Amiens Cedex t, France ..... Fig.2 A-C. Effects of A. aperta venom and BI-1 antisense RNA on cerebellar RNA-directed Ca 2+ channels. A Effect of increasing.
Pfltigers Arch (1993) 423:173-180
Journal of Physiology 9 Springer-Verlag 1993
Cyclic AMP-dependent regulation of P-type calcium channels expressed in Xenopus oocytes F. Fournier*, E. Bourinet, J. Nargeot, P. Charnet CRBM-C.N.R.S. UPR 9008, I.N.S.E.R.M. U249, 1919 Route de Mende, BP 5051, F-34033 Montpellier Cedex, France Received October 22, 1992/Received after revision December 8, 1992/Accepted December 10, 1992
Xenopus oocytes injected with rat cerebellum mRNA, express voltage-dependent calcium channels (VDCC). These were identified as P-type Ca 2+ channels by their insensitivity to dihydropyridines and co-conotoxin and by their blockade by Agelenopsis aperta venom (containing the funnel-web spider toxins: FTX and co-Aga-IV-A). Coinjection of cerebellar mRNA and antisense oligonucleotide complementary to the dihydropyridine-resistant brain Ca 2+ channel, named BI [Mori Y. et at. (1991) Nature 350:398-402] or rbA [Starr T. V. B. et at. (1991) Proc N a t Acad Sci USA 8 8 : 5 6 2 1 5625], strongly reduced the expressed Ba 2+ current suggesting that these clones encode a P-type VDCC. The macroscopic Ca 2+ channel activity was increased by direct intraoocyte injection of cAME This increase in current amplitude was concomitant with a slowing of current inactivation, and was attributed to activation of protein kinase A, since it could be antagonized by a peptidic inhibitor of this enzyme. Positive regulation of P-type VDCC could be of importance in Purkinje neurons and motor nerve terminals where this channel is predominant. Abstract.
Key words: Rat cerebellum - Phosphorylation - Hybrid arrest - Protein kinase A
Introduction
Calcium channels are a family of multisubunit proteins exhibiting marked homology in their structure and primary sequence, but great variety in their pharmacological profile and regulation. In neurons, four major classes of voltage-dependent calcium channels (VDCC), termed T, N, L and P types, have been described, each with * Present address: Laboratoire de Neurobiologie cellulaire, U.ER. des sciences exactes et fondarnentales, 33 rue Saint Leu, F-80039 Amiens Cedex t, France Correspondence to: E Charnet
unique electrophysiological properties, specific inhibitors, regulation by kinases and tissue distribution (for review, see [39, 46, 47]). T-, N- and L-type channels can be blocked by amiloride, dihydropyridines and coconotoxin respectively. The P-type VDCC, which was described recently [23], is insensitive to dihydropyridines and to co-conotoxin, but is blocked by low-molecular-mass fractions of Agelenopsis aperta spider venom (containing the funnel-web spider toxins: FTX and coAga-IV-A). Each neuron possesses a unique set of these channels [2, 9, 11], located at specific sites of physiological importance. It is thus important to understand the properties and regulation of each type of VDCC if one wishes to explain cell behaviour in response to applied stimuli. An important step in this direction has been the molecular cloning of the different VDCC. The pore-forming subunit of the L-type VDCC (al) has been cloned from mammalian skeletal muscle, heart and brain, and several auxiliary subunits (a2-6, t, and 7) have also been obtained. Furthelxnore, several groups have cloned the al subunits of N- and P-type VDCC [8, 30, 31, 41, 43, 52]. Individual channels can now be expressed and studied in Xenopus oocytes or in mammalian cell lines by coinjection of the cRNA of their components or transfection of the corresponding cDNA [8, 30, 51, 52]. These experiments should provide important information about the way each channel works at the molecular level. However, although a tetrameric or trimeric structure seems to be an essential feature of the VDCC, the precise identity of the different subunits forming a complete and functional Ca > channel in a particular neuron is still unknown. Another approach is therefore to inject poly A + RNA from a particular tissue, and to challenge the role of the different subunits using antisense oligonucleotides that lead, via the degradation of a specific messenger, to the repression of a native subunit. Injection of whole-brain mRNA into Xenopus oocytes leads to the expression of a single type of VDCC similar to the P-type channels [21, 22], found at high density in mammalian cerebellar Purkinje cells, through-
174 out the central nervous system [35] and at the motor nerve terminal [48]. The p h a r m a c o l o g y and the electrophysiological properties o f the expressed channel suggest that it might be related to the recently cloned B I [30], or r b A [43], a l subunit. These clones show only 42 % o f amino acid sequence identity with the dihydropyridine receptor f r o m skeletal muscle. Their primary sequences have several putative sites for cAMP/protein kinase A (PKA) phosphorylation. H o w e v e r a c A M P - d e pendent regulation o f these channels has never been reported either in Purkinje cells or after expression in oocytes o f the BI channel. Here we demonstrate that Xenopus oocytes injected with cerebellum R N A express only P-type V D C C closely related to the B I or r b A Ca 2+ channel. We show for the first time that activation o f protein kinase A by cyclic A M P leads to positive regulation o f cerebellar Ptype Ca 2+ channels. Possible functional implications are discussed.
(200 gl) and impaled with electrodes filled with 3 M CsC1 (0.10.5 Ms~). Drugs were applied externally by addition to the superfusate. For intracellular injection, oocytes were impaled with a third or a fourth additional micropipette (3-10 gm tip diameter). The injection volume was 2 % - 5 % of the entire cell volume (assumed to be t gl for an oocyte) and all injected compounds were dissolved in water. To record Ca 2+ channel activity, mRNA-injected oocytes were bathed in the following medium (BAMS, in mM): Ba(OH)2 40, NaOH 50, CsOH 2, HEPES 5, pH adjusted to 7.4 with methanesulphonic acid. Current/voltage curves were obtained by stepping the voltage from - 6 0 mV to 50 mV (5-mV increments) from a holding potential of - 8 0 mV. Steady-state inactivation curves were obtained by holding the oocytes at various conditioning potentials for 2.5 s (from - 6 0 mV to 25 mV, 5-mV increments), and measuring the peak current at a constant test pulse at + 10 mV (400 ms duration). The peak current was then normalized (as percentage) relative to the current at a holding potential of - 8 0 mV and plotted against the value of the holding potential. Voltages for haft inactivation were obtained from the best fit using the equation:
Materials
where I~1(%) is the normalized current, V the conditioning potential, Vo5the potential for half-inactivation, k the slope factor and A represents the non-inactivating current. All results are expressed as the mean value obtained from n oocytes plus or minus the standard error of the mean. Inactivation time courses were fitted to a single exponential function using non-linear regression. All drugs used were from Sigma (Saint Louis, Mo.), except Bay-K8644 and PN200-110, which were kindly provided by Sandoz-France. Stock solutions (10 mM) were made up in ethanol. Funnel-web spider venom (from A. aperta) and co-conotoxin were from Latoxan (Rosans, France) and RBI (Natick, Mass.) respectively. The synthetic protein kinase A inhibitor (rabbit sequence) was from Sigma, and prepared as aliquots of 10 gl at 500 gM in HzO.
and methods
RNA preparation and oligonucleotides. Cerebellum was quickly dissected from 15- to 17-day-old Wistar rats and total RNA was extracted using a phenol/chloroform procedure. Poly A + RNA was then purified by oligo(dT)-cellulose chromatography (type III, Collaborative Research) according to a standard protocol [10, 25]. Finally, mRNA was dissolved in sterile water at a final concentration ranging from 2 ktg/~tl to 4 gg/gl and kept frozen at - 8 0 ~ C. An antisense oligonucleotide was chosen to hybridize with the cytoplasmic loop between repeats II and III of the BI-1 calcium channel [30]. This sequence (corresponding to base pairs 23132333 of the BI-1 sequence, synthesized by Eurogentec S. A., Seraing, Belgium) is not represented in the L-type al subunit from skeletal muscle, heart and brain [12, 30] and only poorly conserved in the al subunit of the N and E types (see Table 1). Stock solutions of oligonucleotide were made at 2 gg/gl and stored at - 2 0 ~ C. For hybrid-arrest experiments, cerehellar mRNA was mixed either with sterile water (for control expression) or with the oligonucleotide (at a ratio of 3/4 RNA and 1/4 water or oligonucleotide), giving a final oligonucleotide concentration of 0.5 ktg/gl.
Oocyte microinjection and maintenance. Adult female Xenopus Iaevis were from the CRBM (CNRS, Montpellier, France). Pieces of the ovary were surgically removed under general anaesthesia (tricaine-MS222, 0.2 %) and dissected in ND96 solution (raM): NaC1 96, KC1 2, MgCI2 2, CaC12 1.8, HEPES 5, pH 7.4 with NaOH. To discard surrounding follicular cells, oocytes were treated for 2 - 3 h with collagenase at 2 mg/ml (type IA, Sigma) in calcium-free medium. Individual fully grown oocytes at stages V and VI were chosen for the experiments. Samples containing 5 0 80 nl poly A + RNA were injected per oocyte using a pneumatic injector. Non-injected oocytes served as controls. Oocytes were kept for 2 - 6 days at 20 -+ 1~ C in ND96 medium supplemented with pyruvate (2.5 raM) and gentamicin (50 gg/ml). The incubation medium was renewed daily.
ElectrophysioIogical measurements. Electrophysiological measurements were performed using the standard two-microelectrode voltage-clamp technique with a TEV-200 Cornerstone amplifier (Dagan Instrument, Mineapolis, Minn.). The holding potential was - 8 0 mV. Stimulation of the preparation and data acquisition were performed using an AT computer interfaced to the amplifier with a Labmaster (Axon Instruments, Burlingame, Calif.). Off-line analysis was performed using the pCLAMP software (version 5.5, Axon Instruments). Oocytes were placed in a recording chamber
I.(%) -
1
+A
Results
M a c r o s c o p i c inward currents were recorded in voltageclamped Xenopus oocytes 3 - 7 days after injection with rat cerebellum m R N A . N e w l y expressed Ca 2+ channel activity was recorded by isolating the inward current using a standard high-Ba 2+ solution ( B A M S , see Materials and methods) where external C1- was replaced by methanesulphonate to suppress the CaZ+-activated C1- current. Outward K + currents were abolished by preincubating oocytes in a Cs+-containing m e d i u m and using CsCl-filled microelectrodes. In these conditions, the only inward current was carried by Ba 2+ ions m o v i n g through Ca 2+ channels expressed after cerebellum m R N A injection (Fig. 1, trace a). This current (IB,) reached an amplitude o f more than 600 n A with a m e a n value o f 123 _+ 36 n A (n = 126). W h e n the average oocyte capacitance was taken into account, the current density was about 0.45 g A / g E In m o s t batches o f oocytes tested, only a single slowly inactivating Ba 2+ current was detected. Sporadically, some batches o f oocytes exhibited dihydropyridine-insensitive endogenous Ca 2+ channels but the amplitude o f the corresponding Ba 2+ current never exceeded 1 0 - 1 5 n A and thus was negligible w h e n c o m p a r e d to the n e w l y expressed Ba 2+ component. The Ba 2+ current recorded after cer-
175
-20
channel blocker in neuroblastoma cells [44], did not affect IBa (n = 3, data not shown). However, a pronounced inhibition was obtained by perfusion of A. aperta venom (I/1000 dilution, see Fig. 1, trace d, n = 15), a mixture of toxins known to contain the Ptype-specific toxins FTX [23] and coAga-IV-A [28]. The remaining IB, after spider venom application could be totally blocked by inorganic CaZ+-channel blockers such as Cd 2+ (final concentration 100 t.tM, n = 27, Fig. 1). The inhibition induced by spider venom was dose-dependent but did not lead to a complete block of IBa, even at the highest concentration tested (63 _+ 8% with 1/1000 dilution, n = 15; see Fig. 2 A). FTX blockade was dependent upon extracellular divalent ion concentration, probably through screening of negative surface charges at or close to the venom-binding sites [22]. When 20 m M B a 2+ was used instead of 40 raM, the spider venom evoked a more potent blockade, and IBa almost completely vanished (81 _+ 8 % inhibition, n = 5, Fig. 2 B, compare with trace d in Fig. 1).
Recently, Moil et al. [30] cloned a Ca 2+ channel (named BI) that was insensitive to dihydropyridine and co-conotoxin, but blocked by spider venom, and highly expressed in Purkinje neurons. They suggested that P channels and BI channels were the same molecular entity [11, 30]. To test this hypothesis further, we performed hybrid-arrest experiments using an antisense oligonucleotide complementary to a specific portion of the cytoplasmic loop between repeats II and III of the al subunit of the BI Ca 2+ channel. As seen in Table 1, this oligonucleotide was complementary to messenger for the BI or rbA channel, but not the L-type or N-type channels. Coinjection of cerebellum mRNA together with this antisense oligonucleotide reduced the expression level of cerebellar calcium channels by 78 _+ 8 % (n = 10; Fig. 2 C, upper part). In the same experiment, the responses obtained by stimulation of glutamate receptors using kainate (1 gM) were not affected by the antisense oligonucleotide (not shown). Coinjection of this antisense oligonucleotide with rat heart mRNA did not affect either the level of expression (n = 4) or the pharmacology of expressed cardiac Ca 2+ channels (Fig. 2 C, lower part), which have been shown to be L-type VDCC [25, 26]. Therefore it is highly probable that cerebellar RNA injection resulted in expression of Ca 2+ channels of the P type, possessing an a l subunit homologous to the BI channel. When about 50 pmol cAMP was microinjected into oocytes, the inward Ba 2+ current, elicited at + 10 mV, slowly increased to reach a new steady level corresponding to 181 + 21% (n = 24, Fig. 3 A, B) of the control IBa amplitude (current before cAMP injection). The Ba 2+ current potentiation occurred immediately, after the injection of cAMP and the steady-state level was reached within 1 0 - 1 5 min (Fig. 3 A). The increase of the IBa peak amplitude developed concomitantly with a marked slowing of the inactivation time course (Fig. 3 B, compare traces a and b). When fitted with a single exponential, the inactivation time constant (at + 10 mV) increased from 193.8_+ 55.6ms (n = 83) to 530 _+ 195 ms (n = 20). The I/V relationships (Fig. 4 A) indicated that after cAMP-induced potentiation neither the threshold potential for activation nor the potential for maximum current was changed. Steady-state inactivation curves before and after cAMP potentiation are shown in Fig. 4 B. After cAMP potentiation, a clear reduction (42 + 3 %) of the "steady-state" inactivation was recorded for short conditioning pulses (2.5 s). The potential for half-inactivation was only slightly affected (from - 7 __ 1 mV to - 1 7 _+ 1 mV after cAMP). The cAMP-stimulated Ba a+ current retained the basic pharmacological properties of the non-stimulated control IBa, i. e. was sensitive only to the spider venom (n = 5, Fig. 3 B, trace c). The effects of the venom on the amplitude of the cAMP-potentiated and control currents were quite comparable (65 +_ 9%, n = 5, and 63 _ 8 %, n = 15 respectively). Bay-K8644 (10 gM) or co-conotoxin was without effect after cAMP action (94.2 _+ 2 %, n = 4, and 104 _+ 2 % of control current, n = 2 respectively). These results indicate that no additional membrane current was revealed following intra-
176
A
C r
RN~ + Antisense AS BI
RNA
60
O .ID ~ r-
Cerebellum
40 20 0
1/I000
1 / 1 0 0 0 0 1/100000
Heart
Ag.A.V. Dilution
B
[
Tt'eet~
g
Fig.2 A-C. Effects of A. aperta venom and BI-1 antisense RNA on cerebellar RNA-directed Ca2+ channels. A Effect of increasing dilution of crude venom on Ba2+ current peak amplitude. The Ba2+ current blockade is expressed as the percentage decrease of the control current before perfusion of the venom. The recording conditions are similar to those of 1. B Spider-venom-induced blockade was more pronounced when the Ba2+ current was recorded in 20 mM (instead of 40 raM) extracellular Ba2+. The other recording conditions are similar to those of Fig. 1. The mean block in 20 mM Ba2+ was 81 _+ 8% (n = 5) for 1/1000 dilution of venom. C Coin-
Ag.AVena-n BayK8644
jection of an oligonucleotide complementary to the BI-1 calcium channel strongly reduced voltage-dependent Ca2+ channels expressed after cerebellum mRNA injection (upper part). Compare traces obtained after coinjection of mRNA with water (left) or with oligonucleotide (right). In this latter case, the remaining current was not affected by spider venom (l/t000 dilution). The same oligonucleotide was without effect when coinjected with heart mRNA (lower part). In this case, currents obtained with or without oligonucleotide were sensitive to Bay-K8644 (10 gM)
Table 1. Comparison of the antisense hybridizing region in the cloned L- (no corresponding sequence), N- [8, 31, 52] and P-type [30,
43] voltage-dependent Ca2+ currents ~ L type N type P type
Rabbit heart Human brain (hbB) Rat brain (rbB) Rabbit brain (BI) Rat brain (rbA) Antisense
5'- . 5'-GCC 5'-GCC 5'-AAG 5'-AAG 3'-TCC
. )s AAG CCT CCT GGA
.
. GCG GCG GCC GCC CGG
. CGC CGC AAG AAG TTC
. ;F'CG TCA TCG TCG AGC
GTG GTA GTG GTG CAC
-3' TGG-3' TGG-3' TGG-3' TGG-3' ACC-5'
a Bold type represents the non-hybridizing bases. Note the selectivity for the P type (100% identity)
cellular cAMP elevation. However, as shown in Fig. 3 B (trace c), the v e n o m could not reverse the cAMP-induced slowing of the inactivation. Furthermore, intraoocyte cAMP injection was without effect on oocytes perfused with cadmium-containing solution (100 I~M, n = 5, data not shown). In the light of these results, we assumed that, in our recording conditions, P-type Ca 2+ channels were the only channels affected by c A M P injection. Additional experiments using compounds known to increase intracellular cAMP concentration indirectly were carried out. External application of 30 gM forskolin (an adenylate cyclase activator) or isobutylmethylxanthine (a non-specific phosphodiesterase inhibitor) resulted in a potentiation of IB,, although this was quantitatively less important than after c A M P injection (n = 4, not shown). The most probable molecular pathway that could account for the cAMP-induced regulation of the expressed Ca > channels is phosphorylation of the channels by protein kinase A. Intracellular application of 10 pmol A-PKi, a specific peptide inhibitor of protein kinase A, completely reversed the effects of cAMP on 1Ba within 5 - 1 0 rain (n = 5, see Fig. 5 A). In the pres-
ence of intracellular A-PKi, not only did the Ba 2+ peak current return to its initial value (before c A M P injection), but also the inactivation time course recovered a shape comparable to the control Ba 2+ current (see Fig. 5 A traces a, b, and c). Moreover, injection of APKi into oocytes before c A M P injection prevented the potentiation of IB, (n = 3, data not shown). These data demonstrate that effects of c A M P on IB~ (on peak amplitude and on the time course of inactivation) were mediated through activation of the protein kinase A by cAME Intracellular c A M P production occurs following stimulation of adenylate cyclase coupled to activation of membrane receptors such as the fl-adrenergic receptor. The fl-adrenergic agonist, isoprenaline, produced a sigrlificant potentiation of the exogenous IB,. The IB, potentiation reached a m a x i m u m for concentrations greater than 10 gM, but was smaller when compared to direct intraoocyte injection of c A M P (155 _+ 17 % of the control current at 10 ~tM, n = 8). The slowing of the inactivation time course was still present (not shown) and could be prevented by pretreatment with the non-selective fi antagonist propranolol (final concentration 10 gM,
177
A~
-100
A cAMP
~ '-100 "200I '~c 30
-200
i
-400 (.3
-300 -500 .6001
I
0
5
I
,
10
15
t
-400 -60
I
20
i
-40
.
-20
0
i
20
.
,
.
40
60
Time (mn)
B B
11
0,8 a
b
g
J 200 ms
Fig. 3 A, B. Cerebellum RNA-directed Ca2+ channels are regulated by cAMR A Intraoocyte injection of cAMP (50 mM final concentration) is indicated by an arrow. The current slowly increases to a new steady level in 10-15 min. The cAMP-stimulated current could still be blocked by spider venom, with a potency similar to the non-stimulated current, and completely blocked by Cd. B Original traces corresponding to the time course shown in A. a, Control trace before cAMP injection, b, Current during the steady-state effect of cAMR Note the slowing of the inactivation decay (see text for values), c, Block induced by spider venom (1/1000 dilution). Note that after inhibition by the venom, the current still displayed the slow inactivation induced by cAMR Current traces were subtracted from the trace obtained after Cd2+ block. All other recording conditions were similar to those of Fig. 1
n = 3, data not shown). The isoprenaline effect on IBa was reversed by intracellular injection of A-PKi (n = 3, see Fig. 5 B). As in the case of c A M E neither o0-conotoxin (10 gM), nor Bay-K8644 (10 gM) or PN200-110 (10 gM) was effective on IBa after the isoprenaline effect (not shown).
Discussion The two major conclusions of this work are that Ca 2+ channels expressed in oocytes after cerebellum m R N A injection are related to the recently described P-type VDCC [23], and are encoded by a messenger similar to BI or rbA VDCC [30, 43], and that these channels are regulated by phosphorylation via activation of the cAMP-dependent protein kinase.
Identification of VDCC expressed from cerebellar RNA FTX-sensitive Ca 2+ channels are predominantly expressed in Purkinje neurons where they constitute the
o~" 0.6 v E 0.4 (_) 0.2 0
t
-60
i
-40
i
-20
0
20
Voltage (mY)
Fig. 4 A, B. Electrophysiological properties of cAMP-stimulated current. A Current/voltage curves before ( I ) and after ([~) injection of cAME The holding potential was - 80 inV. Note that the threshold and the peak of the I/V curves are poorly affected (< 5 mV). B Steady-state inactivation curves before and after cAMP injection (n = 5). Current was recorded from a holding potential of -80 mV and conditioning pulses of variable amplitude were given for 2.5 s. The channel availability was tested at + 10 mV (maximum of the I/V curve). The current was normalized according to the current recorded using a conditioning pulse of - 8 0 mV and plotted against the conditioning voltage. I , Before cAMP injection; [~, during the steady-state effects of cAME The slowing of the inactivation decay resulted in a decrease of steady-state inactivation for relatively short conditioning pulses (2.5 s). The potential for half inactivation was poorly affected (-10 mV and -15 mV for control and cAMP respectively)
more abundant Ca 2+ channel type ( > 90% [11, 28]). However, the major cell type in the cerebellum is the granule cell. There is no d e a r electrophysiological evidence for P-type Ca > current in these cells, although a dihydropyridine-resistant channel has been described [29]. Northern blot hybridization, however, shows that BI m R N A is expressed in granule cells as well as in Purkinje neurons [30]. It was therefore expected that injection of cerebellar m R N A into oocytes would produce such P-type Ca 2+ channels. Other channel types (L and N), present in the cerebellum, should also have been expressed. This was not the case, and a single population of VDCC was expressed that had the pharmacology of the P-type Ca z+ channel [6, 22]. This selective expression could be due to the production of electrically silent L- and N-type Ca 2+ channels, to faulty post-translational modification or to the lack of translation of these VDCC in the oocyte system. Although further experiments are needed before we understand this phenom-
178
A 100 ms 0
ions. For example, the induction of the long-term depression of the synapse between parallel fibres and Purkinje neurons [1, 1 4 - 1 6 , 27] is the consequence of a rise in intracellular Ca 2+ into Purkinje cells. Potentiating the Ca > entry through P channels would therefore modulate this f o r m o f synaptic plasticity.
Acknowledgements. We are grateful to C. Henderson, S. Richard and L. Fagni for helpful comments, E A. Rassendren for cerebellar poly A + RNA and T. W. Song and T. E Snutch for comparison between antisense and Class E al subunit. This work was supported by grants from Association Franqaise contre les Myopathies, and by financial support to E E and E. B. from la Fondation pour
la Recherche M6dicale and le Minist~re de la Recherche et de la Technologie respectively.
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