if FMRFamide were enhancing Cl- current. This possibility was, however, ruled out in experiments in which 88 % of external Cl- was replaced with NO3- or isethionate, bringing Ec1 close to 0 mv, but without effect on the magnitude of inward-current suppression by FMRFamide measured at that potential (two cells). These diverse lines of evidence, as well as other considerations reviewed in the Discussion, appear to rule out modulation of possible contaminating currents as the cause of decreased inward current, and indicate that the effect of FMRFamide application must be a true suppression of Ca2+ current. Effect of membrane potential on Ca2+ current suppression by FMRFamide The possible voltage dependence of the effect of FMRFamide on Ca2+ current was examined by comparison of conventional current-voltage (I- V) relations determined before and after application of the peptide (Fig. 4B; eleven cells). The apparent sharp increase in percentage of inward current suppressed by FMRFamide observed with voltage steps to potentials above about + 50 mv (Fig. 4B) was assumed to be due to development of outward current contamination at these potentials; therefore EK was adjusted to -40 mv (see Methods) and amplitudes of uncontaminated Ca2+ tail currents measured on repolarization to that potential were also plotted (Fig. 4C). At all potentials above about + 10 mv, and allowing for the problem of outward current contamination at very positive potentials just discussed, FMRFamide suppressed a constant fraction, about 15-30% (with puffs of 50 ftm-fmrfamide), of both the Ca2+ tail current and the Ca2+ current during the depolarizing step itself (Fig. 4B and C). At potentials more negative than + 10 mv, however, FMRFamide 575

12 576 V. BREZINA, R. ECKERT AND C. ERXLEBEN often appeared to become markedly less effective, and little or no suppression was usually observed at -20 mv, even though significant Ca2+ current was elicited by steps to that potential (Fig. 4B and C; seven cells). A 25 ms B.j 0 2 pa 50 na 50 ms 2 C ft -- I. od. 14..topploav-4 :i I I 2, 1 50 ms 0 1 pa 1 Fig. 5. FMRFamide has no direct effect on inactivation ofca2+ current (O Na+-TEA-4-AP ASW; 13 C). Voltage steps from -40 to + 1O mv. All records corrected for leakage current. A, Ca2+ current elicited by 250 ms voltage step (trace 1) is suppressed 20 s after 5 s puff of 50,uM-FMRFamide (trace 2), and to an equal extent when the step is preceded 3 s earlier by another step of the same amplitude and duration (trace 3). Cell L6. B, same traces as in Fig. 3A; the FMRFamide-suppressed current (trace 2) has been scaled to the same peak amplitude as the control current (trace 1). C, Ca2+ currents elicited by two 100 ms voltage steps separated by 250 ms (trace 1) are suppressed 20 s after 2 s puff of 50,u1M-FMRFamide (trace 2), and to an equal extent 20 s after 2-5 s puff of 0 Na+- 0 Ca2+-20 Co2+-TEA -4-AP ASW (trace 3); see text. L.u.q. cell. FMRFamide does not directly affect Ca2+ current inactivation A considerable body of evidence indicates that inactivation of Ca2+ current in molluscan neurones is mediated solely or largely by a process dependent on elevation of intracellular Ca2+ (Eckert & Chad, 1984). Consequently, any manipulation that reduces the size of the Ca2+ current, and thus intracellular Ca2+ accumulation (e.g. reduced activating voltage, decreased extracellular Ca2+ concentration, partial inactivation of the Ca2+ current by prior depolarization, or partial block by Ca2+ current blockers such as Co2+ or Cd2+), results in a slower decay of the Ca2+ current (Chad et al. 1984; Eckert & Chad, 1984). Since FMRFamide also reduces the size of the Ca2+ current, it might likewise be

13 SUPPRESSION OF Ca2+ CURRENT expected to produce weaker inactivation of the remaining current. Indeed, when an FMRFamide-suppressed Ca2+ current is scaled to the same peak amplitude as the current recorded before application of the peptide, it can be seen to decay more slowly (Fig. 5B), resulting in a decrease in the fraction of current suppressed by FMRFamide towards the end of the voltage step, as already noted above (Fig. 3E). However, FMRFamide appears to weaken inactivation purely by virtue of its action in reducing the size of the Ca2+ current, and to have no direct effect on the inactivation process itself, for when the Ca2+ current was suppressed to the same extent by other means, such as an inactivating pre-pulse (Fig. 5A; three cells) or a puff of Co2+-containing solution (Fig. 5C; two cells), inactivation was also weakened to the same extent. Ca2+ current inactivation was also evaluated (in two cells) by a twin-pulse paradigm, where inactivation was measured as the percentage decrease in peak Ca2+ current elicited by the second of two identical depolarizing steps, I2, relative to the current during the first step, I, (Fig. 5C). As expected, the effect of FMRFamide in weakening inactivation (in Fig. 5C, trace 1, obtained before application of FMRFamide, I2 is 48% smaller than I,; in trace 2, following a puff of peptide, 12 is only 40 % smaller) was exactly mimicked by puffs of Co2+-containing solution (in trace 3, I2 is 39% smaller than I,)* Secondary depression of Ca2+-dependent K+ current Previously we reported (Brezina et al. 1987) that, in the Aplysia neurones examined here, application of FMRFamide or YGG-FMRFamide has no effect on the Ca2+dependent K+ current, IK(Ca) when this current is activated by direct injection of Ca2+. Thus, when Ca2+ is supplied independently of Ca2+ channels, Ca2+-dependent K+ channels appear not to be modulated by these peptides. However, since the peptides suppress Ca2+ current elicited by depolarization, they should cause a secondary, indirect depression of any current normally activated by the elevation of intracellular Ca2+ concentration resulting from the flow of Ca2+ current. One such Ca2+-dependent current is 'K(ca)' which in previous studies has indeed been shown to be diminished by manipulations that reduce Ca2+ current (Eckert & Ewald, 1982). This prediction, that IK(ca) activated by depolarizing steps should be depressed by FMRFamide, was tested in Na+-free solution (results obtained in Na+-containing solution were similar), but in the absence of K+ current blockers. Under these conditions, depolarizing steps elicited the familiar sequence (Fig. 6A) of inward Ca2+ current with the delayed addition of outward current that had been blocked by TEA and 4-AP in our earlier experiments. Application of FMRFamide decreased the peak amplitude of the initial inward current, corresponding to the suppression of Ca2+ current already described. In addition, however, the FMRFamide puff reduced the subsequent flow of outward current (Fig. 6A). Often seen also was a small outward shift in holding current (Fig. 6A, inset), corresponding to the activation by FMRFamide of IK(s)-like current, as described in our previous paper (Brezina et al. 1987; see also Figs ). The component of depolarization-activated outward current that is suppressed by FMRFamide is associated, on repolarization to -40 mv, with a slow outward tail current (similar to that shown by Meech & Standen (1975) to be due to IK(ca))' which is also suppressed by the peptide (Fig. 6A). Both the tail current in the absence of 19 PHY

14 578 V. BREZINA, R. ECKERT AND C. ERXLEBEN A A / 25 ms 0 1,uA \ _~~~ 1/ \ -~FMRFamide / 1 _A 0-3;iA~~~~~~~~ C/ 0_ 1 pa _ o / V (mv) Fig. 6. FMRFamide suppresses K+ current activated by depolarizing steps (20 C). A, inward, outward and tail currents elicited by voltage step from -40 to + 10 mv (trace 1) are suppressed 20 s after 3 s puff of 50 /im-fmrfamide (trace 2). FMRFamide also elicits a small outward shift in holding current by activating IK(s)-like current (inset). 0 Na+ ASW; cell L3; records not corrected for leakage current. B, leakage-subtracted tail currents following repolarization to -20 (top trace in each panel), -30, -40, -50, -60, -70,-80 and-190 mv (bottom trace) after 00 ms voltage step to + 20 mv. Each trace starts 1-5 ms after the end of the depolarizing step. Top panel shows tail currents before, bottom panel after a broken pipette containing 50 -m-fmrfamide was positioned near the cell. 0 Na+-4-AP ASW (4-AP was included to block the FMRFamide-activated I,(,)-like current); l.u.q. cell. C, I-V plot of tail currents obtained as in B, measured 5 ms after the end of the depolarizing step, recorded in 0 Na+r4-AP ASW (0, *) or 0 Na+-50 K+-4-AP ASW (El, *), with (O, El) or without (0, *) application of FMRFamide as in B. Dashed line indicates holding current. Two l.u.q. cells (note different scales).

15 SUPPRESSION OF Ca2+ CURRENT 579 A 1 C 2 Ca ,4 Co Co2+-TEA 5,6 D A-..2 A-C, 20 ms B co pa V (mv) Fig. 7. The FMRFamide-suppressed K+ current is IK(ca.) Voltage steps from -40 to + 10 mv (20 C). A, early inward (Ca2+) and late outward (mainly K+) currents, elicited by 100 ms voltage step and recorded in 0 Na+ ASW (trace 1), are both suppressed 20 s after 1 s puff of 50,uM-FMRFamide (trace 2). Superfusion of 0 Na+4- Ca2+-20 Co2+ ASW eliminates Ca2+ and Ca2+-dependent currents (trace 3). Application of FMRFamide now produces only a simple increase in outward leakage current through the peptide-activated IK(S)-like conductance (trace 4). This effect of FMRFamide is blocked by superfusion of 0 Na+@- Ca2+-20 Co2+-TEA-4-AP ASW (traces 5,6). Cell L6; records not corrected for leakage current. B, difference currents obtained from pairs of traces shown in A, in each case by subtracting control trace from trace recorded after application of FMRFamide. C, same as traces 1-4 of A, but recorded in cell R15, in which FMRFamide does not enhance the IK(s)-like current. Records corrected for leakage current. D, I-V relations of outward currents measured at end of 100 ms pulses to potentials indicated. Ca2+-dependent component of control outward current, recorded in 0 Na+ ASW (0), is reduced after superfusion of 1,#M-FMRFamide (0). The Ca2+-independent component, measured in 0 Na+-0 Ca2+-20 Co2+ ASW (O), is unaffected by FMRFamide (U; the FMRFamide-activated IK(S)-like current is not seen here because this effect of the peptide desensitizes with sustained bath application: see Brezina et al. 1987). Cell L

16 580 V. BREZINA, R. ECKERT AiVD C. ERXLEBEN FMRFamide, and also that component of the tail current that is suppressed by the peptide, reversed (in l.u.q. cells) in solution containing 10 mm-k+ between -60 and -65 mv (Fig. 6B and C), values typical for the reversal potential of IK(Ca) (Meech & Standen, 1975; Deitmer & Eckert, 1985). Moreover, in solution containing 50 mm-k+, the reversal potential of the tail current recorded both with and without A B 2~~~~~~~~~ 0~~~ :? I // 4 1,uA Qo 25 ms 033iA V (mv) Fig. 8. Effectiveness of FMRFamide in suppressing outward current activated by depolarization is increased after block of IK(A) I'K(V) and IK(S)* Voltage steps from -40 to + 10 mv. Records not corrected for leakage current (20 C). A, top panel shows control current (trace 1) and current 20 s after 1 s puff of 50 /IM-FMRFamide (trace 2), recorded in 0 Na+ ASW. Bottom panel shows control current (trace 3) and current following FMRFamide application (trace 4), recorded in 0 Na+-4-AP ASW. Cell L2. B, I- V relations of outward currents measured at end of 100 ms pulses to potentials indicated, recorded in 0 Na+-4-AP ASW, with (0) and without (0) repeated puffed application of 50,tM- FMRFamide. L.u.q. cell. application of FMRFamide shifted to about -35 mv (Fig. 6 C) approximately as predicted by the Nernst equation, showing that the FMRFamide-suppressed tail current, and thus the FMRFamide-suppressed outward current during the depolarizing step itself, is a K+ current (two cells). The three major depolarization-activated K+ currents described in Aplysia neurones are the classical delayed rectifier, IK(v), the early transient current, IK(A) and 'K(Ca)' of which only the last is Ca2+ dependent (Connor & Stevens, 1971; Neher, 1971; Hermann & Gorman, 1981 a,b). Replacement of extracellular Ca2+ with Co2+ eliminated the depolarization-elicited inward and outward currents that are normally suppressed by FMRFamide (Fig. 7; five cells). In the Co2+-containing solution, FMRFamide produced only a simple increase in outward leakage current through the peptide-activated IK(s)-like conductance; this current could be blocked by further addition of TEA or 4-AP to the bath solution (Fig. 7 A and B; three cells), and was

17 SUPPRESSION OF Ca2+ CURRENT 581 A ms 0-5,A ms 1 pa c I... C) a v 0 Ca US Q C az U) CA) 0) 0~ 0' Percentage suppression -f /Ca Fig. 9. Suppression of Ca2+-dependent K+ current by FMRFamide is proportional to suppression of Ca2+ current (20 C). Voltage steps from -40 to +10 mv. All records corrected for leakage current. A, left panel shows suppression of inward and outward currents elicited by depolarizing step (trace 1) 20 s following 5 s puffof 50 4uM-FMRFamide (trace 2). Right panel (traces 3 and 4) shows that when the duration of a puff of Ca2+-free ASW containing 20 mm-co2+ is adjusted (here 2 s) to give about the same degree of inward current suppression, the degree of outward current suppression is also similar. ASW; cell L6. B, trace 1 shows current elicited by depolarizing step in 0 Na+-4-AP ASW; in this solution the current is primarily the sum of Ca2+ and Ca2+-dependent K+ currents (see text). Superfusion of 0 Na+-TEA-4-AP ASW blocks IK(Ca), allowing measurement of isolated Ca2+ current (trace 2). Subtraction of trace 2 from trace 1 yields isolated IK(Ca) (trace 3; see Eckert & Ewald, 1982). This series can be recorded in the presence of FMRFamide (here 20 s following 10 s puff of 50 /SM-FMRFamide) to allow measurement of the effect of the peptide on isolated IK(Ca) (trace 4). C, plot of percentage suppression of isolated IK(Ca), recorded as in B and measured at the end of the 100 ms depolarizing step, as function of percentage suppression of Ca2+ current integrated over the 100 ms voltage step. The currents were suppressed with 100, 200, 500 ms and 1, 2 and 5 s (in order of increasing suppression) puffs of 50,uM-FMRFamide. L.u.q. cell. not seen at all in cell R15 (Fig. 7C; three cells), in which FMRFamide does not activate the IK(s)-like conductance (Brezina et al. 1987). The Ca2+ dependence of the FMRFamide-sensitive, depolarization-elicited K+ current suggests that it is IK(ca). This identification was supported by the observation

18 582 V. BREZINA, R. ECKERT AND C. ERXLEBEN Aa t t t c.j 2 *1 \ b 2 Ba b c A. Ba-Bc, 50 ms Ba, Bc, 2 MA /4 Bb, 1 5 pa 3 4 Aa-Ac, 20 s C 0 0. (A CL 11) 0~ na C, los /,i ft \. 2 na I, N.. I F... * 0O Time (s) Fig. 10. Three effects of FMRFamide, i.e. direct suppression of Ca2+ current, indirect suppression of Ca2+-dependent K+ current, and direct enhancement of IK(s)-like current, follow similar time courses after a puff of FMRFamide at 20 C (ASW). In Aa-Ac, cell L6 was voltage clamped at -40 mv. Records of control holding current (trace 1 in each of Aa-Ac), and current elicited by 3 s puff of 50 /SM-FMRFamide (arrow, trace 2 in each of Aa-Ac), were obtained alternately. In addition, the membrane potential was stepped to + 10 mv for 100 ms in each control and FMRFamide record, in the latter case at a measured time following application of the peptide. Each of Ba-Bc shows an expansion of the currents elicited by these voltage steps in the corresponding panel in Aa-Ac, either without (trace 3) or with (trace 4) application of FMRFamide (records not corrected for leakage current). To determine the time course of FMRFamide effects, the time between application of FMRFamide and the voltage step was varied. In Aa and Ba, voltage step followed FMRFamide after 1 s; in Ab and Bb, after 20 s; in Ac and Bc, after 50 s. C summarizes results of a similar experiment on a different L6 cell. Time course of enhancement of IK(s)-like current (top), elicited by 2 s puff of 50 4uM-FMRFamide (arrow), is shown on same time scale and aligned with a plot (bottom) of percentage suppression of peak inward current (@), and of outward current measured at end of 100 ms voltage step (0), as a function of time since FMRFamide application. that FMRFamide reduced outward current only in the voltage range corresponding to the 'hump' in the I-V plot (Figs. 7D and 8B) that has been attributed to IK(Ca) (Meech & Standen, 1975). Furthermore, superfusion of solution containing 10 mm-4-ap, which blocks IK(A), IK(V) and the FMRFamide-activated IK(s)-like current but enhances IK(Ca) (Hermann & Gorman, 1981 a; Brezina et al. 1987), increased the percentage depression of outward current by FMRFamide (Figs. 8A and 9B; five cells); this effect presumably resulted partly from the block of peptide-insensitive currents such as IK(A) and IK(v) by 4-AP, partly from the block

19 SUPPRESSION OF Ca2+ CURRENT by the drug of IK(s)-like current, and partly from enhancement of IK(Ca)' and thus of the absolute magnitude of FMRFamide effect on it. In contrast to IK(Ca) IK(V) (which constitutes most of the outward current recorded in Ca2+-free solution; see traces 3 and 4 of Fig. 7 A and C) was unaffected by FMRFamide (five cells). Similarly, the peptide had no effect on IK(A)' which was measured by subtraction of currents elicited by depolarizing steps with and without a 600 ms pre-pulse to -90 mv, or partially isolated in Ca2+-free solution containing 20 mm-co2+ and 100 mm-tea (five cells). Since FMRFamide has no effect on IK(Ca) when this current is elicited directly by injection of Ca21 into the cell (Brezina et al. 1987), the FMRFamide-induced depression seen in the present experiments must arise as a consequence of the suppression of Ca2+ current by the peptide. In this context, it is significant that FMRFamide depressed outward current only in cells (the l.u.q. cells and cells R2 and R15) in which it suppressed Ca2+ current; in cells (L7 and LI 1) in which the peptide did not suppress Ca2+ current, IK(ca) was also unaffected. Moreover, when the extent of suppression of Ca2+ current by FMRFamide was mimicked by puffed application of ASW containing Co2+ (a treatment not expected to directly affect IK(Ca)), the extent of outward-current suppression was also similar (Fig. 9A; two cells), showing that the effect of FMRFamide on IK(ca) can be accounted for by its effect on Ca2+ current. This was also shown by an experiment in which suppression of both Ca2+ current and IK(Ca) was measured directly (Fig. 9B); the amplitude of IK(Ca) suppressed by FMRFamide was linearly related to the integral of suppressed Ca2+ current (Fig. 9C; two cells), a relation found previously to govern the dependence of IK(Ca) on Ca2+ current in these cells (Eckert & Ewald, 1982). Relation between effects of FMRFamide on Ca2+ and IK(s)-like currents In addition to partially suppressing Ca2+ current, application of FMRFamide and YGG-FMRFamide increases IK(s)-like current (Brezina et al. 1987) in most of the same cells. The two peptidergic effects have certain features in common (see Discussion), suggesting that both may be mediated by the same peptide receptor and a common intracellular mechanism such as a GTP-binding protein and/or a second messenger cascade, if indeed these are involved (see below). If the Ca2+ and IK(s)-like conductance changes induced by FMRFamide were closely coupled through common intermediate steps, the rise and fall of the two effects might occur with a similar time course. On the other hand, distinctly dissimilar time courses would indicate separate intermediate mechanisms. To examine this, the extent of suppression of Ca2+ and Ca2+-dependent K+ currents was measured at various times during the rise and fall of the peptide-induced IK(s)-like current (Fig. 1OA and B). At 20 C, the suppression of Ca2+ and Ca2+-dependent K+ currents was sometimes induced with a shorter latency following a puff of peptide than was the enhancement of IK(S)-like current, but in most experiments the time course of the two effects was indistinguishable (Fig. 10 C; six cells). As the temperature was reduced to 13 C, the time course of both effects slowed to the same extent (Fig. 11; five cells). Q10 for the time to peak of the IK(s)-like current, and therefore also the maximal suppression of Ca2+ current and IK(Ca)' is 2-2 (Brezina et al. 1987), higher than expected for a diffusion-limited process. The low Qlo expected for such a process was 583

20 584 V. BREZINA, R. ECKERT AND C. ERXLEBEN observed, in contrast, when Ca2+ current was suppressed by puffs of Ca2+-free ASW containing 20 mm-co2+ (Fig. 11 B; two cells). Mechanism of Ca2+ current suppression by FMRFamide If, as we have argued in the case of enhancement of IK(s)-like current by FMRFamide (Brezina et al. 1987), high Qlo suggests mediation by a metabolic process A ~ ~ 20 C 1 5 s / ~~~~~~13 OC 10 na B 45-1/0==U '- c 0~ ~ ~~~~~~ Time (s) Fig. 11. Reduction in temperature slows the time course of activation of IlK(s)-like current and suppression of Ca2+ current by FMRFamide to an equal extent (ASW). Data obtained as in Fig. 10 (but voltage steps to + 20 mv), and plotted as in Fig. 10 C. A, IK(s)-like current elicited at -40 mv by 2 s puffs of 50,uM-FMRFamide (arrow) at 20 'C and 13 'C. B, percentage suppression of peak inward current by 1 s puffs of 50 /zm-fmrfamide (M) or of Ca2+-free ASW containing 20 mm-co2+ (M), at 13 'C. Two l.u.q. cells. such as a second-messenger system, then this is true also of the suppression of Ca2+ current by the peptide. We carried out a number of experiments to test the possible involvement of known second-messenger systems. Enhancement of IK(S)-like current by FMRFamide is blocked by elevation of intracellular adenosine 3',5'-cyclic monophosphate (cyclic AMP) by means of application of forskolin (an activator of adenylate cyclase), RO (an inhibitor of phosphodiesterase) or serotonin (a neurotransmitter that elevates cyclic AMP in some of these cells), or by direct injection of cyclic AMP into the cell (Brezina et al. 1987). However, neither forskolin (50 jtm; eight cells) nor serotonin (1 mm; two cells), both applied in the presence of 100,tM-RO , had any effect on suppression of Ca2+

21 SUPPRESSION OF Ca2+ CURRENT 585 Aa,Ab,25s Ba 3 7 na 4 Aa j 2 g* b 4-1 ~~~~~~1 2..Ḃa, 0 7 pa Bb, 0 3,uA 30 ms Fig. 12. Cyclic AMP injection blocks FMRFamide-induced IK(s)-like current but not suppression of Ca2+ current (20 C). Procedure and numbering of traces as in Fig. IOA and B. Currents (not leakage-subtracted) in Ba and Bb were elicited by voltage steps from -40 to + 10 mv, before and 20 s after 2 s puff of 50 /sm-fmrfamide (arrows in Aa and Ab). Experiment was carried out in 0 Na+ ASW, to eliminate the Na+-dependent inward current normally elicited by cyclic AMP injection (Brezina et al. 1987), containing 0-25 % DMSO. Aa and Ba, before cyclic AMP injection. Ab and Bb, following superfusion of 100 /LM-RO and cyclic AMP injection (10 min, 0 3,uA ionophoretic current). L.u.q. cell. current by FMRFamide in the l.u.q. cells or even in cell R2, in which these treatments were particularly effective in preventing enhancement of IK(s)-like current. Fig. 12 shows the same result in the case of direct ionophoretic injection of cyclic AMP into the cell (six cells). Similarly, pressure injection of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in the presence of 100 /tm-ro (five cells), and superfusion of 1 mm-sodium nitroprusside (five cells), an agent reported to increase cyclic GMP but not cyclic AMP levels in some tissues (Schultz, Schultz & Schultz, 1977), was without effect on the FMRFamide-induced suppression of Ca2+ current. Phorbol esters, activators ofprotein kinase C (Nishizuka, 1984), mimic Ca2+ current

22 586 V. BREZINA, R. ECKERT AND C. ERXLEBEN B ms A, 2jA B, 0-4 pa C Il\ C. ao 0-3 I GTP-y-S I Time (min) Fig. 13. Injection of GTP-y-S mimics the effect of FMRFamide on Ca2+ current (20 C). A and B show currents elicited by voltage steps from -40 to + 10 mv before (trace 1) and 20 s after 3 s puff of 50 /SM-FMRFamide (trace 2), and before (trace 3) and after ionophoretic injection of GTP-y-S (trace 4). Once GTP-y-S has been injected, a puff of FMRFamide has no further effect (trace 5). A, ASW; l.u.q. cell. Records not corrected for leakage current. GTP-y-S was injected for 5 min (08,uA ionophoretic current). B, 0 Na+-TEA-4-AP ASW; cell L3. Records corrected for leakage current. GTP-y-S was injected for 10 min (0-1,uA ionophoretic current). C, plot of peak amplitude of Ca2+ currents (not corrected for leakage current) elicited by regular voltage steps to + 10 mv. 50,tM-FMRFamide was applied in 5 s puffs (arrows). GTP-y-S was injected (0 5 #ua ionophoretic current) during the time shown. 0 Na+-TEA-4-AP ASW; l.u.q. cell. modulation in several preparations including Aplysia neurones (DeRiemer, Strong, Albert, Greengard & Kaczmarek, 1985; Rane & Dunlap, 1986; Strong, Fox, Tsien & Kaczmarek, 1986). Experiments to be described elsewhere (V. Brezina, in preparation) showed that enhancement of IK(s)-like current by FMRFamide can be blocked by phorbol esters. However, suppression of Ca2+ current by the peptide was

23 SUPPRESSION OF Ca2+ CURRENT unchanged (in six cells) following superfusion of 2 /am solution of each of the phorbol esters 4/J-phorbol 12-myristate 13-acetate (PMA, also called TPA), 4,/-phorbol 12/, 13a-dibutyrate (PDB) and 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA). Another recently proposed second messenger is inositol trisphosphate (JP3; Berridge & Irvine, 1984). IP3 was injected ionophoretically or by pressure, and had no effect on the FMRFamide-induced IK(S)-like current or on suppression of Ca2+ current by the peptide (five cells). Finally, modulation of Ca2+ channels in other preparations, whether or not second messengers are involved, appears to be mediated by guanosine 5'-triphosphate (GTP)-binding proteins (Gilman, 1984), since application of the non-hydrolysable GTP analogues 5'-guanylylimidodiphosphate (GppNHp) and guanosine 5'-O-(3-thiotriphosphate) (GTP-y-S) mimics or potentiates the modulatory effect (Breitwieser & Szabo, 1985; Shibata, Northup, Momose & Giles, 1986; Lewis & Weight, 1986) while guanosine 5'-O-(2-thiodiphosphate) (GDP-,8-S) blocks it (Holz, Rane & Dunlap, 1986). In order to find out whether suppression of Ca2+ current by FMRFamide is also mediated by GTP-binding proteins, we injected GppNHp, GTP-y-S and GDP-,/-S ionophoretically or by pressure. Injection of GTP-y-S mimicked, in the l.u.q. cells and cells R2 and R15, the effect of FMRFamide on Ca2+ and outward K+ currents elicited by depolarizing steps: both currents were suppressed (Fig. 13A; seventeen cells). However, GTP-y-S had no effect, except to increase the outward leakage current due to activation of IK(s)-like conductance (see below), on currents remaining in Ca2+-free solution containing 20 mm_co2+ or on IK(Ca) elicited by injections of Ca2+ into the cell (two cells). Thus, while the depolarization-elicited K+ current suppressed by GTP-y-S in Fig. 13A is IK(ca)' its suppression by GTP-y-S, like that by FMRFamide, is secondary to suppression by both agents of Ca2+ current. The isolated Ca2+ current, recorded in Na+-free solution containing 200 mm-tea plus 5 mm-4-ap, was suppressed by o (but never completely) following injection of GTP-y-S (Fig. 13B and C; twelve cells); the Ca2+-dependent K+ tail current recorded in this solution following repolarization to -40 mv at 20 C (see above) was also suppressed (Fig. 13B). Suppression of Ca2+ current by GTP-y-S, unlike the parallel effect of FMRFamide, was slow to develop (5-20 min) and irreversible, and was observed even without prior exposure of the cell to FMRFamide, although, when the peptide was puffed onto the cell before suppression of Ca2+ current by GTP-y-S was fully developed, its effect also became irreversible. Once GTP-y-S had fully suppressed Ca2+ current, FMRFamide had no further effect (Fig. 13 C). In addition to suppressing Ca2+ current, GTP-y-S also mimicked the other primary effect of FMRFamide, namely activation by the peptide of IK(s)-like current (Brezina & Eckert, 1986). In contrast to GTP-y-S, injection of its adenosine analogue adenosine 5'-O- (3-thiotriphosphate) (ATP-y-S; three cells) had no effect on Ca2+ current or on its suppression by FMRFamide. Injection of GppNHp had, in some cells, effects similar to GTP-y-S, suppressing both Ca2+ and Ca2+-dependent K+ currents. However, these effects of GppNHp were usually much smaller than those of GTP-y-S, and FMRFamide was still able to reversibly suppress the remaining current (ten cells). Injection of GDP-,#-S had no clear effect on Ca2+ current or on its suppression by FMRFamide (fifteen cells); while the effect of the peptide sometimes decreased following injection of GDP-,d-S, this also happened in some control cells, particularly 587

24 588 V. BREZINVA, R. ECKERT AND C. ERXLEBEN with the repeated applications of FMRFamide over prolonged periods that were used to test the effect of GDP-fl-S. Another probe of GTP-binding protein function is pertussis toxin (PTX; also called islet-activating protein, IAP), which ADP-ribosylates and inactivates one or more of these proteins (Ui, 1984; Sternweis & Robishaw, 1984). PTX has recently been Aa Ba 1-40 mv 1,2 \- 45 mv Aa, Ba, 5 s b Ab, 10 ms Bb, 80 ms b \ -4 0 mv - Fig. 14. Shortening of spikes by FMRFamide (20 C). Aa and Ba show voltage trace recorded before (trace 1) and following 3 s puff of 50 /tm-fmrfamide (arrow, trace 2). 4 ms current pulse was used to evoke an action potential (not resolved in Aa and Ba; expanded in Ab and Bb) 20 s following the puff of peptide (trace 4) or at the corresponding time in the control record (trace 3). Membrane potential was brought by current injection to -40 mv before each trace was recorded; when, as in Aa, FMRFamide elicited a hyperpolarization, the potential was returned to -40 mv just before the action potential. A, ASW. B, TEA-4-AP ASW. Cell R2. used to block suppression of Ca2+ current by neurotransmitters in chick dorsal root ganglion neurones (Holz et al. 1986) and cultured mammalian pituitary cells (Lewis & Weight, 1986). We found that neither the enhancement of IK(s)-like current nor the suppression of Ca2+ current by FMRFamide was affected by incubation of the ganglion in PTX (1 jug/ml) for up to 12 h at C (six cells). FMRFamide shortens action potentials We showed earlier (Brezina et al. 1987) that activation of IK(S)-like current by FMRFamide hyperpolarizes the l.u.q. cells and cell R2, and thus suppresses spiking. When action potentials were nevertheless elicited by current injection, however, they were reversibly shortened by application of FMRFamide (Fig. 14), as previously noted by Abrams, Castellucci, Camardo, Kandel & Lloyd (1984). In the absence of K+ current blockers, puffs of 50 1tM-FMRFamide shortened spikes by 5-15 o

25 SUPPRESSION OF Ca2+ CURRENT (Fig. 14A; five cells); this effect resulted presumably partly from activation of IK(s)-like current by FMRFamide (compare Klein & Kandel, 1978,1980; Abrams et al. 1984), but also partly from suppression of Ca2+ current by the peptide, since spikes were shortened, by %, even when TEA and 4-AP were used to block the IK(S) -like current and its associated FMRFamide-induced hyperpolarization (Fig. 14B; five cells). 589 DISCUSSION Our results show that external application of the molluscan neuropeptide FMRFamide, or of its derivative YGG-FMRFamide, leads to suppression of Ca2+ conductance in certain neurones (the l.u.q. cells L2-L4 and L6, and cells R2 and R15) in the abdominal ganglion of Aplysia californica. Analysis of this effect is complicated, however, by the fact that in all of these cells except cell R15 (see below) the peptides also enhance, in the absence of K+ current blockers, a K+ current (Brezina et al. 1987) that resembles the 'S' current IK(S) described in Aplysia neurones (Siegelbaum et al. 1982). Although at a holding potential of -40 mv this peptide-induced current is small (usually < 20 na in the l.u.q. cells; see Figs ), and can be seen in low-gain records such as that in Fig. 6A only as a small outward shift in holding current (Fig. 6A, inset), depolarization increases the outwardly rectifying IK(s)-like current markedly. Enhancement of this current by FMRFamide thus increases the apparent outward leakage current (seen isolated in Fig. 7A, traces 3 and 4) flowing during a depolarizing step. In the absence of K+ current blockers, the extra component of leakage current sums with other currents elicited by the depolarization and obscures the true magnitude of the effect of FMRFamide on them. Thus, the reduction of inward current by the peptide seen in records such as those in Fig. 7A (traces 1,2), and in Fig. 10 is larger than the actual peptidergic suppression of Ca2+ current, and the reduction of outward current seen in these Figures is smaller than the actual depression of Ca2+-dependent K+ current, as revealed by the records in Fig. 8A. In this experiment the FMRFamide-enhanced IK(s)-like current (as well as FMRFamide-insensitive K+ currents) was blocked with 10 mm-4-ap (Brezina et al. 1987), unmasking peptidergic suppression of inward current of an extent more in agreement with measurements on isolated Ca2+ current (Fig. 1 A), and a more pronounced suppression of outward current, which in 4-AP-containing solution is mainly IK(Ca) (although the suppression of outward current was probably increased not only by elimination of the opposing FMRFamide-induced IK(S)-like current by 4-AP, but also by a direct enhancement of IK(Ca)' and thus of the absolute magnitude of FMRFamide effect on it, by the drug as reported by Hermann & Gorman, 1981 a). It might be argued that the reduction of inward current examined in Figs. 1-5 is not in fact due to suppression of Ca2+ current by FMRFamide, but rather to a summing, in the way just described, of peptide-activated IK(S)-like current, or of another contaminating current, with a Ca2+ current of constant amplitude. This possibility is ruled out by a number of observations. (i) We have shown previously (Brezina et al. 1987; compare also traces 3,4 and 5,6 of Fig. 7A) that the FMRFamide-enhanced IK(s)-like current is blocked effectively by the high concentrations of TEA (200 mm) and 4-AP (5 mm) that were present in

26 590 V. BREZINA, R. ECKERT AND C. ERXLEBEN the bath solution throughout all experiments involving isolation of Ca2+ current. Accordingly, in these experiments FMRFamide never elicited the outward shift in holding current, or the increased leakage current during voltage steps, that would indicate an enhancement by the peptide of IK(s)-like current. In contrast, these effects were regularly seen in the same cells in the absence of K+ current blockers (see above). Furthermore, suppression of Ca2+ current by FMRFamide was unaffected by injection into the cell of TEA+ and Cs+, which might be expected to block any remaining IK(s)-like (Brezina et al. 1987) and other K+ currents. (ii) FMRFamide suppresses Ca2+ current in cell R15 (Fig. 7C), in which it appears to have no effect on IK(S)-like current (Brezina et al. 1987; see below). (iii) The effect of FMRFamide on inward current, together with the current itself, is eliminated by replacement of bath Ca2+ with Co2+ (Fig. 2B), suggesting that the effect of the peptide is on Ca2+ current or a Ca2+-dependent current; in contrast, the peptide-enhanced IK(s)-like current is Ca2+ independent (Fig. 7A and B; Brezina et al. 1987). The possibility that FMRFamide might be acting not on Ca2+ current, but on a Ca2+-dependent current is ruled out by injection of the Ca2+ chelating agent EGTA, which does not affect the peptidergic suppression of inward current (Fig. 3 C). (iv) FMRFamide is equally effective in suppressing Ca2+ current elicited by depolarizing steps, as well as Ca2+ tail currents on repolarization, when these currents are recorded under conditions expected to null the contribution to the total current of K+, H+ and Cl- currents, and thus null also any possible effects of FMRFamide on them. (v) The FMRFamide-suppressed fraction of inward current is always proportional (ignoring the complications arising from Ca2+-dependent inactivation of the Ca2+ current) to the control Ca2+ current, even when the latter varies in size as a result of inactivation during the voltage step or with steps to different potentials (above + 10 mv; see below). (vi) FMRFamide blocks a number of processes that are normally activated by elevated intracellular Ca2+ resulting from the flow of Ca2+ current; thus, FMRFamide weakens Ca2+-dependent inactivation of the Ca2+ current itself, suppresses activation of IK(Ca) and suppresses the Ca2+-dependent K+ tail current resistant to TEA and 4-AP often seen on repolarization to -40 mv at high temperatures (Deitmer & Eckert, 1985; Fig. 1 A, inset). Since FMRFamide does not directly affect at least two of these processes, namely Ca2+ current inactivation (Fig. 5) and IK(Ca) activation (Brezina et al. 1987), all of these effects can be assumed to arise as a consequence of suppression of Ca2+ current by the peptide. FMRFamide eliminates the same fraction of control Ca2+ current at all potentials above + 10 mv, and does not directly affect the time course of the remaining current. The effect of FMRFamide thus resembles other instances of Ca2+ current modulation by neurotransmitters in various preparations, for example cardiac myocytes (Reuter & Scholz, 1977), dorsal root ganglion neurones (Dunlap & Fischbach, 1981; Forscher & Oxford, 1985; Macdonald & Werz, 1986), and indeed by FMRFamide in Helix neurones (Colombaioni, Paupardin-Tritsch, Vidal & Gerschenfeld, 1985). Change in the number of functional Ca2+ channels has been suggested as the mechanism of some of these effects (Reuter & Scholz, 1977; Dunlap & Fischbach, 1981; Bean, Nowycky & Tsien, 1984; but see Cachelin, de Peyer, Kokubun & Reuter, 1983). In the Aplysia

27 SUPPRESSION OF Ca2+ CURRENT neurones examined here it apears that, in addition, not all of the Ca2+ current is susceptible to suppression by FMRFamide (Fig. 1 C), possibly because the current flows through two or more different classes of Ca2+ channels, only one of which is affected by the peptide. This may also explain the incomplete suppressibility of Ca2+ current by GTP-y-S (Fig. 13B and C) and, assuming the different classes of Ca2+ channels to be activated at different potentials, the reduced effectiveness of FMRFamide in suppressing Ca2+ current at potentials below + 10 mv. Two or more types of Ca2+ channels have been reported in Aptysia neurones (Chesnoy-Marchais, 1985; Strong et al. 1986). Several results suggest that the two direct effects of FMRFamide, namely suppression of Ca2+ current and enhancement of IK(s)-like current, are mediated by the same receptor and perhaps by a common intracellular mechanism. (i) FMRFamide elicits both effects in all abdominal ganglion cells examined that are at all sensitive to the peptide, with the only exception of cell R15, in which we did not observe the peptide-enhanced IK(s)-like current (Fig. 7C; Brezina et al. 1987). It appears, however, that in tissue culture even cell R15 is capable of responding to FMRFamide with the hyperpolarization attributable to enhanced IK(s)-like conductance (Ambron, Lloyd, Flaster & Schacher, 1985). (ii) The concentrations of FMRFamide required to elicit the two effects are similar (apparent KD- 10,UM; Fig. 1 C). (iii) Both effects are elicited equally well by FMRFamide and YGG-FMRFamide, and not at all by FMRF or methionine-enkephalin (Brezina et at. 1987). (iv) The two effects follow the same time course at both 20 C and 13 C (Figs. 10 and 11). The relatively high sensitivity to temperature (Q1o 2 2) of both effects suggests that they may be mediated by a metabolic process such as a second-messenger system. We have been unable, however, to demonstrate mediation of Ca2+ current suppression by any of the known second-messenger systems, those involving cyclic AMP, cyclic GMP, Ca2+ (suppression is not eliminated by injection of EGTA), inositol trisphosphate and protein kinase C. (v) Both effects are mimicked by injection of GTP-y-S into the cell. The result that GTP-y-S, like FMRFamide, suppresses Ca2+ current, and at the same time blocks further effect of the peptide, suggests that FMRFamide may normally suppress Ca2+ current by activating a GTP-binding protein. Alternatively, GTP-y-S (whether or not through a GTP-binding protein) and FMRFamide may act independently on the Ca2+ current; the block of FMRFamide effect by GTP-y-S would then simply be a consequence of the suppression of all FMRFamide-sensitive Ca2+ current by GTP-y-S. The former interpretation is strongly supported by the observation that GTP-y-S mimics the effect of FMRFamide on IK(s)-like current as well as on Ca2+ current (Brezina & Eckert, 1986), while the lack of clear effectiveness of GDP-,f-S (which might be expected to block effects of FMRFamide without mimicking them) argues for the latter interpretation. While the same may be said for the effectiveness ofgtp-y-s even in the absence of applied FMRFamide (compare Breitwieser & Szabo, 1985), it has recently become clear that presence of agonist is not required in order for GTP-y-S to activate GTP-binding proteins in several preparations (Shibata et al. 1986; Lewis & Weight, 1986; Kurachi, Nakajima & Sugimoto, 1986). Similarly, the inability of pertussis toxin to block the effects of FMRFamide may simply mean that the putative GTP-binding protein mediating actions of the peptide is not ADPribosylated by PTX; such GTP-binding proteins are known (for example G., the 591

28 592 V. BREZINA, R. ECKERT AND C. ERXLEBEN protein that activates adenylate cyclase; see Gilman, 1984; Sternweis & Robishaw, 1984) and may even mediate Ca2+ current suppression in some preparations (Shibata et al. 1986). It should also be noted that PTX must, to be effective, bind to a receptor on the cell surface and, after crossing the cell membrane, be dissociated into its subunits and its intra-chain disulphide bonds reduced by possibly specific intracel-, (YGG-)FMRFamide Ca2'-dependent K+ channel Fig. 15. Schematic diagram showing the probable mechanism of the major effects of FMRFamide and YGG-FMRFamide in the l.u.q. cells and cells R2 and R15 (but in R15 the peptides have no effect on IK(s)-like current), as described in this paper and by Brezina et al. (1987). The Na+-dependent inward current sometimes induced by the peptides (Brezina & Eckert, 1986; Brezina et al. 1987) is not shown. For further explanation see text. lular processing enzymes (Katada, Tamura & Ui, 1983; Ui, 1984). Aplysia neurones may thus lack some of these components, as apparently do other cell types (Katada et al. 1983; Ui, 1984). Fig. 15 presents a graphical summary of the most likely mechanism of FMRFamide action in the Aplysia neurones examined here, based on the conclusions reached in this paper and by Brezina et al. (1987). Thus, FMRFamide and YGG-FMRFamide are shown to bind to a single type of extracellular receptor coupled to a GTP-binding protein. A common second-messenger system then mediates both increased activity of IK(s)-like channels (an effect that is counteracted by phosphorylation of these channels or associated proteins by cyclic AMP-dependent protein kinase) and decreased activity of Ca2+ channels, and thus decreased intracellular accumulation of Ca2+ and decreased activity of Ca2+-dependent K+ channels.

29 SUPPRESSION OF Ca2+ CURRENT The effect of FMRFamide on these Aplysia neurones resembles neurotransmitter effects in many preparations (for example, Williams & North, 1985; Jacklet & Acosta-Urquidi, 1985; Cherubini & North, 1985; Kretz, Shapiro, Bailey, Chen & Kandel, 1986) in that simultaneous actions of the peptide on IK(S)-like and Ca2+ currents synergistically modulate (in this case, inhibit) electrical activity of the cell. Thus, enhancement of IK(S)-like current leads to hyperpolarization, less frequent spiking (Brezina et al. 1987), and, together with the suppression of Ca2+ current, to shortening ofthose spikes that are nevertheless generated (Fig. 14 A). Spike shortening then presumably results in decreased transmitter release (see Klein & Kandel, 1980; Abrams et al. 1984) since the Ca2+ current necessary for release flows for a shorter time; additionally, of course, FMRFamide will directly reduce the amplitude of this current, assuming that its action in the presynaptic terminal at all resembles its effects in the cell body. The likely reduction of transmitter release by FMRFamide suggests a role for the peptide in counteracting presynaptic facilitation (Brezina et al. 1987), or as an agent of presynaptic inhibition (compare Kretz et al. 1986). This work was supported by NSF grant BNS , USPHS grant ROI NS08364, and by USPHS National Research Service Award GM REFERENCES ABRAMS, T. W., CASTELLUCCI, V. F., CAMARDO, J. S., KANDEL, E. R. & LLOYD, P. E. (1984). Two endogenous neuropeptides modulate the gill and siphon withdrawal reflex in Aplysia by presynaptic facilitation involving cyclic AMP-dependent closure of a serotonin-sensitive potassium channel. Proceedings of the National Academy of Sciences of the U.S.A. 81, AMBRON, R. T., LLOYD, P., FLASTER, M. S. & SCHACHER, S. (1985). FMRF-amide in neuron R2 of Aplysia: evidence for its role as a second neurotransmitter. Society for Neuroscience Abstracts 11, 483. BEAN, B. P., NOWYCKY, M. C. & TSIEN, R. W. (1984).,B-Adrenergic modulation of calcium channels in frog ventricular heart muscle. Nature 307, BERRIDGE, M. J. & IRVINE, R. F. (1984). Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312, BOER, H. H., SCHOT, L. P. C., VEENSTRA, J. A. & REICHELT, D. (1980). Immunocytochemical identification of neural elements in the central nervous system of a snail, some insects, a fish, and a mammal with an antiserum to the molluscan cardio-excitatory tetrapeptide FMRF-amide. Cell and Tissue Research 213, BREITWIESER, G. E. & SZABO, G. (1985). Uncoupling of cardiac muscarinic and,-adrenergic receptors from ion channels by a guanine nucleotide analogue. Nature 317, BREZINA, V. & ECKERT, R. (1986). Modulation of K and Ca currents by FMRFamide in Aplysia neurons is mimicked by a non-hydrolyzable GTP analog. Society for Neuroscience Abstracts 12, BREZINA, V., ECKERT, R. & ERXLEBEN, C. (1987). Modulation of potassium conductances by an endogenous neuropeptide in neurones of Aplysia californica. Journal of Physiology 382, BREZINA, V., ERXLEBEN, C. & ECKERT, R. (1985). FMRF-amide suppresses calcium current in Aplysia neurons. Biophysical Journal 47, 435a. BYERLY, L., MEECH, R. & MOODY, W. (1984). Rapidly activating hydrogen ion currents in perfused neurones of the snail, Lymnaea stagnalis. Journal of Physiology 351, CACHELIN, A. B., DE PEYER, J. E., KOKUBUN, S. & REUTER, H. (1983). Ca2+ channel modulation by 8-bromocyclic AMP in cultured heart cells. Nature 304, CHAD, J., ECKERT, R. & EWALD, D. (1984). Kinetics of calcium-dependent inactivation of calcium current in voltage-clamped neurones of Aplysia californica. Journal of Physiology 347, CHERUBINI, E. & NORTH, R. A. (1985).,s and K opioids inhibit transmitter release by different mechanisms. Proceedings of the National Academy of Sciences of the U.S.A. 82,

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