Sweatt et al 1989 This FMRFamide channel opening ac tion has two d - PDF document

1 Sweatt et al. 1989). This FMRFamide chan
Sweatt et al. 1989). This FMRFamide channel opening ac- tion has two distinct components. 1) In basal conditions, FMRFamide increases the S-K+ channel opening probabil- ity (Belardetti et al. 1987) through activation of the 12-li- poxygenase pathway of arachidonic acid (Piomelli et al. 1987). The active metabolite of this pathway in l Piomelli et al. 1989) to increase the opening of the S-K+ channel (Bela’rdetti et al. 1989). This opening action does not involve a phosphorylation or a dephosphorylation reaction (Belardetti et al. 1989; Buttner et al. 1989), and it is thought to be direct. 2) In the presence of elevated CAMP levels, FMRFamide completely antagonizes the closing action of CAMP and reopens the S-K+ channels (Belardetti et al. 1987). There is evidence that this antagonistic of FMRFamide is also me- diated by a lipoxygenase pathway of arachidonate metabo- lism (Volterra and Siegelbaum 1988), which ultimately leads to dephosphorylation of the S-K+ channel (Ichinose et al. 1990; Sweatt et al. 1989). However, the mechanism that leads to this dephosphorylation is not METHODS Cell culture ApZysia calijhxica were purchased from the Aplysia Research Facility at the University of Miami (Miami, FL), kept in a refriger- ated ( 15°C) aquarium, and fed with fresh red gracyllaria. Solutions The cells were superfused with artificial sea water (ASW), con- taining (in mM) 460 NaCl, 10 KCI, 55 MgCl,, 11 CaCl,, and N-2-hydroxyethylpiperazine- N ‘-2-ethanesulfonic acid (HEPES), brought to pH 7.6 with NaOH. All

2 the solutions were filtered through a 0.
the solutions were filtered through a 0.2~pm filter before use. TEA containing sea water was obtained by replacing the appropriate amount of NaCl with either TEA-Cl or TEA-Br (Fluka, Buchs, Switzerland). Chemicals The components for the ASW and the CAMP analogues were purchased from Sigma Chemical (St. Louis, MO), FMRFamide from Peninsula Laboratories (Belmont, CA), 5-HT from Calbio- them (San Diego, CA), Drug application The CAMP analogues and the transmitters were stored dry at -25 OC. Arachidonic acid was stored in toluene under and at -70°C. Stock solutions of the CAMP analogues and the transmit- ters in ASW were prepared at the beginning of each day and were kept in ice. An aliquot from the arachidonic acid solution was dried under gentle argon flux in a glass tube immediately before use; then 200 ~1 of ASW were added, and the mixture was soni- cated for 15 s before the application. CAMP or 5-HT were man- ually pipetted into the experimental bath as concentrated aliquots from the stock solution. Their concentrations, as given in the text and figures, are their final bath concentrations. FMRFamide and arachidonic acid were pressure-applied from a wide-mouthed pi- FMRFAMIDE INHIBITION BY SEROTONIN 1849 pette positioned near the neuronal cell body; the concentrations of these compounds refer to Voltug6-wlamp recordings Intracellular microelectrodes were pulled from WPI (New Ha- ven, CT) thin-wall glass capillaries and were filled with 2.5 KC1 to resistances Da tu anahis . Current were played back on chart paper.

3 We measured the peak current responses t
We measured the peak current responses to the drug applications in the outward and in the inward directions (Fig. 1). using a holding current of A + FMRFa 0 FMRFa zero for all our measurements. This analysis is based on two as- sumptions: I) that the S-K’ already opens and closes spontane- ously at the resting S current, in the absence of FMRFamide or arachidonic acid, was 100 % A2 -I [FMRF a] A + 8b-CYCLIC AMP 0 8b-CYCLIC AMP I3 I +5-HT [5-HTl 0 S-HT FIG. 1. Drawings showing the method used to mea- sure, normalize, and plot the dose-response relationship of FMRFamide (or arachidonic acid, A) and 5-HT (or 8b- CAMP, B). See METHODS for explanations. -I- FMWa 0 FMRFa I 81 ! m ril s T obtained by plotting the normalized responses to each concentra- tion (AZ), which were measured before the application of FMRFamide or arachidonic acid. The RESULTS S-K+ conductance is the dominant target oj’regulation at the resting potential level Three K+ currents are modulated by’ FMRFamide and 5-HT in Aplvsia mechanosensory neurons: the S-K+ current, Ik(& and IktI/). We exploited their different sensi- tivities to TEA to confirm that, under our experimental conditions, the S-K+ conductance is the dominant target of the regulation by FMRFamide and 5-HT. The macroscopic S-K+ current response to serotonin at -35 mV is insensitive l i. At the single-chan- nel level, external TEA reduces the elementary S-K+ chan- nel current, i ( Kd = 90 mM), but also increases its probabil- ity of opening, P, ( Shuster and Siegelbau

4 m 1987 ). Because 5-HT decreases N, the
m 1987 ). Because 5-HT decreases N, the number of active channels (Siegel- baum et al. 1982), the microscopic mechanism of action of TEA explains its lack of effect at macroscopic level, as pro- posed by Shuster and Siegelbaum ( 1987). Whatever the interpretation, this experiment indicates that, under our ex- perimental conditions, the IKCCaJ AlNSW A2+ IOOmM TEA IOpM 5-HT IOpM S-HT SOOpA 40sec +50mM TEA ------------------- - ------es -- 5OOpM FMRFo NSW 63 ---------m-e. 50OpM FMRFo 0.1 pM 5-HT 500pM FMRFo 5OOpM FMRFo O.lpM 5-HT FIG. 2. Effect of TEA on responses to 5-HT and FMRFamide and their interaction. Macroscopic current recordings from 2 cells (4 and B, respectively ). Horizontal bars underlying records mark drug applications. A I and .4,: inward current in the two analogues. Selected examples of this interaction are shown in Fig. 3. Pressure applications of FMRFamide ( l- 500 PM, Fig. 3, A,, B,, and B3) produced an outward current accompanied by increased slope conductance, which reflects the increased opening of the S-K+ channels induced by the peptide. Bath application of either CAMP analogue (Fig. 3, AZ, B,, and B4) produced an inward current with decreased slope conductance, generated by the closure of the same channel that is opened by FMRFamide. If, in the presence of 1.4 PM 8cpt-CAMP, 1 PM FMRFa- mide is applied (AZ), an outward current equal to the sum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . .

5 . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . but, at 5 and . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8b _ 8b _ 1 10 100 1000 and 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . not the 1 10 1001000 and 6 . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i 1 10 . . broad range but not on a but not 1854 R. SHI AND F. BELARDETTI 25pM AA 500 pA 40 set J 25pM AA IpM 5-HT FIG. 8. Inhibitory actions of 5-HT on the response to a fixed concentration of arachidonic acid ( 25 PM). Same symbols and explanations as in Fig. 7. Five cells were used. For each cell, the responses were normalized to Buttner et al. 1989). Under opposite conditions, high FMRFamide doses can completely overcome the channel- closing action of CAMP-dependent phosphorylation, as shown in Figs. 3 4. This second action probably is mechanistically distinct because it requires dephosphoryla- tion of the channel (Ichinose et al. 1990; Sweatt et al. 1989). Arachidonic acid metabolism probably mediates both actions

6 ofFMRFamide 25pM AA 25bM AA 25pM AA
ofFMRFamide 25pM AA 25bM AA 25pM AA O.OlpM S-HT IOOpM AA 100~ M AA 0.01 p looy.M AA 500 pA 40 set I l / /n Cl 4 v 4 / -./ C2 pzz”. . ..o... . 0 FIG. 9. Dose-response relationship B, 100 PM) before (A, and B, ), during (A, and B2), and after ( A3 and B,) application of 0.01 PM 5-HT. Horizontal bars mark drug applications. Holding potential, -35 mV. Step volt- age commands to -40 ARACHIDONIC ACID (PM) FMRFAMIDE INHIBITION BY SEROTONIN 1855 (0.01 PM, Fig. 9). We reasoned that if 5-HT inhibits the release of arachidonic acid, then when arachidonic acid is exogenously applied, the site of the CAMP-independent in- hibition by 5-HT would be bypassed. In this case, high con- centrations of arachidonic acid, by stimulating the dephos- phorylation mechanism (Sweatt et al. 1989)) would fully DISCUSSION Antagonism of’the CAMP action by FMRFamide only preliminary such as such as P. E. FMRFAMIDE INHIBITION BY SEROTONIN ROSOLOWSKY, M. Products of heme-catalyzed transformation of the arachidonate derivative, 12-HPETE, open S-type K+ channels in Aply- siu. Neuron 3: 497-505, 1989. BELARDETTI, F., KANDEL, E.R., AND~IEGELBAUM, S.A.Neuronalinhibi- tion by the peptide FMRFamide involves opening of S-K+ channels. Nutrrre Lond. 325: 153- 156, 1987. BELARDETTI, F., SCHACHER,S.,ANDSIEGELBAUM, S. A.Actionpotentials, macroscopic and single channel currents recorded from growth cones of Apivsia neurones in culture. J. Physiol. 374: 289-3 1991. BELARDETTI, F. AND SIEGELBAUM, S. Up- and down-modulation of single K+

7 channel function by distinct second mes
channel function by distinct second messengers. Trends Neu- rosci. 1 1: 232-238, 1988. BLUMENFELD, H., SPIRA, M.E., KANDEL, E.R., ANDSIEGELBAUMJ. A. Facilitatory and inhibitory transmitters modulate calcium influx during action potentials in Aplysia sensory neurons. Neuron 5: 487-499, 1990. BOYLE, M. B., KLEIN, M., SMITH, S. J., AND KANDEL, E. R. Serotonin increases intracellular Ca2+ transients in voltage-clamped sensory neu- rons of Aplysin cal[/brnicu. Proc. Natl. Acud. Sci. USA 8 1: 7642-7646, 1984. BRAHA, O., DALE, N., HOCHNER, B., KLEIN, M., ABRAMS, T. W., AND KANDEL, E. R. Second messengers involved in the two processes of presynaptic facilitation that contribute to sensitization and dishabitua- tion in .4plysiu sensory neurons. Proc. Nutl. Acad. Sci. USA 87: 2040- BREZINA, V., ECKERT, R., AND ERXLEBEN, C. Modulation ofpotassium conductances by an endogenous neuropeptide in neurones of Aplysia culjfiwnicu. J. Physiol. Lond. 382: 267-290, I987a. BREZINA, V., ECKERT, R., AND ERXLEBEN, C. Suppression of calcium current by an endogenous neuropeptide in neurones of Aplysia cal[jbr- nica. J. Physiol. Land. 388: 565-596, 1987b. BUTTNER, N.,SIEGELBAUM,S. A., AND VOLTERRA, A. Direct modulation of Aplysia S-K+ channels by a 12-lipoxygenase metabolite of arachi- donic acid. Nuturc Lond. 342: 553-555, 1989. COTTRELL,G. A., DAVIES,N. W., ANDGREEN, K.A.Multipleactionsofa molluscan cardioexcitatory neuropeptide and related peptides on identi- fied Ncb neurones. J. Physiol. Lond. 356: 3 15-333, 1984. EDMONDS, B. W., KLE

8 IN, M., DALE, N., AND KANDEL, E. R. Con
IN, M., DALE, N., AND KANDEL, E. R. Contribution of two types of calcium channels to synaptic transmission and plasticity. Scicncc Wush. DC 250: 1 142- 1 147, 1990. HARTZELL, H. C. AND FISCHMEISTER, R. Opposite effects of cyclic GMP and cyclic AMP 273-275, 1986. HERMANN, A. AND GORMAN, A. L. F. Effects of tetraethylammonium on potassium currents in a molluscan neuron. J. Gen. Physiol. 78: 87-l 10, 1981. ICHINOSE, M., ENDO, S., AND BYRNE, J. H. Role ofprotein phosphatases in FMRFamide-, serotonin-, and CAMP-dependent modulation of mem- brane currents in sensory neurons of Aplysia. Sot. Neurosci. Ahstr. 16: 187, 1990. KANDEL, E. R. AND SCHWARTZ, J. H. Molecular biology of learning: mod- ulation of transmitter release. Science Wash. DC 2 18: 433-443, 1982. KLEIN, M., CAMARDO, J., AND KANDEL, E. R. Serotonin modulates a specific potassium current in the sensory neurons that show presynaptic facilitation in ~4plysicr. Proc. Natl. ,Jcad. Sci. USA 79: 57 13-57 17, 1982. KRAMER, R. AND LEVITAN, I. B. Activity-dependent neuromodulation in /lplwia neuron R 15: intracellular calcium antagonizes neurotransmit- ter responses mediated by CAMP. J. Neurophys. 1075-1088, 1990. LEVITAN, I. B. Modulation of ion channels in neurons and other cells. Annu. Rw. Neurosci. 1 1: 1 19- 136, 1988. LOTSHAW, D. P. AND LEVITAN, I. B. Reciprocal modulation of calcium current by serotonin and dopamine in the identified Aplysia neuron R15. Brain Res. 439: 64-76, 1988. OCORR, K. AND BYRNE, J. H. Membrane responses and changes in CAMP

9 levels in Aplysia sensory neurons produ
levels in Aplysia sensory neurons produced by serotonin, trypta- mine, FMRFamide and small cardioactive peptide ( SCPs) . Neurosci. Lett. 55: 113-l 18, 1985. PIOMELLI, D. AND GREENGARD, P. Lipoxygenase metabolites of arachi- donic acid in neuronal transmembrane signalling. Trends Phurmucol. Sci. 11: 367-373, 1990. PIOMELLI, D., SHAPIRO, E., ZIPKIN, R., SCHWARTZ, J.H., ANDFEINMARK, S. J. Formation and action of 8-hydroxy- 11,12-epoxy-5,9,14-icosatri- enoic acid in Apl-ysia: a possible second messenger in neurons. Proc. Natl. Acad. Sci. USA 86: 172 I- 1725, PIOMELLI, D., VOLTERRA, A., DALE, A., SIEGELBAUM, sensory cells. Nature Lond. 328: 38-43, 1987. PRICE, D. A. AND GREENBERG, M. J. Structure of a molluscan cardioex- citatory neuropeptide. Science Wash. DC 197: 670-67 1, 1977. SACKTOR, T. C. AND SCHWARTZ, J. H. Sensitizing stimuli cause transloca- tion of protein kinase C in Aplysia sensory neurons. Proc. N&l. Acad. Sci. USA 87: 2036-2039, 1990. SCHAAD, N., SCHORDERET, M., AND MAGISTRETTI, P. J. Prostaglandins and the synergism between VIP and noradrenaline in the cerebral cor- tex. Nuture Lond. 328: 637-640, 1987. SCHACHER, S. AND PROSHANSKY, E. Neurite regeneration by Aplysia neu- rons in dissociated cell culture: modulation by Apfysia hemolymph and the presence of the initial axonal segment. J. Neurosci. 3: 2403-2413, 1983. SCHWEITZER, P., MADAMBA, S., AND SIGGINS, G. R. Arachidonic acid metabolites as 1990. SHI, R. AND BELARDETTI, F. Interplay of 5-HT and FMRFa cascades on the S-K+ current in Aplysiu. S

10 ot. Neurosci. Ahstr. 16: 794, 1990. SHUS
ot. Neurosci. Ahstr. 16: 794, 1990. SHUSTER, M.J., CAMARDO, J.S., SIEGELBAUM,~. A., ANDKANDEL, E.R. Cyclic AMP-dependent protein kinase closes the serotonin-sensitive K + channels of Aplwvsia sensory neurones in cell-free membrane patches. Nature Land. 3 13: 392-395, 1985. SHUSTER, M. AND SIEGELBAUM, S. Pharmacological characterization of the serotonin-sensitive potassium channel of Aplysia sensory neurons. J. Gcn. Physiol. 90: 587-608, 1987. SIEGELBAUM, S. A., CAMARDO, J. S., AND KANDEL, E. R. Serotonin and cyclic AMP close single K+ channels in Aplysiu sensory neurones. Nu- ture Land. 299: 4 13-4 17, 1982. STERNWEIS, P. C. AND PANG, I.-H. The G-protein channel connection. Trends Neurosci. 13: 122- 126, 1990. SWEATT, J.D., VOLTERRA, A., EDMONDS, B., KARL, K.A., SIEGELBAUM, S. A., AND 1989. VOLTERRA, A. AND SIEGELBAUM, S. Role of two different guanine nu- cleotide-binding proteins in the antagonistic modulation of the S-type K+ channel by CAMP and arachidonic acid metabolites in Aplysia sen- sory neurons. Proc. Nutl. Acad. Sci. USA 85: 78 10-7845, 1988. WALSH, J. P. AND BYRNE, J. H. Modulation of a steady-state Ca2+-acti- vated, K+ current in tail sensory neurons of Aplysia: role of serotonin and CAMP. J. Neurophysiol. 6 1: 32-44, 1989. WALTERS, E. T., BYRNE, J. H., CAREW, T. J., AND KANDEL, E. R. Mecha- noafferent neurons innervating tail of Aplysiu. I. Response properties and synaptic connections. J. Nwrophysiol. 50: 1522- 1542, YAU, K.-W. AND BAYLOR, D. A. Cyclic GMP-activated conductance

11 of retinal photoreceptor cells. nnnu.
of retinal photoreceptor cells. nnnu. Rev. Neurosci. 12: 289-327, 1989. Vol. 66. No. 6, Dcccmbcr I99 I. /‘YIIZ~CY/ Serotonin Inhibits the Peptide FMRF’amide Response Through a Cyclic AMP-Independent Pathway in AplySia RIYI SHT AND FRANCESCO BELARDETTI Department of Pharmacology, University of’ Texas Southwestern Medical School, Dallas, Texas 75235 SUMMARY AND CONCLUSIONS I. The S-K+ conductance was isolated by voltage-clamping near the resting potential pleural mechanosensory neurons of &l~.siti in culture. This background conductance is modulated in opposite directions by two distinct, transmitter-controlled second- messenger cascades: it is enhanced by the peptide FMRFamide through the 12-lipoxygenase pathway of arachidonic acid, and it is decreased by serotonin (5HT) through adenosine 3’,5’-cyclic monophosphate ( CAMP)-dependent phosphorylation. 2. The dose-dependent activating effect of FMRFamide (0.0 l- 500 ,uM) on the S-K conductance was measured in the presence and the absence either of 1- 100 PM 8-bromo-CAMP ( 8b-CAMP, a membrane-permeable and hydrolysis-resistant analogue of CAMP), or of 0.01-0.1 PM 5-HT. 3. When 8b-CAMP was applied, it produced a slow inward current INTRODUCTION Receptors modulate the activity of ion channels and thus neuronal excitability through a variety of biochemical cas- cades (for recent reviews, see Belardetti and Siegelbaum 1988; Levitan 1988; Piomelli and Greengard 1990; Stern- weis and Pang 002x077/9 1 so C opyright KJ 199 The American Physiological Soc