Pfliigers Arch (1992) 421:247-255

Journal of Phys,ology 9 Springer-Verlag1992

Protein kinase C regulation of cardiac calcium channels expressed in Xenopus oocytes E. Bourinet, F. Fournier, P. Lory, P. Charnet, and J. Nargeot Centre de Recherches de BiochimieMacromoleculaire, CNRS UPR 9008, INSERM U 249, Route de Mende, BP 5051, F-34033 Montpellier, Cedex, France Received October 7, 1991/Received after revision February 7, 1992/Accepted March 4, 1992

Abstract. L-Type cardiac Ca 2+ channels expressed in Xenopus oocyte were studied following rat heart ribonucleic acid, messenger (mRNA) injection. We demonstrate that exogenous Ca 2 + channels are sensitive to intracellular regulation by protein kinase C (PKC). This was performed by using two types of PKC activators [phorbol esters and a structural analogue of diacylglycerol (DAG)] and a specific peptidic inhibitor. Ca 2 + channel modulation resulted in an initial increase of the inward current, without any modification of the voltagedependent properties, and a second delayed phase, specifically observed with phorbol esters, characterized by a progressive decrease in current amplitude. Concomitantly, a reduction of membrane capacitance, reflecting a reduction of the total membrane surface area, was observed. We suggest that this phenomenon underlies the irreversible decrease of the expressed Ba 2+ current via sequestration of Ca 2 + channels and/or PKC. We also demonstrate that regulation of cardiac mRNA-directed Ca 2 + channels by PKC activators was strictly dependent on intracellular Ca 2+ concentration, and was partially additive with cyclic-adenosine-monophosphate-(cAMP) dependent regulation. Key words: Cardiac calcium channel - Xenopus oocyte - Protein kinase C - Phorbol esters - Diacylglycerol analog - Down-regulation - Intracellular Ca - cAMP

Introduction Phosphorylation is the main regulatory process involved in the modulation of cardiac voltage-dependent calcium channels (VDCCs; for review see [40]). Extracellular stimuli (neurotransmitters and hormones) are linked to channel phosphorylation by: (i) membrane receptor activation Offprint requests"to: J. Nargeot

and, (ii) production of second messengers such as cy'clic adenosine monophosphate (cAMP), diacylglycerol (DAG) and Ca 2 + which can, in turn, activate different protein kinases. These phosphorylations eventually lead to an increase in the opening probability of VDCCs, resulting in a potentiation of the inward Ca 2 + current (Ic,). The fi-adrenergic stimulation of Ic,, acting via cAMP, is undoubtedly the best established example of these types of regulation; but other membrane receptors acting through different pathways, also seem to play an important role in regulating Ic~. Recently, rat heart protein kinase C (PKC) has been purified and three distinct isoforms, a fi (I and II) and 7, have been characterized [35]. PKC activation was shown to mimic angiotensin II effects in modulating positively VDCCs in voltageclamped neonatal cardiomyocytes [10]. This potentiation, obtained with phorbol ester treatment, seemed to be correlated to an initial increase in channel opening detected during cell-attached patch-clamp recordings [22]. On the other hand, inhibitory effects on Ic, [24] and contraction [42] could be obtained using DAG analogs or phorbol esters. So, the molecular mechanism through which PKC modulates cardiac function is not clear. Ca 2 + channels are oligomeric membrane proteins consisting in a main pore-forming subunit (~1) associated with other accessory subunits ( e 2 - 6 , fl and 7, [4]). Cardiac el and fi subunits have been cloned from rabbit heart and consensus sequences for protein kinases A and C [18, 28] have been found both in al and fl subunits. It is not known, however, which sites are of physiological importance, and whether kinases A and C act on the same site(s) through different pathways. As previously reported, PKC-mediated potentiation of Ic~ occurs over a time course of several minutes and requires appropriate stable recording conditions, difficult to obtain on contractile cells such as cardiac myocytes. These conditions are obtained here using the oocyte expression system where it was shown that following heart ribonucleic acid, messenger (mRNA) injection, cardiac Ba 2 + currents (IBa,C) can be recorded for a long period ( > i h) without any marked run-down [26]. In addition,

248 this expression system presents other attractive features to investigate intracellular regulation since: (i) the oocyte is particularly amenable to modification o f the intracellular m e d i u m before and during the time course o f the experiment and, (ii) c o n t r a r y to p a t c h - c l a m p investigation on isolated myocytes, current recording on oocytes can be p e r f o r m e d w i t h o u t buffering intracellular Ca a § a procedure able to m a s k Ca 2 § regulations. In this report, two different kinds o f P K C activators: p h o r b o l esters [phorbol 12-myristate 13-acetate ( P M A ) and p h o r b o l 12,13-dibutyrate (PDBU)] and a structural analog o f D A G , [l-oleyl 2-acetyl-rac-glycerol (OAG)], and the specific peptidic P K C inhibitor (C-PKi) were used. We d e m o n s t r a t e that expressed cardiac Ca 2 § channels are positively regulated by P K C . Following an initial potentiation o f IB,,c, P K C activators induced a declining phase, which, in the case o f P M A , completely abolished IBa,C. Possible mechanisms accounting for this delayed phase are proposed. Moreover, lines o f evidence indicate that P K C - m e d i a t e d effects are strongly dependent on intracellular Ca 2 +. Finally, it is s h o w n that prior application o f P K C did not prevent, but reduced, an increase o f IB~,c by c A M P injection. These results suggest that the p h o s p h o r y l a t i o n sites o f P K C and P K A , which lead to a similar functional effect m a y not occur at the same site.

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Fig. 1 A - F . Properties of cardiac Ca 2+ channels expressed in Xenopus oocytes. A Typical trace showing a Ca2+-dependent CIMaterials and methods mRNA extraction and oocyte injection. Total RNA was extracted from hearts of 13- to 16-day-old-rat using a modified guanidium/ CsC1 method [7]. After two successive cycles of ethanol precipitation, RNA was subjected to an oligo-dT cellulose affinity chromatography (type III collaborative research) according to standard procedures. Purified Poly A + mRNA were dissolved in sterile water at final concentrations ranging t - 4 m g / m l and kept frozen at - 8 0 ~C. Oocytes were dissected away from tricaine methane sulphonate-anaesthetized female Xenopus Laevis (from Centre de Recherche en Biochimie Macromoleculaire CRBM, Montpellier, France) and prepared as reported elsewhere [26]. Stage V and VI oocytes were injected with 50 nl of polyA + mRNA using a micrometer-driven micropipette (Drummond Scientific, Broomall, Pa., USA). Uninjected oocytes served as control. After injection, oocytes were maintained for 2 - 6 days at 20~ in a medium containing (ND96, in raM): NaCI 96, KC1 2, MgC12 2, CaC12 1.8, 4-(2hydroxyethyl)-l-piperazineethanesulphonic acid (HEPES) 5, pH 7.4 with NaOH; and supplemented with 50 gg/ml gentamicin and 50 gg/ml soybean trypsin inhibitor. Incubation medium was renewed daily.

Electrophysiological measurements. Electrophysiological measurements were performed using the standard two microelectrode voltage-clamp technique with the TEV-200 Cornerstone amplifier (Dagan instrument, Minneapolis, Minn., USA). In most experiments the holding voltage was - 80 inV. Stimulation of the preparation, data acquisition and analysis were performed using the pCLAMP software (version 5.5, Axon instruments, Burlingame, Calif., USA). Oocytes were placed in a recording chamber (200 gl) and impaled with 3 M KCl-filled electrodes (0.1-0.5 Mr2). Drugs were applied externally by addition to the superfusate (gravitydriven superfusion). For intracellular injection, oocytes were impaled with a third additional micropipette (3-10 ~tm in tip diameter). The injection volume was 2--5% of the entire cell volume (1 gl) and all injected compounds were prepared in water. To record Ca 2 + channel activity, oocytes were routinely tested in the following medium (BAMS, in mM): BaOH 40, NaOH 50, KOH 2, HEPES 5,

current [Icl(ca)]in a rat heart polyA + RNA-injected oocyte bathed in normal physiological medium (ND96, see Methods). The holding potential was stepped from - 8 0 mV to + 20 mV for 5 s. B - F Main characteristics of expressed Ba 2+ currents (IBa,c) in rat heart polyA+RNA-injected occytes bathed in BaMS solution (see Methods). B IB,,c elicited by a 400-ms pulse to + 10 mV from a holding potential of - 80 mV. C Current/voltage relationship for the peak current. D Steady-state inactivation curve of IB,,C- IB,,C were evoked by test pulses to + 10 mV after 7.5-s pre-pulses to various holding potentials. The peak current recorded during the test pulse (I) is normalized against the peak current recorded from -100 mV of holding potential (Im~), and plotted as a function of the pre-pulse potential. E Current trace recorded before (filled circle) and after application (open circle) of 10 gM Bay-K 8644. F Current trace recorded before (filled circle) and after application (open circle) of 2 IxM nicardipine pH adjusted to 7.4 with methane sulphonic acid. Ca z +-activated C1currents [/el(ca)]were measured in a saline solution of the following composition (in mM): NaC196, KC12, CaC12 1.8, MgCI2 2, HEPES 5, pH adjusted to 7.4 with NaOH. Drugs were all purchased from Sigma (St Louis, Mis., USA) except the peptidic protein kinase inhibitors used. C-PKi and A-PKi, respectively 13 and 18 aminoacid peptides, were synthesized at the CRBM (CNRS, Montpellier, France).

Results W h e n an a p p r o p r i a t e depolarizing step c o m m a n d was applied to heart m R N A - i n j e c t e d oocytes in a n o r m a l physiological bathing m e d i u m (ND96), a transient outw a r d current reaching an amplitude o f 3 0 0 - 1 0 0 0 n A could be elicited (Fig. 1 A). This large current described in previous studies as a Ic~(c,), is mainly supported by e n d o g e n o u s C1- channels but p r e d o m i n a n t l y depends on Ca a + entry t h r o u g h newly expressed cardiac V D C C s [9].

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Fig. 2. Current/voltage relationship of IBa,C before (filled circles) and after (open circles) injection of cAMP (10 pmol). Membrane potential was - 80 mV, test pulses, 400 ms in duration and 5 mV in increment, were applied every 10 s. Inset traces correspond to the maximum current amplitude elicited with a + 5 mV test pulse before and after cAMP injection

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C In order to directly investigate Ca 2+ channel activity, C1- ions were suppressed from the external medium and Ba 2 + (40 mM) was used as the charge carrier. Under these conditions and with an appropriate voltage step, an inward monophasic Ba 2 + current, reflecting exogenous cardiac VDCCs activity and reaching an amplitude up to 200 hA, could usually be observed (Fig. I B), In our hands, only a slowly inactivating component of Ba 2 § current was detected and heart RNA-injected oocytes never distinctly displayed the additional transient component of expressed Ba 2+ current previously described in some reports [13, 27]. Sporadically, some batches of oocytes exhibited dihydropyridine insensitive endogenous Ca 2 § channels, but the amplitude of the corresponding Ba 2 + current never exceeded 10 nA. Typical current/voltage relationships of IBa,C are presented in Fig. 1 C. The voltage threshold for inward IB,,c activation occurred at - 2 5 mV and the maximal amplitude appeared at + 10 inV. Voltage-dependent inactivation of IB~,c was tested by using the classical double pulse protocol with a conditioning pulse of 7.5 s (Fig. 1 D). These data revealed a half-inactivation potential for IB,,c near - 3 0 mV. Finally, pharmacological investigations clearly indicate that IB,,c was sensitive to organic Ca a + channel modulators such as dihydropyridines since IB,,c was potentiated by BAY-K 8644 (final concentration: 10 ~tM, Fig. 1 E), and partially blocked by nicardipine (final concentration: 2 gM, Fig. 1 F). In light of these basic electrophysiological and pharmacological characteristics, it may be stated, in agreement with previous reports [26, 30] that exogenous VDCCs expressed in oocytes are similar to cardiac Ltype Ca 2 § channels. In native mammalian cardiac cells, L-type VDCCs are known to be positively modulated by the c A M P / P K A pathway. This regulation which occurs through phosphorylation of Ca 2 § channels and leads to a marked increase of the macroscopic/ca, has been extensively described in the case of the stimulation of/q adrenergic receptors in cardiomyocytes [40]. Superfusion of cardiac mRNA-injected oocytes with isoprenaline (final concentration: 10 gM) produced a potentiation of expressed cardiac IB,,c (data not illustrated). Moreover, as illus-

Fig. 3 A - C. Effect of three different types ofphorbol esters on IBa,c. A Filled circle, control current; open circle, current after perfusion of 300 nM phorbol 12-myristate 13-acetate (PMA). B Filled circle, control current; open circle, current after perfusion of 300 nM phorbol 12,13-dibutyrate (PDBU). C Filled circle, control current; open circle, current after a 30 nm perfusion of 4c~-phorbol 12,13-

didecanoate (4c~-PDD, 1 gM). Note the absence of potentiation. Current traces were elicited by a 400-ms pulse to § 10 mV from a holding potential of -80 mV every 10 s

trated in Fig. 2, IB,,c could be directly stimulated by intraoocyte injection of cAMP (final concentration: 10 gM, n = 10) without significant modification of the voltagedependent features of IB,,c. Therefore, the apparent preservation of the cAMP-directed regulation for IBa,Cfurther confirmed that after expression in X e n o p u s oocytes, Ltype Ca 2 + channels retained most of their native properties. This led us to undertake a characterization of the P K C modulation of cardiac Ca 2 § channels expressed in the oocyte. First approach utilized the potent P K C activators,/% phorbol esters. As depicted in Fig. 3,300 nM superfusion of either P M A or P D B U (Fig. 3A and B respectively) caused a significant increase in IB,,c amplitude (146 + 18% of control, n = 18, for P M A and 140 _+ 15% of control, n = 10, for PDBU). Inversely, 4~-phorbol 12,13didecanoate (4ctPDD) treatment (final concentration: 1 ~tM), a phorbol ester ineffective in stimulating P K C did not give rise to any effect on IBa,C (n = 5) indicating that the action of P M A or P D B U is specifically mediated through activation of P K C (Fig. 3 C). This potentiation by phorbol esters occurred within a few minutes and was generally followed by an irreversible and progressive depression of IB,,c. Similar to phorbol esters, structural analogs of the cellular PKC activator DAG, such as OAG, enhanced IB,,c. However, results illustrated in Fig. 4A indicate that addition of 2 gM OAG to the external solution produced a significant but stable increase

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Fig. 5. Effect of a peptidic protein kinase C (PKC) inhibitor on the potentiation induced by a phorbol ester. In control conditions, rat heart polyA + RNA-injected oocytes responded to PMA perfusion (1 gM) by an increase in I~,,c (open square). However, when such oocytes are preinjected with a peptidic PKC-inhibitor (C-PKi, 3 pmol, 5 rain before), PMA failed to increase IBa,C (open circle). Currents were evoked by a 400-ms test pulse from - 8 0 mV to 10 mV every 15 s

Membrane volfoge (mV) Fig. 4 A, B. Effect of an analog of diacylglycerol (DAG) on IBa,C. A Current traces recorded before (filled circle) and after (open circle) application of 2 gM 1-oleyl 12-acetyl-rac-glycerol (OAG). B Current/voltage relationship for IB~,C obtained before and after OAG perfusion. Currents were recorded by stepping the membrane potential from - 8 0 mV to various test potentials (using a 5-mV in' crement) for 400 ms, every 10 s, in BAMS solution. Currents were leak-subtracted on line using the P/5 procedure. The test potential used in A was + 5 mV

in IB,,C amplitude (139 ___19%, n = 10). Current/voltage relationships (Fig. 4 B) revealed that the voltage dependence of I~,,c remained unchanged in the presence of OAG. Data not illustrated in this report also indicated that voltage-dependent inactivation of IB,,c was not modified. With the aim of confirming the direct involvement of PKC in the previous experiments, a specific inhibitory peptide of PKC (C-PKi), corresponding to a "pscudosubstrate" for PKC [16], was employed. This peptide traps inactive PKC preventing its activation by phorbol esters. Preinjection of C-PKi (3 pmol, 5 rain before) prevented PMA-induced variations of IB,,c (Fig. 5, n = 4). In addition, the basal current itself remains unchanged by the C-PKi pretreatment. Other PKC inhibitors such as staurosporine (10 gM external concentration) and H7 (100 gM external concentration) antagonized PKC effects but with a lower potency than C-PKi (data not illustrated). To rule out potential parallel activation of PKC and cAMP/PKA, control experiments were conducted. As expected, potentiations induced by PKC activators (300 nM PMA or 2 gM OAG) were not affected when oocytes were preloaded with the specific peptidic inhibitor of PKA: A-PKi (1 gM final concentration, n = 2). These experiments clearly indicate that PKC was directly responsible for the modulation of the expressed VDCC activity.

PKC is known to be present in mammalian heart, but also in J(enopus oocyte, where several isoforms have been characterized [23]. The pseudosubstrate (C-PKi) used in this work corresponds to a conserve sequence of the amino terminal part of all the PKC isoforms cloned today. Considerable homology at the level of the pseudosubstrate sequence exists between the different isoforms [17], so the C-PKi is highly specific for PKC, but cannot be used to differentiate the different isoforms. It is noteworthy that the high concentration of PKC activators necessary to activate the protein kinase might be related to two phenomena: (i) the type of isoform activated since it is well known that the different isoforms display different sensitivity to phorbol esters and analogs of DAG and, (ii) the intracellular Ca 2+ concentration, since activation of all three cardiac isoforms, c~fl and 7, has been shown to be Ca 2§ dependent. It was then of interest to test the Ca 2+ dependence of the PKC effects on IB,,C. In this way, free cytosolic Ca 2+ was chelated by loading the oocyte with 1,2bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 100 pmol injected per oocyte, final concentration of 100 gM). It was assumed that the steady-state effect of BAPTA was fully achieved when Icl(c,), a natural index of cytosolic Ca 2+, had completely vanished (Fig. 6A1 inset). In these conditions, OAG (2 gM) did not induced any stimulation of IB,,c (Fig. 6A1). The OAG-induced increase of IB.,c falls from 37 _+ 17% to 3 + 5% in BAPTA-injected oocytes (Figs. 6A2, 7 oocytes from the same donor in each case). This latter observation strongly supports evidence for a Ca 2 § requirement for functional PKC effects, and thus the hypothesis of activation of a Ca 2 § isoform of PKC. It should be noted that our recording conditions used extracellular Ba 2 + instead of Ca 2§ as the main permeating ion, to prevent activation of the endogenous C1current. Thus the normal intracellular Ca a+ concen-

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Fig. 6 A, B. Ca 2 + dependence of PKC-induced potentiation of IB~,c. A 1 0 A G was without effect when rat heart polyA + RNA-injected oocytes are preloaded with BAPTA (100 pmol per oocyte). Filled cirele, control current before application of OAG; open circle, current trace 15 rain after perfusion of OAG (2 gM). Inset traces show the effect of BAPTA injection on Icl(c~) recorded in ND96 solution (see Methods). The arrow points toward the current trace recorded after the injection. Note the complete inhibition of Ic~(e,). A2 Effect of preinjection of BAPTA on OAG-(2 gM) induced potentiation of IB,,c. Results are presented as % of increase in control current before OAG perfusion. IB~,c and Ic~c,> were evoked by test pulses to + 10 or + 20 mV from a holding potential of - 80 mV. Test pulses have respective durations of 400 ms and 5 s. B Effects of PMA (10 nM) on IB,,c in N D 96 solution. I~,,c was first recorded in BAMS solution (filled circle). Then the oocyte was perfused with normal N D 96 solution, and PMA was applied. An increase of Ic~(c,) was recorded (inset) reflecting the variations of voltage-dependent Ca 2 + channels (VDCC) activity. Indeed when the perfusion was switched back to BAMS a marked increase in ln,,c amplitude was obtained (open

circle)

tration was probably lowered. To test whether these conditions could decrease the PKC-induced potentiation of /ca, we first recorded a control IB,,C in BAMS solution and then switched to a Ca2+-containing solution (ND 96), and followed the Ca 2 + channel potentiation (induced by PKC activators) by measuring the Ca 2 +-activated C1conductance (see Fig. 6 B, inset). At the steady-state effect we switched back to the BAMS solution and measured the increase in IB,,c (Fig, 6 B). Interestingly, for a concentration of PMA as low as 10 nM, ineffective in previous conditions, a significant potentiation of Ba 2+ current could be obtained, suggesting again that the PKC isoform(s) involved it, the Ca 2+ channel potentiation were Ca 2 + dependent. These isoforms have been found in Xenopus oocytes as well as in rat cardiocytes. However,

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Fig. 7A, B. Time-course of IBa,c potentiation induced by PMA, PDBU or OAG. A Upperpart, open square, effect of 300 nM PMA; open circle, effect of 300 nM PDBU; lowerpart, open triangle, effect of 1 ~tM OAG;filled triangle, evolution of the control current without PKC activator. Drug applications started at time "0 min" and currents were evoked by a 400-ms test pulse from - 8 0 mV to + i0 mV every 15 s. Results are presented as % of control current recorded at time zero before drug application. Note the different time-courses, and the low run-down of the control current. B Membrane capacitance is affected by PKC activators. Average variation (_+ SEM) of membrane capacitance induced by OAG (n = 2) and PMA (n = 3). Variation of the membrane capacitance in control conditions, without PKC activators, is also shown (n = 3). Results are presented as % of membrane capacitance at time zero. Capacitance is calculated as the time integral of capacitive current (similar to those shown in B) recorded by a 10-mV voltage step over the value of this voltage step (Cm = Q/U). Holding potential was - 8 0 mV

cardiac isoforms (type I, II, III, or 7, fl, e [32, 35]) while being Ca 2 + dependent, can still be stimulated (at least in vitro) in the absence of Ca 2 + [with added ethylenglycol bis-(fl-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA)], which suggests that either they are not transcribed in the oocytes or they possess a different Ca 2+ requirement after translation in the oocyte. The temporal evolution of the IBa,C amplitude with different PKC activators is depicted in Fig. 7. Application of P M A (300 nM) clearly induced a transient increase of IBa,Cfollowed by a progressive decline of the current back to the basal level within 15 - 20 rain after the start of the agonist superfusion (n = 10). With longer P M A exposure, IBa,C completely vanished (n = 10). On the contrary, the time course of the effect of PDBU (300 nM) on IBa,c appeared to be more sustained since return to the initial

252 value occurred only within 3 0 - 3 5 min (n = 5). However, this PDBU-evoked decline of IBa,C could be accelerated with an over-application of P M A without any transient increase (result not illustrated). The lower part of Fig, 7A indicates that the potentiation induced by OAG was not followed by a marked reduction of IBa,C as routinely observed with phorbol esters. In the presence of OAG (1 gM), only a slow decrease could be observed (n = 5). Moreover, this decline oflBa,C approximately followed the slight natural "run-down" exhibited by the unstimulated control IB,,c ( 5 - 1 0 % of decrease after 1 h, n = 5). Membrane protein turnover may be regulated by internalization mechanisms. In the oocyte, this phenomenon could be amplified by phorbol ester treatment. For example, N a / K adenosine triphosphatase (ATPase) probed with radioactive ouabain, was seen to progressively disappear from the plasma membrane in PMA-treated oocytes [41]. As shown in Fig. 7B, P M A (300 nM) induced a marked reduction of membrane capacitance whereas OAG (1 gM) was less effective (80 _+ 5% for PMA, 20 _+ 20% for OAG). We could not demonstrate any marked change in membrane capacitance on unstimulated oocytes for 1 h (less than 10% change from 0.21 to 0.19 btF, n = 3). The characteristics of the decrease in current amplitude and membrane capacitance, induced by the different PKC activators (PMA, OAG), suggest that the two phenomena could be related (see Discussion). cAMP-dependent regulation is an important pathway for cardiac Ca 2-- channel potentiation, and molecular cloning as well as biochemical techniques have identified distinct sites for PKA and PKC. These data suggest that both kinases act by phosphorylating the channel at different sites, while electrophysiological recording evidenced a similar effect: a potentiation in the current amplitude. However the additivity of the effects of the two kinases has never been tested. Shown in Fig. 8 is the combined effects ofintracellular injection of cAMP and superfusion of PMA. In Fig. 8 A, cAMP was first injected as described in Fig. 2. When the maximal effect was reached, P M A (300 nM) was applied and induced a further increase of IBa,c amplitude. It should be noted that while cAMP markedly slowed the inactivation time course, the addition of PMA produced very little change in the kinetics of [Ba.c. The reverse experiment is shown in Fig. 8 B, where cAMP clearly increased IB,,c after a previous potentiation by PMA. The declining phase observed with P M A was still present after cAMP potentiation and lead to an underestimation of cAMP effects. Similarly to the experiment shown in Fig. 7A, inactivation time course was more sensitive to the application of cAMP than PMA. In any case the combined application of cAMP and PMA always produced an increase smaller than the algebraic sum of the increases resulting from the application of the two agents alone. In both cases, however, /Ba,Ccould still be increased by application of Bay-K 8644 (10 ~M). Discussion

In agreement with previous reports [9, 26], basic electrophysiological and pharmacological features of

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Time (rain) Fig. 8A, B. Cummulative effects of PKA and PKC stimulation. A Left side, time course of the effect of an intraoocyte injection of cAMP (same conditions as in Fig. 2). At the steady-state effect, PMA (300 nM) was perfused and induced an additional increase in current amplitude. Note that Bay K 8644 (10 gM) was still able to elevate current amplitude. Right side, current traces corresponding to control, cAMP, PMA and Bay K 8644 conditions. Amplitude scale bar is identical to the y axis of the graph. B Same experiments as in A, but PMA was perfused before cAMP injection. Note the declining phase observed with PMA, before cAMP injection. IB,,C were recorded using a test pulse to + 10 mV (400 ms duration) from an holding potential of -80 mV

native cardiac Ca 2 + channels appeared to be preserved after reconstitution in the oocyte plasma membrane. This is consistent with the detection in oocytes of a 20-pS acquired unitary conductance [30] corresponding to the value generally described for L-type VDCC in mammalian cardiac cells. Furthermore, the expected cAMP/ PKA modulation pathway was also retrieved for the reconstituted Ca 2+ channels. These results validated the oocyte model and led us to use this system to study VDCC regulation mediated by PKC. Such investigations are more difficult to carry out on native cells since patchclamp experiments on contractile cells require intracellular Ca 2+ buffering. Our results provide evidences for L-type cardiac VDCCs modulation by PKC following expression in Xenopus oocytes. Although endogenous VDCCs seem to be also regulated by PKC [5], our results can be unambiguously ascribed to modulation of expressed cardiac VDCCs since: (i) all results presented here were recorded from batches of oocytes possessing very small endoge-

253 nous Ic, (less than 10 nA) and, (ii) the amplitude of the PKC-stimulated endogenous Ba 2 § current in oocytes expressing unusually high currents (not used in this study) never exceeded 10% of the averaged amplitude of the expressed cardiac current. An increase of I~,.c was routinely obtained with two different classes of PKC activators (phorbol esters and structural analogs of DAG). A lack of effect on IB,,c with the inactive phorbol ester 4uPDD indicates that PMAand PDBU-mediated effects are related to a specific activation of PKC. One major characteristic of the IB,,c modulation by PKC activation was a potentiation of the inward current without significant alteration of both inactivation decay and voltage dependency. Consistently, in rat neonatal cardiomyocytes, 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of/Ca does not affect the voltage dependence of either the transient or steady components of this current [10]. Furthermore, no modification of the cadmium-insensitive outward current was ever recorded in the presence of PKC activator in the external solution. This builds a strong argument for a direct effect of PKC on VDCCs, probably through phosphorylation processes. In order to further confirm the implication of PKC activation in the observed effects, a specific inhibitor (CPKi) was employed. This synthetic peptide derives from that initially described by House and Kemp [16] and corresponds to a sequence of the regulatory domain of PKC. On the basis of previous reports [17], it acts as a potent substrate antagonist and leads to a specific inhibition of this enzyme under in vitro assay conditions. Nevertheless, C-PKi has recently been shown to be effective in living cells such as fibroblasts, neurons and even isolated aorta [12, 15, 36]. In our study, micromolar concentrations of C-PKi completely prevented PKC activation as suggested by the lack of IBa,Cpotentiation even with a submaximal concentration of PMA (1 gM). In contrast, the specific PKA inhibitor failed to be effective on PMA- (300 nM) and OAG- (2 gM) induced responses. Thus, PKC-induced potentiation of IBa.c cannot be explained by a secondary stimulation of the cAMP/PKA pathway like the "cross-talk" between second messenger systems recently described in NCB20 and NIH 3T3 cells [141. PKC was originally characterized as a Ca2+/DAG/ phospholipid-dependent protein kinase. But recently, several new members of the PKC family whose activities are dependent on DAG/phospholipid but independent of Ca 2 +, have been identified [32]. We examined this Ca 2§ dependence by using intracellular BAPTA to reduce cytosolic free Ca 2§ Under these conditions IBa,c was founded to be insensitive to OAG, suggesting a strong Ca 2 § dependence of the PKC isotype(s) involved in cardiac Ca 2 § channel modulation. These results imply that, in oocytes bathed with BAMS solution, the basal cytosolic Ca 2 + concentration is sufficient to allow PKC activation. However, effective phorbol ester concentration (for PKC activation) were lowered by one order of magnitude when PKC activation was performed in ND96 (Ca 2 § solution) prior to IBa.Crecording in BAMS (Ca2§ solution). This could explain the

relatively large concentration of PKC activators required to obtain significant effects. This Ca 2§ dependence of PKC stimulation is physiologically relevant since activation of membrane receptors linked to phospholipase C (~1 adrenergic, muscarinic, angiotensin II and P2Y purinergic receptors) lead to a rise in concentration of both PKC-activating cofactors: Ca 2 § and DAG. Long-term effects of PKC on Ba 2 § currents varied with the different types of activators used in this study. It could be stated that phorbol esters produced a transient increase of IBm.c, by contrast with OAG, turning to complete inhibition of the current, especially when PMA is employed. Various hypotheses may be raised to explain the declining phase. First, a down-regulation of the PKC generally obtained with phorbol esters in numerous cells [11]. The long-lasting PKC translocation induced by phorbol esters, but not by OAG, could create an irreversible depression of cellular PKC concentration [29] and account for the irreversible and marked depression of IBm,c, observed in our study with PMA. In addition, it was suggested that this down regulation was partially supported by calpain activity [1]. However, our attempts to demonstrate this hypothesis by inhibition of the calpain activity using calpastatine has been unsuccessful (not shown). Second, activation of phosphatases or proteases, directly acting on VDCC function, and associated with a decrease in channel availability [2, 3, 19, 37]. Third, activation of endocytosis by phorbol ester treatment in Xenopus oocyte which could trigger internalization of membrane proteins accompanied with dramatical disappearance of membrane microvilli as described for the Na/ K ATPase [41]. This proposition is more likely since our results pointed out a reduction of membrane capacitance which could be related to the reduction of cell surface area. This original phenomenon could promote VDCC internalization and could be responsible, at least in part, for the IBa,C collapsing phase observed with phorbol esters. Indeed, temporal similarities between membrane capacitance and Ba 2 + current decreasing phase reinforce this hypothesis. Occurrence of such an unspecific mechanism implies that other channels should undergo this type of down-regulation. In light of these results, one may ask if reduction of the capacitance is due to PKC activation or to a direct effect of/~-phorbol esters on plasma membrane. The fact that 4ePDD is unable to reduce membrane capacitance [41] strongly favours the first hypothesis. However an unspecific effect of pphorbol esters on plasma membrane cannot be completely ruled out with these experiments. It should be noted however that PKC induced inhibition of other channel activity (potassium and sodium channels; [8, 31]) may occur through mechanisms where oocyte capacitance is unaffected. In any case, the biphasic effect of phorbol esters observed on cardiac Ca 2 § channels is not specifically detected on oocytes. A similar regulation, including the declining phase, has been recorded on cardiac cells [22], although no precise mechanism has been proposed. Such effects of PKC have also been reported in the case of B cell activation [29]. Experiments performed on oocytes injected with rat cerebellum R N A show that neuronal

254 Ica also possess this delayed declining phase after P M A application, indicating that long-term effects of P K C are not specific to cardiac Ca 2 + channels ([20] and unpublished data). As previously shown, expressed Ca z + channels can be positively regulated by c A M P [25]. This potentiation is concomitant with a slowing of the inactivation time course and can be blocked by specific P K A inhibitors. Our results show that P M A can still enhance IBa,Cfollowing the application of a maximally effective dose o f cAMP. PMA, either before or after c A M P injection, increased IBa,C without modification of the inactivation time course. Such a result favours the hypothesis that the two phosphorylation pathways produced different modifications of the channel gating [22, 33]. However they both increase Ca 2 + channel amplitude through related mechanism leading to an increase in channel opening probability. Completely different mechanisms for potentiation, such as an increase in the number of active channels and an increase in the opening probability of active channels, should have resulted in a total additivity of the combined effects of P M A and cAMP. The partial additivity of these effects leads to a scheme where phosphorylations are effective on a limited pool of channels whose activity reach an asymptotic value. The causes of this asymptote might be due to: (i) a probability of opening reaching unity or, (ii) a complete occupation of the potential phosphate acceptors. The additional increase of IBa,C by Bay-K 8644 suggests that channel opening probability can be further enhanced after action of both kinases. The emerging functional picture of the cardiac V D C C would give a phosphorylation area with distinct - but spatially grouped - sites for P K A and PKC. Occupation of these sites could increase the opening probability via electrostatic interaction as originally proposed by Perozzo and Bezanilla [34] for the delayed K + channel and, for some of them, influence the inactivation process. P K A and P K C activities are controlled by different m e m b r a n e receptors activation. The impossibility for a unique pathway to maximally increase Ca z + channel activity prevent the cardiac myocyte of physiological blindness during overstimulation of one of its regulation mechanism. Moreover P K C pathway, through c~ adrenergic receptors, m a y play an important role in situations where the effectiveness of the fl adrenergic stimulation is reduced as in the failing heart [6]. These different effects might probably be explain by the location of the phosphate group on the channel. The molecular structure o f the Ca 2 § channel from skeletal muscle has been resolved, and consensus sequence for P K A and P K C phosphorylation sites have been found on the cd and • subunits [18, 38, 39]. U p to now, ~1 and /? are the only cardiac subunits cloned [23] and they possess similar phosphorylation sites. There are indications for the presence of at least two more cardiac isoforms of the fl subunit [18]. However the ultimate functional targets of the P K C , ~1 and/or t , are still unknown. It has recently been shown [21] that c A M P could potentiate Ca 2 + channel activity only when the fl subunit was coexpressed with el, but whether /? was phos-

phorylated or just necessary for the phosphorylation of the el subunit was not resolved. Our work provide the basis for future investigations directed at identifying the role of these various subunits in P K C regulation using either hybrid-arrest experiments or direct expression of the cloned - and mutated - subunits (when available) in oocytes.

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