Voltage-dependent modulation of calcium current by GTPγS and dopamine in cultured frog pituitary melanotrophs

Neuroscience Letters, 138 (1992) 216 220 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Voltage-dependent modulation of calcium current by GTPyS and dopamine in cultured frog pituitary melanotrophs Jack A. Valentijn, Estelle Louiset, H u b e r t Vaudry and Lionel Cazin European Institute for Peptide Research, Laboratory of Molecular Endocrinology, CNRS URA 650, UA INSERM, University o[ Rouen. Mont-Saint-Aignan (France) (Received 20 November 1991; Revised version received 28 January 1992; Accepted 28 January 1992)

Key words: Calcium current; Dopamine; GTP~,S; G protein; Voltage dependency; Pituitary melanotroph: Patch-clamp Dopamine (1 ,uM) reversibly scaled down barium current through high-voltage activated (HVA) calcium channels but had little effect on the time course of current activation in cultured frog melanotrophs. Intracellular perfusion with guanosine-5'-O-(3-thiotriphosphate) (GTPyS; 100 ,uM) sustained the effect of dopamine. Moreover, GTPyS drastically slowed down the current activation kinetics. The latter effect was in part reversed by dopamine. A conditioning prepulse to +70 mV facilitated the current in GTP?'S-dialyzed cells but not in cells exposed to dopamine. These results suggest the existence of a dual G protein-mediated mechanism for reducing HVA calcium current.

High- and low-voltage activated (HVA and LVA) calcium currents in vertebrate neurons and endocrine cells are negatively regulated by various neurotransmitters including dopamine [4, 10-12, 17, 20, 21, 23], noradrenaline [1, 7, 9, 12, 18], y-aminobutyric acid (GABA) [3-5], acetylcholine [3], as well as by neuropeptides such as somatostatin [1, 8, 18], luteinizing hormone-releasing hormone (LHRH) [2] and enkephalin [18]. The inhibitory action of these regulatory signals consists in a run-down of the amplitude and, in many cases, a slow-down of the activation time course of calcium currents [1, 2, 5, 12]. Similar effects can be induced by internal perfusion of cells with the non-hydrolyzable GTP-analogue guanosine-5'-O-(3-thiotriphosphate) (GTPyS)[2-5, 8, 10, 13, 15, 16, 21,23], which irreversibly activates G proteins. In some models, the receptor-operated as well as the GTP?'S-induced inhibition of HVA calcium current can be reversed by a brief but strong depolarization [2, 5, 16]. This interesting finding led to the suggestion that G proteins may directly interact with calcium channels in a voltage-dependent manner [19]. We have recently shown that dopamine inhibits HVA calcium currents in cultured frog melanotrophs [20]. The Correspondence: J.A. Valentijn, European Institute for Peptide Research, Laboratory of Molecular Endocrinology, CNRS URA 650, UA INSERM, University of Rouen, 76134 Mont-Saint-Aignan, France.

inhibition was sustained by internally perfused GTP),S [21]. In the present study, we compared the effects of dopamine on HVA calcium channels to those of GTPyS and investigated their voltage-dependence. Patch-clamp recordings were carried out at room temperature in the whole-cell configuration [6] on 3- to 12day-old cultured frog melanotrophs. Cell cultures were prepared from enzymatically dispersed frog neurointermediate lobes, as described in detail elsewhere [20]. Half an hour before electrophysiological recording, the culture medium was replaced with a bathing solution containing (in mM): NaC1 112, tetraethylammonium (TEA)CI 20, BaC12 10, HEPES 15, tetrodotoxin 0.001 (pH adjusted to 7.4 using TEA-OH). Soft glass patch electrodes of 2 4 M£2 resistance were made on a vertical pipette puller (David Kopf Instruments, USA) and heatpolished on a microforge. The electrodes were filled with an intracellular solution containing (in mM): CsCI 100, TEA-C1 20, CaCI2 1, MgC12 2, ethyleneglycoltetraacetic acid (EGTA) 10 and HEPES 10 (pH adjusted to 7.4 using TEA-OH). In several experiments, GTPyS-tetralithium salt (100/,tM) was added to the intracellular solution. Dopamine (1/IM) was applied by means of a glass pipette using a fast pressure-ejection system. In some cases, a drop of dopamine solution (1 mM) was administered from a large-tipped pipette to the bath, giving a final concentration of 1 /~M. Throughout all experiments, the maximum effect of dopamine was considered. After the establishment of the full dopamine effect, the

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Fig. 1. Dopamine (DA) inhibited HVA barium currents through calcium channels ( Vh -80 mV). Top, the superimposed traces of barium current at 0 mV were recorded before (control, C), during (DA) and after (recovery, R) exposure of the cell to dopamine (1/IM). The activation time course of each trace was fitted by the sum of two exponentials, yielding r~:0.8 ms, r2:3.0 ms, A=25%, B=75% for C, r~- 1.0 ms, r2=5.2 ms, A=61%, B=39% for DA and vt=0.9 ms, v2=4.1 ms, A=42%, B=58% for R. Bottom, the F V relationship of peak barium current was obtained from the same cell, in the absence (©) or in the presence (@) of 1 /~M dopamine. The inset shows the relative inhibition induced by dopamine as a function of the membrane potential. Note that stronger inhibition occurred at more depolarized potentials.

b a t h i n g s o l u t i o n was r e n e w e d using a peristaltic p u m p . A l l reagents were p u r c h a s e d f r o m S i g m a ( U S A ) . C u r r e n t signals were r e c o r d e d with an E P C - 7 p a t c h c l a m p amplifier (List Electronics, F R G ) a n d digitally s t o r e d on v i d e o t a p e at a s a m p l i n g rate o f 44 k H z using a pulse c o d e m o d u l a t o r (Sony, J a p a n ) . D e p o l a r i z i n g test pulses were a p p l i e d with a p r o g r a m m a b l e s t i m u l a t o r (Biologic, F r a n c e ) . T h e h o l d i n g p o t e n t i a l was - 8 0 mV. C a p a c i t i v e c u r r e n t s were c o r r e c t e d as m u c h as possible using the a n a l o g circuitry o n the amplifier. T h e residual c a p a c i t i v e transients (<0.1 ms) were b l a n k e d . Typical settings for the series resistance a n d c a p a c i t a n c e were 6 M~2 a n d 4 pF, respectively. Off-line, d a t a were digitized

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Fig. 2. In some cells, dopamine (1 ,uM) induced a slight slow-down of the activation time course of HVA barium current through calcium channels at 0 mV (Vh -80 mV). Recordings a (control) and b (dopamine) (top) correspond to the time points marked a and b in the graph (bottom). The curves fitted to the current traces gave r~=O,5ms, r~-2.8 ms, A=30%, B=70% for a and vt=3.2 ms, r2=22.0 ms, A=62%, B=38% for b.

at s a m p l i n g times o f 100/Is per p o i n t using a L a b m a s t e r TL-1 D M A m o t h e r b o a r d interfaced with an I B M c o m patible, 80286 m i c r o p r o c e s s o r - b a s e d c o m p u t e r (H210, Hermes). T h e d a t a were a n a l y z e d with the help o f p C l a m p 5.5.1 software ( A x o n I n s t r u m e n t s , U S A ) . L e a k a g e was d e t e r m i n e d f r o m c u r r e n t s e v o k e d by hyperp o l a r i z i n g pulses to - 1 6 0 inV. A passive O h m i c c o m p o n e n t , c o r r e s p o n d i n g to the m e a s u r e d l e a k a g e current, was s u b t r a c t e d f r o m the recordings. F o r the I/V relationship, the l e a k a g e currents were p r o p e r l y scaled. O u t w a r d c u r r e n t s e v o k e d b y d e p o l a r i z i n g prepulses were n o t l e a k - s u b t r a c t e d . Curve-fitting p r o c e d u r e s e m p l o y e d a m u l t i p l e least squares regression a l g o r i t h m . D a t a are p r e s e n t e d as the m e a n + S.D. T h e a c t i v a t i o n time course o f c o n t r o l b a r i u m currents, e v o k e d b y d e p o l a r i z i n g pulses t o O mV, was best described by the sum o f two e x p o n e n t i a l s with time constants v~=0.6+0.2 ms a n d r2=2.7+_0.4 ms (Figs. 1 a n d 2). T h e fast a n d slow c o m p o n e n t s were o b s e r v e d in all cells tested (n--16) a n d their relative a m p l i t u d e s A, B were 30% a n d 70%, respectively. T h e presence o f each corn-

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Fig. 3. Internai perfusion with GTPyS (100/~M) caused a slowing of the activation time course Of HVA barium current at 0 mV ( Vh --80 mV). This effect was partially reversed by dopamine (DA; 1 /~M). The recordings a, b and c (top) correspond to the time points marked a, b and c in the graph (bottom). During the first minute following the establishment of the whole-cell configuration, the activation time course of barium current was similar to that observed in control cells (trace a). Thereafter, a slowing down of the current activation due to the dialysis with GTPyS rapidly appeared (trace b). The slowing effect was accompanied by a decrease in the relative amplitude (B/(A+B)) of the slow activational component (~2). Brief exposure of the cell to dopamine caused a current depression and a speed up of r2 without changing its relative amplitude (trace c). The effect of dopamine was irreversible. The double exponential curves fitted to the current traces yielded rj=0.8 ms, r2=4.5 ms, A=28%, B=72% for a, r1=4.2 ms, r2=69.3 ms, A=64%, B=36% for b and r1=8.5 ms, r2=34.7 ms, A=62%, B=38% for c. Current amplitudes were measured at their peak and expressed as a ratio of the initial amplitude at 0 s (I0).

ponent was independent of the holding potential, which ranged between -90 and -40 mV (data not shown). Dopamine drastically reduced the amplitude of barium current through HVA calcium channels in all cells. The current depression was voltage-dependent, stronger inhibition occurring at more depolarized potentials (Fig. 1). Conversely, it has been shown in bullfrog neurons that the reduction of calcium current induced by noradrenaline was stronger at more hyperpolarized potentials [1]. As for the control currents, the time course of activation at 0 mV was approximated by a double exponential curve. A slowing down of the activation kinetics was observed in only a small number of cells (5 out of 16) (Fig. 2). For these cells, the curves fitted to the

currents yielded time constants with peak values r~=3.6_+0.6 ms and z'2=24.5_+2.0 ms, and the relative amplitude of the slow component decreased from 70% to 40%. As shown in Fig. 2, the slow-down of the activation time course was completely reversible. In the remaining cells, i.e. where dopamine did not slow down the activation kinetics, the time constants were similar to those of control currents (r~=0.9+0.4 ms and "/'2=3.4_+0.8 m s ) . However, as in the cells where barium current was slowed by dopamine, the relative amplitude of r2 also decreased from 70% tot 40%. Barium current at 0 mV, recorded with pipettes containing GTPyS (n= 14) displayed, during the first minute following the establishment of the whole-cell configuration, activation kinetics comparable to those of control cells, giving time constants ~h=0.8+0.3 ms and r2=3.9+1.3 ms, A=30% and B--70%. After this lag period, the duration of both time constants rapidly increased, with values as high as r~---4.4+0.5 ms and r2--64.2+6.9 ms, A=65% and B--35% (Fig. 3). Thus, the slowing down of the activation time course of barium current caused by GTPyS was much more pronounced than that induced by dopamine. With internal GTPyS, the current amplitude gradually declined, and a complete depression appeared within 10 min of whole-cell recording. As long as there was a current flowing, the slowdown phenomenon could be observed. Interestingly, dopamine-exposure to GTPyS-dialyzed cells resulted in a speed-up of the slow activational component whereas the fast component was further slowed down (Fig. 3). The exponential curves fitted to the current traces, recorded with intracellular GTPyS following application of dopamine, yielded time constants q=8.4+0.9 ms and r2=34.9+3.8 ms, A=60% and B=40%. Dopamine caused an accelerated current depression as well (Fig. 3). The current slow-down due to internal GTPTS fully recovered when the test pulse was preceded by a 10 msprepulse to +70 mV. This recovery was also characterized by a facilitation, i.e. an increase in current amplitude (Fig. 4). In fact, a prepulse to +40 mV was already sufficient to reverse the GTPyS-effects. However, an identical prepulse to +70 mV could not counteract the inhibition induced by dopamine (Fig. 4), even when the prepulse duration was augmented to 40 ms or when the waiting time for the test pulse was reduced up to 0 ms (not shown). Similarly, a prepulse had no effect on unmodulated control barium current (Fig. 4). Several findings presented in this study indicate, that dopamine and GTPyS have a distinct action on HVA calcium channels in cultured frog melanotrophs. (i) Dopamine had little effect on the current kinetics, whereas GTPyS drastically slowed down the current activation. The lack of effect of dopamine on calcium current kine-

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tics has also been observed in adult rat sensory neurons [4], rat melanotrophs [17, 23] and lactotrophs [11]. (ii) Dopamine partially reversed the GTP~,S-mediated increase in the duration of the slow phase of current activation. This observation cannot be ascribed to preferential blocking of the slow component by dopamine, since its relative amplitude was not altered following drug-exposure. (iii) A conditioning prepulse to +70 mV faci-

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litated the barium current in GTPyS-dialyzed cells but not in cells under the influence of dopamine. Similar results have been obtained for GTPyS compared to dopamine, baclofen and acetylcholine in adult rat sensory neurons [3, 4]. Given the rapidity of occurrence of the facilitation phenomenon in GTPyS-perfused cells (40 ms following the conditioning prepulse), it seems unlikely that this effect could depend on an enzymatic process such as a phosphorylation/de-phosphorylation. These data rather support the existence of a voltage-dependent, allosteric interaction between G proteins and calcium channels, as suggested by various authors [2, 5, 9, 13, 15, 16, 19]. The lack of effect of a prepulse on unmodulated barium current excludes the existence of a tonic inhibition of calcium channels by G proteins under control conditions. These data are in agreement with our previous finding that the G proteins involved in the dopamine-induced inhibition of electrical activity possess a low basal activity [21]. Taken altogether, these results indicate that GTPyS unmasks a current component which is normally not seen. The inability of a depolarizing prepulse to reverse the inhibition induced by dopamine suggests that there may exist another G proteinmediated mechanism for the reduction of calcium currents in frog melanotrophs. This alternative pathway may involve a second messenger/protein kinase system. However, in other cell models it has been shown that a prepulse is capable of reversing the effect of neurotransmitters, thereby excluding the involvement of a second messenger [2, 5, 16]. We have recently demonstrated that alterations in cellular cAMP-levels do not modify the dopamine-induced run-down of HVA calcium current in frog melanotrophs [21]. Similar data have been described in rat lactotrophs [10] suggesting that the adenylate cyclase/protein kinase A system does not play a pivotal role in the inhibitory pathway. Possible candidates could be the phospholipase C- and phospholipase A2-1inked second messenger cascades, since both are known to mediate the action of dopamine on D2 receptors [14, 22].

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Fig. 4. A I0-ms conditioning prepulse (PP) to +70 mV facilitated HVA barium current at 0 mV in a cell dialyzed with GTP?'S (100 p M ) (middle), but was unable to facilitate a control current in a second cell (top) or to reverse a current depression induced by dopamine (DA; 1 /IM) in a third cell (bottom). Trace C refers to control and R to recovery throughout the recordings. Top, the curves fitted to the current traces gave r~=l.0 ms, z'2=4.8 ms, A=28%, B=72% for C and r~=l.l ms, ~2=3.5 ms, A-31%, B=69% for PP. Middle, curve fitting yielded r~=4.3 ms, r2-61.5 ms, A-67%, B=33% for C and rL-0.9 ms, r2=5.2 ms, A=32%, B - 6 8 % for PP. Bottom, r,=0.7 ms, r2=3.2 ms, A-24%, B=76% for C, r~=0.9 ms, r2~-2.9 ms, A--59%, B--41% for DA, r,=0.7 ms, r2-2.6 ms, A - 6 5 % , B=35% for D A + P P and r t - 0 . 9 ms, r2=4.1 ms, A--34%, B=66% for R.

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Complementary studies are in progress to further elucidate the mechanisms of action of dopamine on calcium current. In conclusion, our data provide evidence that dopamine and GTPyS differentially modulate HVA calcium channels in cultured frog melanotrophs. These differences may reflect distinct pathways by which calcium channels are regulated. This work was supported by grants from the Institut National de la Sant6 et de la Recherche M6dicale (894015), the European Economic Community (ST2J0468C), the Minist6re de la Recherche et de la Technologie (R6seau Europ6en de Laboratoires) and the Conseil R6gional de Haute-Normandie. The authors wish to thank Mrs. Catherine Buquet for excellent technical assistance. 1 Bean, B.P., Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence, Nature, 340 (1989) 153 156. 2 Elmslie, K.S., Zhou, W. and Jones, S.W., LHRH and GTP-~'-S modify calcium current activation in bullfrog sympathetic neurons, Neuron, 5 (1990) 75 80. 3 Formenti, A. and Sansone, V., Inhibitory action of acetylcholine, baclofen and GTP-y-S on calcium channels in adult rat sensory neurons, Neurosci. Lett., 131 (1991) 267 272. 4 Formenti, A., Sansone, V. and Mancia, M., Inhibitory action of baclofen, dopamine and GTP-analogues on calcium channels in adult rat sensory neurons, Eur. J. Neurosci., Suppl. 4 ( 1991 ) 3199A. 5 Grassi, F. and Lux, H.D., Voltage-dependent GABA-induced modulation of calcium currents in chick sensory neurons, Neurosci. Lett., 105 (1989) 113 119, 6 Hamill, O.E, Marty, A., Neher, E., Sakmann, B. and Sigworth, F.G., Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches, Pfl~igers Arch, Eur. J. Physiol., 39 (1981) 85-100, 7 Holz IV, G.G., Rane, S.G. and Dunlap, K., GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels, Nature, 319 (1986) 670-672. 8 Lewis, D.L., Weight, F.F. and Luini, A., A guanine nucleotidebinding protein mediates the inhibition of voltage-dependent calcium current by somatostatin in a pituitary cell line, Proc. Natl. Acad. Sci. USA, 83 (1986)9035 9039. 9 Lipscombe, D., Kongsamut, S. and Tsien, R.W., a-Adrenergic inhibition of sympathetic neurotransmitter release mediated by

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modulation of N-type calcium-channel gating, Nature, 340 (1989) 639-642. Lledo, P.M., Israel, J.M. and Vincent, J.D., A guanine nucleotidebinding protein mediates the inhibition of voltage-dependent calcium currents by dopamine in rat lactotrophs, Brain Res., 528 (1990) 143-147. Lledo, EM., Legendre, E, Zhang, J., Israel, J.M. and Vincent, J.D., Dopamine inhibits two characterized voltage-dependent calcium currents in identified rat lactotroph cells, Endocrinology, 127 (1990) 990-1001. Marchetti, C., Carbone, E. and Lux, H.D., Effects ofdopamine and noradrenaline on Ca channels of cultured sensory and sympathetic neurons of chick, Pfltigers Arch. Eur. J. Physiol., 406 (1986) 104111. Marchetti, C. and Robello, M., Guanosine-5'-O-(3-thiotriphosphate) modifies kinetics of voltage-dependent calcium current in chick sensory neurons, Biophys. J., 56 (1989) 1267-1272. Piomelli, D., Pilon, C., Giros, B., Sokoloff, P., Martres, M.-P. and Schwartz, J.-C., Dopamine activation of the arachidonic acid cascade as a basis for DjD2 receptor synergism, Nature, 353 (1991) 164-167. Scott, R.H. and Dolphin, A.C., Activation o f a G protein promotes agonist responses to calcium channel ligands, Nature, 330 (1987) 760-762. Scott, R.H. and Dolphin, A.C., Voltage-dependent modulation of rat sensory neurone calcium channel currents by G protein activation: effect of a dihydropyridine antagonist, Br. J. Pharmacol, 99 (1990) 629-630. Stack, J. and Surprenant, A., Dopamine actions on calcium currents, potassium currents and hormone release in rat melanotrophs, J. Physiol., 439 (1991) 37 58, Surprenant, A., Zhen, K.-Z., North, R.A. and Tatsumi H., Inhibition of calcium currents by noradrenaline, somatostatin and opioids in guinea-pig submucosal neurones, J. Physiol., 431 (1990) 585 608. Swandulla, D., Carbone, E. and Lux, H.D., Do calcium channel classifications account for neuronal calcium channel diversity?, Trends Neurosci., 14 (1991) 46-51. Valentijn, J.A., Louiset, E., Vaudry, H. and Cazin, L., Dopamineinduced inhibition of action potentials in pituitary melanotrophs is mediated through activation of potassium channels and inhibition of calcium and sodium channels, Neuroscience, 42 (1991) 29-39. Valentijn, J.A., Louiset, E., Vaudry, H. and Cazin, L., Dopamine regulates the electrical activity of frog melanotrophs through a G protein-mediated mechanism, Neuroscience, 44 ( 1991 ) 85-95. Vallar, L. and Meldolesi, J., Mechanisms of signal transduction at the dopamine D 2 receptor, Trends Pharmacol. Sci., 10 (1989) 74-77. Williams, P.J., MacVicar, B.A. and Pittman, Q.J., Synaptic modulation by dopamine of calcium currents in rat pars intermedia, J. Neurosci., 10 (1990) 757-763.