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Originally published In Press as doi:10.1074/jbc.M313284200 on January 13, 2004

J. Biol. Chem., Vol. 279, Issue 15, 14619-14630, April 9, 2004
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Ca2+ and Phosphatidylinositol 4,5-Bisphosphate Stabilize a G{beta}{gamma}-sensitive State of CaV2 Ca2+ Channels*

Matthieu Rousset{ddagger}§, Thierry Cens{ddagger}, Annie Gouin-Charnet¶, Frédérique Scamps||, and Pierre Charnet{ddagger}**

From the {ddagger}Centre de Recherche de Biochimie Macromoléculaire, CNRS-FRE 2593, 1919 Route de Mende, 34293 Montpellier, Centre CNRS-INSERM de Pharmacologie-Endocrinologie U469, rue de la Cardonille, 34094 Montpellier, and ||INSERM U583, Place Eugène Bataillon, 34095 Montpellier, France

Received for publication, December 5, 2003 , and in revised form, January 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Direct interactions between G-protein {beta}{gamma} subunits and N- or P/Q-type Ca2+ channels mediate the inhibitory action of several neurotransmitters in the brain. Membrane potential, channel phosphorylation, or auxiliary subunit association tightly regulate these interactions and the consequent inhibition of Ca2+ current. We now provide evidence that intracellular Ca2+ concentration and phosphoinositides play a stabilizing role in this direct voltage-dependent inhibition. Lowering resting cytosolic Ca2+ concentration in Xenopus oocytes expressing CaV2Ca2+ channels strongly decreased basal as well as phasic, agonist-dependent inhibition of Ca2+ channels by G-proteins. Decreasing phosphoinositide levels also suppressed G-protein inhibition and completely occluded the effects of a subsequent injection of Ca2+ chelator. Similar regulations are observed in mouse dorsal root ganglia neurons. Alteration of G-protein block by these agents is independent of protein phosphorylation, cytoskeleton dynamics, and GTPase or GDP/GTP exchange activity, suggesting a direct action at the level of the Ca2+ channel/G{beta}{gamma}-protein interaction. Moreover, affinity binding experiments of intracellular loops of the CaV2.1 Ca2+ channels to different phospholipids revealed specific interactions between the C-terminal tail of the channel and phosphoinositides. Taken together these data indicate that a Ca2+-sensitive interaction of the C-terminal tail of P/Q channels with the plasma membrane is important for G-protein regulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Voltage-gated Ca2+ channels are the key transducers that couple cellular excitability to several processes including contraction, secretion, and gene regulation. As such, they are tightly regulated by several direct mechanisms that finely tune, either positively or negatively, the spatiotemporal characteristics of the Ca2+ signal to adapt it to the cellular demand and to prevent, via elaborate safety feedback mechanisms, Ca2+ overload and cellular damage. The first of these mechanisms is triggered by Ca2+ ions themselves, which through binding to channel-associated proteins (1–6) or to the Ca2+ channel itself (7) negatively or positively couple Ca2+ entry to channel activity. Direct binding of Ca2+ ions to CaV1.2 or CaV2.1 {alpha}1Ca2+ channel-tethered calmodulin (1, 2, 5, 8–10) has recently been shown to lead to Ca2+-dependent channel inactivation and facilitation.

Intracellular signaling pathways, activated after G-protein-coupled receptor stimulation, are also important dual regulators of Ca2+ influx, either directly (via the G{beta}{gamma} dimer) or indirectly via phosphorylation of the channel {alpha}1 and/or {beta} subunits (11, 12). G{beta}{gamma} dimers are able to directly interact with several domains of the {alpha}1 CaV2.1, CaV2.2 or CaV2.3 subunits, including the loop connecting domains I to II and the N- and C-terminal tails of the channel, thus forcing the channel into a reluctant state of low open probability (13–16). Protein kinase C-dependent phosphorylation of the channel, however, is known to facilitate channel activity partly by removing G{beta}{gamma} inhibition (15).

Recently, a third type of dual regulation has been characterized on CaV2.1 voltage-gated Ca2+ channels. In this latter case, phosphatidylinositol 4,5-bisphosphate (PIP2),1 a well known regulatory phospholipid for potassium channels, has been suggested to bind directly to two sites of the CaV2.1 channel, preventing Ca2+ current rundown but also promoting voltage-dependent inhibition of channel activity (17). Because G{beta}{gamma}-dependent regulation of many channels and enzymes has been shown to be under the control of both Ca2+ ions and PIP2 directly, through specific binding of PIP2 on the protein or indirectly via activation of enzymatic cascades, we asked whether similar cross-regulations may exist in the case of the G{beta}{gamma}-induced inhibition of CaV2 Ca2+.

We describe here a new feedback mechanism by which a decrease in the intracellular Ca2+ and/or PIP2 concentrations may alter the direct inhibition of the CaV2 Ca2+ channel subfamily by G-proteins. When the Ca2+ chelating agent BAPTA was injected into Xenopus laevis oocytes expressing CaV2.1 or CaV2.2 Ca2+ channels, a marked increase in current amplitude developed consecutive to the removal of the tonic inhibition of the channel imposed by free G-protein {beta}{gamma} dimers. This increase was independent of channel phosphorylation or GTPase or GDP/GTP exchange activities but was occluded by injection of anti-PIP2 antibody or by treatment with phosphoinositide-kinase inhibitor. Injection of anti-PIP2 by itself produced an increase in current amplitude similar to BAPTA, suggesting that both Ca2+ and PIP2 are necessary for G-protein regulation. BAPTA and anti-PIP2 strongly reduced the GTP{gamma}S-induced Ca2+ current facilitation in mouse DRG neurons. Binding assays revealed a direct interaction between the C-terminal tail of the channel and the phosphoinositides. We thus propose that stabilization of a G{beta}{gamma}-sensitive state of Ca2+ channel may require direct interaction with membrane phosphoinositides and may represent one of the mechanisms by which PIP2 down-regulates Ca2+ channel activity. Cytosolic Ca2+, acting at the PIP2 and/or the channel level, is an important cofactor.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Oocyte Preparation—BAPTA (Sigma), nitro-BAPTA, di-Br-BAPTA, di-methyl BAPTA, and DM-nitrophen (Molecular Probes Inc., Eugene, OR) were prepared at 100 mM in HEPES 10 mM (pH 7.2 with CsOH) for on-line injection into oocytes. In Fig. 1, BAPTA was loaded with 20% Ca2+. DAMGO (Sigma) was prepared daily at 10 µM from a frozen stock solution at 10 mM. H9 (1 mM), H89 (100 µM), staurosporine (3 µM), and cytochalasin B (20 µg/ml) were prepared at the mentioned concentration and added to the incubation medium overnight. Genistein (50 µM), paclitaxel (30 µM), colchicine (20 µM), and wortmannin (10 µM) were added to the incubation medium 1–3 h before recordings were made. All of these drugs were also included in the recording Ba2+-containing solution at the mentioned concentration. Anti-PIP2 antibody (Echelon Biosciences Inc., Salt Lake city, UT) was injected into oocytes giving a final estimated dilution of 1:50. In the experiments describing the effects of GTP{gamma}S, the G{alpha}o G-protein subunit cDNA was coinjected with the Ca2+ channel subunit cDNA.



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  		FIG. 1.
BAPTA increases CaV2.1 Ca2+ currents. A, time course of the effects of BAPTA. Ba2+ currents were recorded in oocytes injected with the CaV2.1+{alpha}2-{delta} subunit cDNAs during 400-ms depolarizations to +10 mV from a holding potential of –80 mV. BAPTA (estimated final concentration 2–4 mM) was injected at the time marked by an arrow (n = 90). Typical current traces (labeled a, b, c) recorded at different times after injection of BAPTA are shown on the right. Note the marked increase of the Ca2+ current recorded after BAPTA injection. B, left, the averaged steady-state effect of an injection of BAPTA was strongly decreased when BAPTA was preloaded with Ca2+. Recordings were obtained on oocytes expressing the CaV2.1 and the {alpha}2-{delta} Ca2+ channel subunits. The effect of BAPTA was quantified by dividing the peak current measured at the steady state effect of BAPTA by the amplitude of the last current recorded before injection of BAPTA (I/Icontrol). Recording conditions were the same as described in A. n = 13 and 7 for BAPTA and BAPTA + Ca2+, respectively. *, the two means are statistically different (p < 0.05). Right, effect of injection of different calcium chelators with increasing affinity for Ca2+ (based on Kd dissociation constants from Molecular Probes Inc.). Each chelator was injected at a concentration of 100 nM in oocytes expressing CaV2.1 and the {alpha}2-{delta} Ca2+ channel subunits. The steady-state effect on Ba2+ current amplitude was measured as described in A. Half-maximum effect was obtained for chelators with a Kd for Ca2+ of around 0.5 µM, giving an estimated free Ca2+ concentration of 15–20 nM. C, effects of BAPTA on oocytes injected with different combinations of Ca2+ channel subunit cDNAs. Recording conditions and injection of BAPTA are the same as those shown in A. The amplitude of the increase induced by BAPTA (I/Icontrol) was measured at the steady state of the effect (at time = 7 min; shown in A). n = 90, 7, 8, and 9 for CaV2.1, CaV2.2, CaV1.2, and CaV2.3, all with {alpha}2-{delta}, and n = 5, 10, 8, 5, and 5 for CaV2.1 + {alpha}2-{delta}, CaV2.1, CaV2.1 + {alpha}2-{delta} + {beta}1, CaV2.1 + {alpha}2-{delta} + {beta}2, CaV2.1 + {alpha}2-{delta} + {beta}3, and CaV2.1 + {alpha}2-{delta} + {beta}4.

 
X. laevis oocyte preparation and injection were performed as described previously (18). Each oocyte was injected with 5–10 nl of a cDNA mixture containing the {alpha}1 + {alpha}2-{delta} + {beta} cDNAs at {approx}0.3 ng/nl with a ratio of 1:2:3. When one or more of these cDNAs were omitted, cDNA concentrations were kept constant by the addition of the appropriate volume of deionized water. Oocytes were kept for 2–4 days at 18 °C under gentle agitation before use.

Electrophysiology—Whole-cell Ba2+ currents were recorded under two electrode voltage clamp using the GeneClamp 500 amplifier (Axon Instruments, Union City, CA). Current and voltage electrodes (less than 1 megohm) were filled with 3 M KCl, pH 7.2, with KOH. Ba2+ current recordings were performed using the following external solution (in mM): BaOH 10, tetraethylammonium hydroxide 20, N-methyl-D-glucamine 50, CsOH 2, HEPES 10, pH 7.2, with methanesulfonic acid. Currents were filtered and digitized using a Digidata-1200 interface (Axon Instruments) and were subsequently stored on a Pentium-based personal computer using version 6.02 pClamp software (Axon Instruments). Around 10–30 nl of BAPTA buffer (in mM) (BAPTA-free acid 100, CsOH 10, HEPES 10, pH 7.2, CsOH) was injected into the oocytes (10 p.s.i., 150 ms) during the course of the experiment at the times indicated.

Ba2+ currents were recorded during a 400-ms test pulse from –80 mV to +10 mV. Current amplitudes were measured at the peak of the current. Comparisons of averaged amplitudes between batches were always performed on currents measured on the same number of days following injection. Comparisons between experiments were made by normalizing all average amplitudes with respect to the control current amplitude set at 100%. Isochronal steady-state inactivation curves (2.5 s of conditioning voltage followed by a 400-ms test pulse to +10 mV) were fitted using the following equation,

(Eq. 1)
where I is the current amplitude measured during the test pulse to +10 mV for conditioning voltage steps varying from –80 to +50 mV, Imax is the current amplitude measured during the test pulse for a conditioning step to –80 mV, Vin is the potential for half-inactivation, V is the conditioning voltage, kin is the slope factor, and Rin is the proportion of non-inactivating current. Current to voltage curves were fitted using the following equation,

(Eq. 2)
where I is the current amplitude measured during depolarizations varying from –80 to +50 mV, Imax is the peak current amplitude measured at the maximum of the current-voltage curve, G is the normalized macroscopic conductance, Erev is the apparent reversal potential, Vact is the potential for half-activation, V is the value of the depolarization, and k is a slope factor.

Inactivation kinetics were estimated by fitting Ba2+ current decay with two exponential components using the following equation,

(Eq. 3)
where I is the current amplitude, t is time, {tau}1, {tau}2, A1, and A2 represent the time constants and amplitudes of the two components, and C is a constant. The proportion of the slow time constant (%{tau}2) is the ratio A2/(A1 + A2).

Mouse DRG Neurons—Adult female Swiss mice (6 to 12 weeks old) were killed by CO2 inhalation followed by cervical dislocation, and neuron cultures were established from lumbar dorsal root ganglia as described previously (19). Dissociated neurons were plated on poly-D-L-ornithine (0.5 mg/ml)-laminin (5 µg/ml)-coated glass coverslips at a density of 2500 neurons/well and were incubated in an incubator with a humidified 95% air, 5% CO2 atmosphere. Experiments were performed on neurons of 30–40-µm diameter at room temperature (20–24 °C) after 12–24 h in culture. Ba2+ currents were recorded in the whole-cell mode using the Axopatch 200B amplifier and Clampex (version 8) software (Axon Instruments) with the following extracellular solution (in mM): BaCl2 5, TEA-Cl 140, HEPES 10, glucose 10, pH adjusted to 7.4, with Cs-OH. Pipettes (4–6 meghoms) were filled with CsCl 145, GTP{gamma}S 1, MgATP 2, HEPES 10, and either EGTA 1, or BAPTA 10; pH was adjusted to 7.3 with CsOH. In experiments with anti-PIP2, the antibody was diluted at 1:30 in the EGTA-containing pipette solution. Signals were filtered at 2 kHz and sampled at 5 kHz. The experimental paradigms were essentially the same as in oocytes (see Fig. 7). Usually, current recordings started 1–2 min after patch rupture. Currents were measured at their peak amplitudes.



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  		FIG. 7.
BAPTA and anti-PIP2 remove GTP{gamma}S-induced facilitation in DRG neurons. A, Ba2+ current facilitation recorded in whole-cell voltage-clamped mouse DRG neurons in primary culture. Currents were recorded during a 250-ms test depolarization to 0 mV (P1), and facilitation (P2) was evoked by the same depolarization preceded by a 50-ms pre-depolarization to +80 mV. Control, currents recorded under control conditions (intracellular medium containing 1 mM EGTA and 1 mM GTP{gamma}S); BAPTA, same intracellular medium, but EGTA was replaced with 10 mM BAPTA; anti-PIP2, control intracellular medium with anti-PIP2 antibody diluted at 1:30. After equilibration with the intracellular solution, facilitation was recorded only under control conditions. Calibration: 1, 1, and 0.5 nA and 100 ms for control, BAPTA, and anti-PIP2 traces, respectively, B, time course of GTP{gamma}S-induced facilitation. Three DRG neurons were dialyzed with the three different intracellular solutions (control, BAPTA, or anti-PIP2; see panel A), and facilitation was evaluated as the ratio of current amplitudes recorded during P2 and P1 (I2/I1). Time zero represents the first recording after going into the whole-cell mode (usually 1–2 min after patch rupture). I2/I1 > 1 denotes the existence of a current facilitation.

 
Baclofen-stimulated GTP Binding Assay—Rat brain membranes were prepared as described (20) and further diluted in the following solution (in mM unless specified otherwise): Tris/HCl 50, BAPTA 10, 1, 0.1, or 0.01, MgCl2 5, dithiothreitol 10, GDP 0.01, GTP{gamma}S35 0.5–1 nM, and leupeptin 0.01 mg/ml to reach a total volume of 100 µl at a protein concentration of 50 µg/ml. Baclofen (300 µM) was added for 1 h at 30 °C. Each sample was then filtered on a glass fiber C (Millipore) and submitted to scintillation counting. Values were corrected for basal GTP incorporation obtained under similar conditions without added baclofen. Experiments were performed in triplicate.

All values are presented as means ± S.E. of n experiments. Statistical significance was determined by analysis with the non-paired Student's t test (0.05 level).

Phospholipid Binding Assays—Phospholipids immobilized on nitrocellulose strip (PIP-strips P-6001, Echelon Biosciences Inc., Salt Lake City, UT) were first blocked for 90 min with bovine serum albumin dissolved at 3% in binding buffer according to the manufacturer's recommendations. Binding of CaV2.1 intracellular loops was performed overnight at room temperature using 50 µl of in vitro translated 35S-labeled intracellular loop (T7 TNT-coupled reticulocyte lysate, Promega) diluted in 1 ml of binding buffer. After three washes with bovine serum albumin, binding was visualized with a Thyphoon phosphorimaging device.

The following cDNA were used (GenBankTM accession numbers are given): CaV1.2 ({alpha}1C), M67515 [GenBank] ; CaV2.1 ({alpha}1A), M64373 [GenBank] ; CaV2.2 ({alpha}1B), D14157 [GenBank] ; CaV2.3 ({alpha}1E), L15453 [GenBank] ; {alpha}2-{delta}1, M86621 [GenBank] ; {beta}1b, X61394 [GenBank] ; {beta}2a, M80545 [GenBank] ; {beta}3, M88751 [GenBank] ; {beta}4, L02315 [GenBank] ; µ opioid receptor, NM013071.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Injection of the Ca2+ chelator BAPTA (100 mM in 10 mM HEPES, pH 7.2; estimated final concentration of BAPTA, 2–5 mM) into oocytes expressing the CaV2.1 {alpha}1A + {alpha}2-{delta} Ca2+ channel subunits induced a slow increase in Ba2+ current amplitude that reached a steady state in 5–6 min with a time constant of 1.3 ± 0.03 min (n = 90, Fig. 1A). On average, this increase was 2.8 ± 0.2-fold (n = 90) and appeared independently of the inhibition of the endogenous Ca2+-activated Cl– current, because it could be recorded from batches of oocytes in which the contamination by this current was almost negligible (see current traces in Fig. 1 for example). Concomitant with the increase in current amplitude, a significant acceleration of the inactivation kinetics was also recorded (see current traces in Fig. 1A) without marked modifications in either the activation or the inactivation parameters (see supplemental material). Loading BAPTA at 20% with Ca2+ (mixture of 100 mM BAPTA and 20 mM CaCl2) greatly reduced the capacity of BAPTA to increase CaV2.1 Ba2+ currents (Fig. 1B, left). Injection of a panel of different calcium chelators (nitro-BAPTA, dibromo-BAPTA, dimethyl-BAPTA or DM-nitrophen) with different dissociation constants for Ca2+ clearly produced an increase in current amplitude that was related to their affinity for Ca2+ (Fig. 1B, right), indicating that the action of BAPTA and other chelators was more likely mediated by their capacity to reduce resting cytosolic Ca2+ rather than by buffering the Ca2+ signal or by unspecific pharmacological effects.

We next analyzed the effects of the intracellular injection of BAPTA on oocytes expressing different types of voltage-gated Ca2+ channels and different combinations of auxiliary subunits (Fig. 1C). Although a similar increase (2.5–3-fold) was recorded when BAPTA was injected into oocytes expressing the CaV2.2 subunit, expression of CaV1.2 or CaV2.3 (brain isoform (21)) severely depressed the effects of BAPTA. Moreover, although removing the {alpha}2-{delta} subunit cDNA from the injection mix did not significantly modify the effect of BAPTA (Fig. 1C, bars labeled CaV2.1 and CaV2.1+{alpha}2-{delta}), co-expression of an auxiliary Ca2+ channel {beta} subunit ({beta}1, {beta}2, {beta}3, or {beta}4) markedly reduced the increase in current amplitude recorded following injection of BAPTA. Taken together, these results suggested that Ca2+ chelation was only effective on G-protein-sensitive (P/Q and N-type) Ca2+ channels expressed in a configuration that allowed a strong inhibition by G-proteins (i.e. without expression of a {beta} subunit).

In control conditions, Ba2+ currents recorded from oocytes expressing the CaV2.1 + {alpha}2-{delta} subunits have been shown to be under a tonic inhibition by endogenously active G-proteins (22, 23). We thus characterized the involvement of the G-proteins in the potentiation induced by BAPTA. GDP{beta}S (a GDP analog that blocks GDP/GTP exchange and thus favors the inactive trimeric state of the G-protein), when injected into oocytes expressing the CaV2.1 + {alpha}2-{delta} Ca2+ channel subunits, induced a >2.5-fold increase in Ba2+ current amplitude, as expected for the removal of the tonic inhibition (Fig. 2A and Ref. 22). Interestingly, this injection prevented any effect of a subsequent injection of BAPTA (Fig. 2A). Inversely, GDP{beta}S, when injected after BAPTA, was clearly less effective (Fig. 2A, right panel), thus suggesting that the two substances were acting on the same regulatory pathway. CaV2.1 + {alpha}2-{delta}-expressing oocytes incubated 3 h in the presence of pertussis toxin (PTX) (at 0.1 µg/µl) displayed Ba2+ current amplitudes that were 3–4-fold larger than those recorded under control conditions (Fig. 2B, left). The effects of an injection of BAPTA in these PTX-treated oocytes were reduced by ~70% (Fig. 2B, right), further demonstrating that a tonic G-protein-dependent regulation of the CaV2.1 Ca2+ channel was present under control conditions and was necessary to record the effects of BAPTA.



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  		FIG. 2.
BAPTA removes a G{beta}{gamma}- and voltage-dependent block of CaV2.1. A, left, time course of the effects on current amplitude (quantified as I/Icontrol; see Fig. 1) of successive injections of GDP{beta}S (2 nM/oocyte) and BAPTA into oocytes expressing the CaV2.1 {alpha}1 and {alpha}2-{delta} subunits. Right, quantification of the steady-state effects of consecutive injections of GDP{beta}S and BAPTA (n = 9) or BAPTA and GDP{beta}S (n = 9). Note the lack of additivity of the two effects. B, left, averaged peak current amplitudes recorded in control and PTX-treated oocytes (injected with ~4 ng of PTX, 3 h before the recordings). A >3-fold increase in current amplitude is recorded after PTX treatment. *, significantly different from control (p < 0.05, n = 5). Right, although PTX treatment or co-expression of G{alpha}i or G{alpha}o G-protein subunits significantly reduced the effects of an injection of BAPTA (n = 5, 5, and 6, respectively), co-expression of the G{beta}1{gamma}2 G-protein subunits had the opposite effect, suggesting a facilitating role of the active G{beta}1{gamma}2 dimer (n = 6). All recording conditions were the same as described in A. C, top traces, steady-state effects of an injection of BAPTA on the prepulse-dependent facilitation of Ba2+ currents recorded in oocytes expressing the CaV2.1 {alpha}1 and {alpha}2-{delta} subunits. Ba2+ currents recorded during a 200-ms depolarization to +10 mV were facilitated when preceded by a prepulse of 50 ms to +100 mV (compare trace without prepulse (P1) with trace after a prepulse (P2)). Injection of BAPTA (two left traces) completely suppressed this facilitation. Bottom traces, facilitation in control conditions after an injection of GDP{beta}S and after a subsequent injection of BAPTA. To clearly show the effects on the voltage-dependent facilitation independently of the increase induced by the injection of BAPTA or GDP{gamma}S, recordings have been normalized to display P1 currents of similar amplitude (dotted line).

 
Direct inhibition of the CaV2.1 Ca2+ channel upon activation of G-protein-coupled receptors occurs mainly through a voltage-dependent pathway via direct binding of the G{beta}{gamma} dimer onto the I–II loop, the N and C termini of the CaV2.1 Ca2+ channel subunit, and also through a less well characterized voltage-resistant (and G{alpha}i subunit-dependent) pathway (24–26). To identify the active arm of the G-protein involved in the effects of BAPTA, similar injections of chelator were performed on oocytes submitted to different levels of tonic G-protein inhibition, by co-injecting with CaV2.1 + {alpha}2-{delta} subunits the G{alpha}o, G{alpha}i, or G{beta}1{gamma}2 G-protein subunits. Over-expression of G{alpha}i or G{alpha}o greatly increased the Ba2+ current amplitude recorded before injection of BAPTA and strongly decreased the effect of a subsequent injection of BAPTA (Fig. 2B, right). Overexpression of G{beta}1{gamma}2 had the opposite effect, giving rise to smaller currents in control conditions but potentiating the effect of an injection of BAPTA by almost 40% when compared with similar injections performed without coexpression of the G{beta}1{gamma}2 subunit (Fig. 2B). These data therefore suggested that an excess of active G{beta}{gamma} subunits tonically inhibited CaV2.1 Ca2+ channels under control conditions. Over-expression of G{alpha}i or G{alpha}o, by quenching free G{beta}{gamma}, strongly decreased this tonic inhibition, whereas overexpression of G{beta}{gamma} had the opposite effect.

The tonic G{beta}{gamma} inhibition of the CaV2.1 channel (24) was voltage-dependent under control conditions, inducing a significant facilitation of the current. This facilitation is clearly seen when comparing current amplitudes recorded during a single depolarization that was preceded, or not, by a strong (50 ms, +100 mV) voltage step (Fig. 2C, compare traces P1 and P2). In BAPTA-injected oocytes, this voltage-dependent facilitation was completely removed, giving way to a small decrease due to channel inactivation (27) during the +100 mV depolarization (Fig. 2C, top traces labeled "after BAPTA"). The same effects were obtained by injection of GDP{beta}S, which, together with the increase in current amplitude, completely suppressed the voltage-dependent current facilitation (Fig. 2C, bottom traces labeled "after GDP{beta}S"). In these GDP{beta}S-injected oocytes, a further injection of BAPTA was without effect on current amplitude (Fig. 2A) and current facilitation (Fig. 2C). These data strongly imply that the chelation of cytosolic Ca2+ was able to remove the voltage-dependent block produced by tonically active G{beta}{gamma} subunits on CaV2.1 subunits.

To gain some insight into the mechanism by which Ca2+ chelation could inhibit G{beta}{gamma}-dependent regulation of the Ca2+ influx, we explored several regulatory pathways that might interfere with the tonic G-protein-dependent inhibition of the Ca2+ channel. Injection of GTP{gamma}S, a GTP analog resistant to hydrolysis, into voltage-clamped CaV2.1 + {alpha}2-{delta}-expressing oocytes produced a marked inhibition of the current amplitude attributed to persistent activation of G-proteins, as seen by the decreased in current amplitude and the augmentation of the prepulse facilitation (Fig. 3A). The subsequent injection of BAPTA completely reversed this inhibition and suppressed the facilitation (Fig. 3A, right), thus discarding any possible involvement of a direct (or indirect, via RGS (28, 29)) Ca2+-dependent activation of the GTPase activity of the G{alpha} subunit. The same argument can be used to exclude any down-regulation of a tonically active Go/Gq-coupled endogenous membrane receptor, suggesting that the action of BAPTA was located downstream of G-protein activation. The similarity between the effects of GDP{beta}S and BAPTA led us to think that the GDP/GTP exchange could be one of the targets by which BAPTA is able to reduce tonic G-protein inhibition. However, BAPTA, at concentrations between 10 µM and 10 mM, was unable to modify the incorporation of GTP{gamma}S into neuronal membrane preparations stimulated by the {gamma}-aminobutyric acid-B receptor agonist, baclofen (300 µM, Fig. 3B), suggesting that the GDP/GTP exchange activity was not Ca2+-dependent and thus restricting the effect of BAPTA to the level of the G{beta}{gamma}-Ca2+ channel {alpha}1 subunit interaction.



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  		FIG. 3.
Removal of the G{beta}{gamma} block by BAPTA is sensitive to the phosphoinositide kinase inhibitor wortmannin. A, time course of the effects on current amplitude of successive injections of GTP{gamma}S (0.4 nM/oocyte) and BAPTA into oocytes expressing the CaV2.1 {alpha}1 and {alpha}2-{delta} Ca2+ channel subunits. Injection of GTP{gamma}S induced a ~35% inhibition of the Ba2+ current that was fully reversed by a subsequent injection of BAPTA (n = 3). Right, facilitation in control conditions after injection of GTP{gamma}S and after a subsequent injection of BAPTA (recording conditions similar to those described for Fig. 2). B, BAPTA does not change GDP/GTP exchange in G{alpha} subunit. Agonist-induced GTP{gamma}S35-incorporation in G{alpha}o-containing rat brain membranes was measured in the presence of 300 µM {gamma}-aminobutyric acid-B receptor agonist, baclofen, and various concentrations of BAPTA (10 µM–10 mM). Specific binding of GTP{gamma}S35 was not modified by BAPTA (n = 3). C, oocytes expressing the CaV2.1 {alpha}1 and {alpha}2-{delta} subunits were incubated overnight with staurosporine (3 µM, n = 7), H9 (1 mM, n = 4); H89 (100 µM, n = 5), or cytochalasin B (20 µg/ml, n = 3) or for 1–3 h with genistein (50 µM, n = 8), paclitaxel (30 µM, n = 5), colchicine (20 µM, n = 5), or wortmannin (1–10 µM, n = 8). Peak Ba2+ currents were then recorded during 400-ms depolarizations to +10 mV from a holding potential of –80mV, and the effect of BAPTA was quantified as I/Icontrol (see Fig. 1B). All experiments were then normalized with respect to the effect of BAPTA on nontreated oocytes determined on the same days in the same batch of oocytes and taken as 100%. The effects of BAPTA were strongly depressed by wortmannin, marginally by paclitaxel (taxol, p < 0.05), and not at all by the other compounds.

 
We then tested several pharmacological tools known to interfere with key signaling proteins that could possibly play a role in these effects. Neither the general serine/threonine kinase inhibitors H9, H89, or staurosporine (respective concentrations 1 mM, 0.1 mM, and 3 µM, Fig. 3C) nor the tyrosine kinase inhibitor genistein (50 µM) had any effect on the potentiation induced by BAPTA, suggesting that none of these pathways was involved. Similarly, drugs that increase or decrease the polymerization state of microtubules (paclitaxel or colchicine at 30 and 20 µM, respectively) or actin filaments (cytochalasin B at 20 µg/ml) were also for the most part ineffective (Fig. 3C), with the exception of paclitaxel, which very slightly, but significantly, decreased the effects of BAPTA. Over-expression of a dead mutant of calmodulin, invalidated on the four EF-hands and unable to bind Ca2+, could not prevent the effect of BAPTA (not shown). Similar results were also found with neuronal Ca2+ sensor protein-1, another Ca2+-binding protein involved in Ca2+ channel regulation,2 thus refuting a participation of these two Ca2+-binding proteins in the sensibility to Ca2+ ions. However, preincubation of the oocytes in a solution containing the lipid kinase inhibitor wortmannin (at 1 to 10 µM) strongly inhibited the effects of BAPTA. At these doses wortmannin was able to block both the PI 3- and PI 4-kinases and was therefore expected to reduce the level of PIP2 in the plasma membrane.

The recent discovery that PIP2 phospholipids are important regulators of the CaV2.1 channel activity (17) and sensitize G-protein-activated K+ channels to G-proteins (30) led us to evaluate the role of PIP2 in the potentiation induced by BAPTA. Injection of an anti-phosphatidylinositol 4,5-bisphosphate antibody (Fig. 4A, anti-PIP2) into oocytes expressing the CaV2.1 and {alpha}2-{delta} subunits clearly and reproducibly increased the Ba2+ current amplitude (recorded at +10 mV) up to a new steady-state level in 4–5 min. Concomitantly, the current facilitation observed under control conditions after a +100 mV depolarization (Fig. 4A, inset, traces labeled "control") completely vanished after the injection (Fig. 4A, traces labeled "anti-PIP2"), effects reminiscent of those of BAPTA and suggesting that PIP2 was also acting on the tonic G{beta}{gamma}-dependent inhibition of the channel. As expected and already seen for BAPTA, pretreatment of the oocytes with PTX completely suppressed the effect of the antibody (Fig. 4, A and B). The effects of the antibody could also be prevented by a prior injection of BAPTA or by co-expression of the {beta}3 subunit (Fig. 4B, left). Conversely, the potentiation induced by injection of BAPTA was dramatically decreased when anti-PIP2 was first injected (Fig. 4B, right) or when PIP2 synthesis was inhibited by prior incubation of the oocytes with the lipid kinase inhibitor wortmannin (Fig. 3C). Altogether, these data strongly supported the hypothesis that intracellular Ca2+ and PIP2 were both necessary for the tonic G{beta}{gamma} subunit-dependent inhibition of the channel, suggesting that, as is the case for K+ channel, favorable membrane-channel interactions occurring via PIP2 and requiring cytosolic Ca2+ are necessary for the normal development of channel-G{beta}{gamma} interaction.



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  		FIG. 4.
Injection of anti-PIP2 antibody occludes the effects of BAPTA. A, left, time course of the effects of an injection of anti-PIP2 antibody on Ba2+ currents recorded in oocytes injected with the CaV2.1+{alpha}2-{delta} subunit cDNAs during 400-ms depolarizations to +10 mV from a holding potential of –80 mV (open squares). Anti-PIP2 (final dilution 1:50) was injected at the time marked by an arrow. Pretreatment with PTX (open circles; see legend for Fig. 3) completely suppressed the potentiation of the current recorded after BAPTA injection. Right, the prepulse current facilitation recorded in control condition ("control" trace) was completely suppressed after injection of anti-PIP2 ("anti-PIP2" trace). The recording conditions and paradigms were identical to those of Fig. 2B. B, effects of anti-PIP2 antibody. Ba2+ currents were recorded under standard conditions (see Fig. 1) on oocytes expressing the CaV2.1 {alpha}1 and {alpha}2-{delta} subunits. Left, injection of anti-PIP2 antibody under these conditions (final dilution 1:50) induced a large increase in current amplitude that was almost completely suppressed by prior injection of PTX or BAPTA or co-expression of the Ca2+ channel {beta}3 subunit. Right, conversely BAPTA was without effect when injected after anti-PIP2 antibody: –, effect of BAPTA in control conditions; +, effect of BAPTA after anti-PIP2 injection.

 
Are these two regulatory elements also necessary for phasic receptor-mediated G-protein regulation of the Ca2+ channel? To answer this question we co-expressed µ opioid receptors with the CaV2.1 and {alpha}2-{delta} subunits and analyzed the effect of BAPTA and anti-PIP2 under phasic activation of G-proteins elicited by the application of a µ opioid agonist. In such oocytes, extracellular application of the µ opioid agonist DAMGO clearly and reproducibly decreased the Ba2+ current amplitude by about 35% in less than a minute (Fig. 5A, and bar graph labeled "before BAPTA"). Injection of BAPTA between two successive applications of DAMGO not only produced the usual current potentiation but also significantly reduced the inhibition induced by a subsequent application of DAMGO (Fig. 5A, bar graph labeled "after BAPTA"). When this injection of BAPTA was performed during the application of DAMGO (Fig. 5B, +DAMGO), the increase in current amplitude provoked by the chelator was statistically larger than under control conditions, as expected for a higher degree of G-protein (G{beta}{gamma}) activation (tonic + phasic inhibition). The complete relief of the Ca2+ current block was confirmed by the lack of current recovery during the washout of DAMGO (Fig. 5B, +BAPTA). The same results were also found when a {beta} subunit was expressed (Fig. 5A, right bar graph), although in this case the inhibition induced by DAMGO was less, as expected when a {beta} subunit is expressed (31). Anti-PIP2 injected under the same conditions, during application of DAMGO, was also able to remove this phasic inhibition in addition to the removal of the tonic inhibition, leading to an over-recovery of the current amplitude (Fig. 5C, left). Indeed, after injection of the antibody, the potentiation of the current facilitation produced by perfusion of DAMGO (and the subsequent activation of the G{beta}{gamma}) was completely suppressed (Fig. 5C, inset, compare traces labeled 1 (before application of DAMGO), 2 (during application of DAMGO), and 3 (after injection of the antibody)), further demonstrating the removal of the voltage-dependent G-protein inhibition.



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  		FIG. 5.
BAPTA and anti-PIP2 remove a phasic G-protein inhibition. A, left, time course of peak Ba2+ current amplitude recorded during two successive applications of the µ opioid agonist DAMGO (10 µM) in oocytes expressing the CaV2.1 {alpha}1 and {alpha}2-{delta} Ca2+ channel subunits, the µ opioid receptor, and G{alpha}o G-protein subunit. Note that the inhibition induced by DAMGO was greatly reduced when BAPTA was injected between the two applications. Right, average inhibition produced by perfusion of DAMGO before (n = 10) or after (n = 10) injection of BAPTA. Injection of BAPTA produced a similar reduction in the DAMGO-induced inhibition when the {beta}4 subunit was co-expressed (histogram labeled "CaV2.1+{alpha}2- {delta}+{beta}4"). B, left, time course of I/Icontrol during perfusion of DAMGO and injection of BAPTA. This is the same experimental procedure as described in A, but BAPTA was injected during the inhibition induced by DAMGO. Perfusion of DAMGO significantly increased the potentiation of the current usually recorded after injection of BAPTA. After injection of BAPTA, the washout of DAMGO did not produce any change in current amplitude, whereas before injection of BAPTA an over-recovery of current amplitude was observed (see also Ref. 59). Right, bar graph showing the average increases in current amplitude induced by BAPTA before (–, n = 5) or during (+, n = 3) perfusion of DAMGO and the current recovery during DAMGO washout without (–, n = 6) or with (+, n = 8) injection of BAPTA. C, peak Ba2+ current amplitude recorded in oocytes expressing the CaV2.1 {alpha}1 and {alpha}2-{delta} Ca2+ channel subunits, the µ opioid receptor, and G{alpha}o G-protein subunit before and during application of the µ opioid agonist DAMGO (10 µM). Left, when the inhibition induced by DAMGO reached a steady state, the anti-PIP2 antibody was injected (arrow), producing an over-recovery of the current amplitude. Right, pre-pulse current facilitation recorded in control condition (1), during DAMGO application (2), and after anti-PIP2 injection (3). The recording conditions and paradigms were identical to those in Fig. 2C. Note that anti-PIP2 completely suppressed the current facilitation induced by perfusion of DAMGO.

 
Direct binding of channel intracellular loops to various membrane lipids was then tested using nitrocellulose-immobilized phospholipids (PIP-strips). The N- and C-terminal tails and intracellular loops 1–2, 2–3, and 3–4 of the CaV2.1 channel were first translated and labeled with [35S]methionine using a commercial kit (TNT system, see Fig. 6A). Binding to phospholipids was then assayed by lipid-protein overlay using the PIP-strip and the pleckstrin homology (PH) domain of phospholipase C{delta} as a positive control for PIP2 binding (32). The PH domain showed a mark specificity of binding for PI(4,5)P2 and PI(3,4,5)P3 (Fig. 6B) as described previously (32). Specific binding to PI, PIP, and PIP2 was only detected for the C terminus and to a lesser extent the N terminus and loop 1–2 of the channel; the 2–3 and 3–4 loops displayed no specific binding to any of the phospholipids spotted on the strip (see Fig. 6B). The lipid binding specificity of the C terminus was clearly different from that of the phospholipase C{delta}-PH domain, indicating that binding of PIP2 to the C terminus does not involve a classical pleckstrin homology domain. Consistently, no such domain was found in this region (not shown).



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  		FIG. 6.
The C terminus of Cav2.1 binds to phosphoinositides. A, SDS-polyacrylamide gel of in vitro translated N and C termini (N-ter, C-ter), intracellular loops 1–2, 2–3, and 3–4 (L1–2, L2–3, L3–4) of the CaV2.1 Ca2+ channels, and the PH domain of phospholipase C{delta} (PH (32)) labeled with [35S]methionine. In each case a clear band was seen at the expected molecular mass of 20, 10, 51.7, 15, 58, and 6.8 kDa for the PH domain, the N and C termini, and loops 1–2, 2–3, and 3–4, respectively. N.P., non-programmed TNT reticulocyte. B, lipid-protein overlays were made using PIP-strips and 50 µl of in vitro translated, 35S-labeled channel intracellular loops and the PH domain of phospholipase C{delta} as shown in A. Significant interactions with lipids (100 pmol/spot) were obtained only for the PH domain and the C terminus of CaV2.1. Note that different specific bindings to phosphoinositides were obtained for the PH domain and the C terminus. On the left is a schematic diagram of the different lipids spotted on the membrane.

 
We further investigated whether Ca2+and/or PIP2 regulated Ca2+ current facilitation in native neurons, where G-protein inhibition of {beta} subunits-containing Ca2+ channels only occurs after activation of G-protein. In mouse DRG neurons, the modulation of Ca2+ channels by endogenous G-proteins, resulting in a voltage-dependent current facilitation, can be elicited by intracellular perfusion of GTP{gamma}S via the patch pipette (see Fig. 7A). This type of facilitation developed slowly after patch rupture, reflecting the progressive voltage-dependent inhibition of the channel that follows activation of G-proteins by diffusion of GTP{gamma}S into the cell (Fig. 7B, see time course of Control). The addition of BAPTA (10 mM, Fig. 7) or anti-PIP2 (1:30 dilution, Fig. 7) in the patch pipette clearly suppressed the normal development of this facilitation as was also the case in Xenopus oocytes when the {beta}4 subunit was expressed (see Fig. 5A, right). Under these two conditions, a strong voltage step (+80 mV, 50 ms) preceding the test depolarization was unable to elicit any current facilitation, even at later times, after equilibration of the pipette solution into the cells, suggesting that BAPTA, just as PIP2, strongly reduced the phasic inhibition of {beta} subunit-containing Ca2+ channels that follows activation of G-proteins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cytosolic Ca2+, a Cofactor for G-protein Inhibition—In Xenopus oocytes, the tonic inhibition of expressed CaV2.1 channels by G-protein {beta}{gamma} dimers, first reported by Roche and Treistman (22) and later reproduced by many authors (27, 33, 34), was characterized by an increase in the basal current amplitude recorded after PTX treatment, injection of GDP{beta}S, or preconditioning depolarizations, which could be suppressed upon coexpression of the Ca2+ channel {beta} subunit (23, 31, 34). The potentiation of the current amplitude, recorded following injection of BAPTA, was resistant to a large spectrum of protein kinase inhibitors but was completely suppressed by PTX treatment prior injection of GDP{beta}S or co-expression of a Ca2+ channel {beta} subunit. Moreover, factors that promoted G-protein-dependent inhibition (GTP{gamma}S, co-expression of the G{beta}1{gamma}2 subunits) also increased the effects of an injection of BAPTA. We thus conclude that BAPTA was indeed able to suppress this tonic inhibition. Unchanged current-voltage and inactivation curves, increase in the inactivation kinetics, and suppression of the prepulse-sensitive current facilitation were also confirmatory of the removal of a tonic or phasic G-protein inhibition (23, 35), allowing us to discard nonspecific effects of Ca2+ chelation on membrane integrity. The absence of an effect of BAPTA when injected after GDP{beta}S suggested no additional role in these conditions. Moreover, a direct pharmacological action of BAPTA molecules on Ca2+ channels seems very unlikely because loading BAPTA with 20% Ca2+ almost completely prevented the effects of the injection (36). The estimated free Ca2+ concentrations reached under control conditions (50 nM) and after injection of BAPTA (<15 nM (37, 38)) placed the lower limit of the free Ca2+ concentration necessary for the inhibitory action of G{beta}{gamma} between these two values and suggested that resting Ca2+ concentration, rather than peak Ca2+ concentration reached during channel opening, was the relevant regulatory factor for the action of G-proteins. Of course, these values may be regarded as crude estimates, because specific subcellular distributions of BAPTA, especially in the submembrane space where its action is expected to be most critical in the case of the G{beta}{gamma} inhibition, would lead to lower values for the Ca2+ concentration in these particular compartments.

Mechanism for the Action of Ca2+ and PIP2—The G-protein G{beta}{gamma} subunits have been shown to interact directly with several parts of the CaV2.1, CaV2.2, and CaV2.3 subunits. Although the intracellular loop connecting domains I and II seems to provide two major sites (15, 16), partly in competition with the {beta} subunit, the N- and C-terminal tails of the channel provide further secondary interaction/regulatory sites essential for the functional effects (14, 33, 35, 39). The proposal, from reinhibition kinetic studies (40), that the binding of only one G{beta}{gamma} subunit is sufficient to block channel conduction, led to the hypothesis that these four binding sites might come together to form a single G{beta}{gamma} binding pocket with several interacting zones cooperating to bind the G{beta}{gamma} dimer, as described for the G protein-activated K+ channel (41, 42). The analysis of the G{beta}{gamma} interactions with these binding domains, taken independently, thus far has not revealed any Ca2+ dependence (14, 16). Although the role of Ca2+ in these interactions will need to be tested more rigorously now, it is conceivable that whereas these sites may not individually require Ca2+ to bind G{beta}{gamma} subunits, the global functional conformation of the G{beta}{gamma} binding pocket may be under the stabilizing influence of Ca2+ ions. In this scenario, Ca2+, rather than playing a direct role in the G{beta}{gamma} inhibition, would spatially organize the four G{beta}{gamma} binding sites dispersed on the primary structure of the CaV2.1 subunit into one single functional binding pocket and/or would also assure its proper location for interacting with freely moving, membrane-tethered, active G{beta}{gamma} subunits.

Experimental evidence supporting this hypothesis includes the insensitivity of the effects of BAPTA to the use of different kinase or cytoskeleton inhibitors, clearly rejecting the participation of a possible Ca2+-induced modification of the phosphorylation state of the channel or any change in the interactions with the cytoskeleton and suggesting a more direct role for Ca2+. It should be noted that down-regulation of tonic protein kinase C activity by injection of BAPTA should decrease channel activity (11) and increase inhibition by G-proteins (15, 43), i.e. exactly the opposite of what was recorded here (Fig. 1).

The fact that injection of anti-PIP2 was also able to remove the tonic block of the channel imposed by the G-proteins and that it could occlude the effects of BAPTA strongly suggested that the two molecules were acting through the same mechanism. PIP2, via their positive action on membrane-protein interactions, regulate the activity of various ions channel including M currents (44), ATP-sensitive K+ channels (45), K+ACh channels (46), inward rectifier K+ channels (47, 48), but also the Ca2+-permeable channel TRPM7 (49). Binding of PIP2 to specific sites on the channel protein leads to prevention of Mg-ATP-dependent rundown of IRK+ channels (47) or stabilization of a G{beta}{gamma}-sensitive state of K+ACh channels (46). These sites include multiple cationic residues interspersed with hydrophobic residues and display less selectivity than the PH domains of phospholipase C-{delta} (47, 50, 51). In the case of the PH domain, the existence of proximal EF-hand motifs confer to this interaction a Ca2+ sensitivity (52) similar to the one we report here in the case of the CaV2.1-type Ca2+ channel. The marked phosphoinositide-C terminus interactions shown in Fig. 6 strongly suggest the existence of one or more PIP2 binding sites on this tail of CaV2.1 channels. The poor specificity of this (or these) site(s) for phospholipids fits with the fact that no sequence homologous to known phospholipid binding sites (53) is found in this tail. It is also strongly similar to the specificity of activation of the G-protein activated K+ channels or other channels by phosphoinositides (51, 54). For these channels, as for Ca2+ channels, specific activation by PIP2 could thus be provided by two factors: 1) PIP2 is a trivalent ion at pH 7, thus increasing electrostatic interactions with cationic residues of the channel; and (2) PIP2 is much more abundant than phosphatidylinositol 1,4,5-trisphosphate (PIP3) in biological membrane (see Ref. 51 for review). The existence of specific membrane compartments enriched in PIP2 ("lipid raft") may also favor specific channel localization and activation.

Taking into account the usual electrostatic nature of PIP2-protein interactions (55), one may thus postulate that this site involves clusters of positive charges such as those located close to secondary binding sites for G{beta}{gamma} subunits in the distal part of the C terminus (RRRGRPR or RRRDRSHR). This tail also possesses an EF-hand positioned just downstream of the last S6 segment. Each of these sequences is a good candidate for future mutagenesis studies to identify more precisely the determinants of the sensitivity to Ca2+ and PIP2.

In a recent work by Wu et al. (17), PIP2 was shown to exert a dual antagonistic action on Ca2+ channels, preventing rundown but also promoting a reluctant mode of activity of low open channel probability via, as suggested by the authors, two separate PIP2 binding sites, called S and R, respectively, with different affinities. Although the recording conditions used in this study were different from ours (expression of {beta} subunits and lack of G-protein stimulation), the promotion of a reluctant mode of activity by PIP2 (at the R site) is clearly reminiscent of G{beta}{gamma}-dependent stimulation of the current amplitude recorded here, after anti-PIP2 antibodies or BAPTA injections. Such injections in whole oocytes may not be sufficiently effective to decrease the high affinity interaction between PIP2 and the S site necessary for channel activity. Starting from our results on channel-phosphoinositide interactions, further studies in macropatches using a truncated C terminus should help to confirm the existence and localization of these R and S sites.

The precise mode of action of Ca2+ ions on this interaction remains to be elucidated but could include electrostatic effects favoring channel or G{beta}{gamma}-phospholipid interactions (52), direct binding to the channel, and via allosteric mechanisms, the formation of a putative PIP2-binding domain on the channel (as described for Na+ ions on G protein-activated K+ channels (48)) or induction of PIP2 aggregation (56). Drawing a more precise picture of the molecular mechanisms involved in the effects of BAPTA seems premature, but testing the Ca2+-dependence of this channel-PIP2 interaction will surely constitute a key result to confirm our hypothesis and will open new perspectives for future research on the precise molecular mechanisms of G-protein action on voltage-gated Ca2+ channels. We also emphasize the fact that although this study was performed on the CaV2.1 Ca2+ channels, looking at Fig. 1, we suspect similar results will be found with CaV2.2 N-type Ca2+ channels. Whether G-protein-sensitive isoforms of the CaV2.3 channels (24) are also sensitive to this form of Ca2+-dependent regulation remains to be determined.

Can Ca2+ Work without Calmodulin?—Direct binding of Ca2+ ions to cytosolic or membrane proteins is known to trigger a variety of processes, including Ca2+/calmodulin-dependent inactivation and facilitation of the CaV2.1 {alpha}1 subunit and activation of protein kinase C or phospholipase C (for review see Refs. 10 and 57). At least three putative binding sites for Ca2+ have been identified on the CaV2.1 subunit: the C-terminal E-F hand motif of the CaV2.1 {alpha}1 subunit and the two lobes of the calmodulin, previously shown to be constitutively tethered to the channel (3). However these three sites do not seem to bind Ca2+ in resting conditions but rather require channel opening and the subsequent elevation of the resting concentration. Our preliminary results show that a mutant calmodulin, incapable of binding Ca2+, cannot inhibit the effects of BAPTA and thus suggest that these two sites are not involved in the effect of BAPTA. These data, however, do not completely obliterate a role for calmodulin, because the constitutive binding of calmodulin on the {alpha}1 subunit has its own Ca2+ dependence with a Kd in the range of concentrations that is expected to be found when Ca2+ is reduced following injection of BAPTA (13 nM (8)), a process that does not rely on the two lobes of calmodulin. This leaves thus open the interesting possibility that the Ca2+-dependent constitutive binding of apo-calmodulin on the channel may be necessary for channel-PIP2 interaction and G{beta}{gamma} inhibition. Clearly Ca2+ binding to the EF-hand also remains a possibility.

Endogenous Ca2+ buffers at synaptic nerve terminals are fast chelating, moving proteins that work with an efficiency equivalent to ~1–2 mM BAPTA (58). They have been proposed to moderate fast exocytosis by restricting, both temporally and spatially, the spread of the Ca2+ signal, thus limiting neurotransmitter release to the active zone in the millisecond time-scale. We show here that any drop of this tightly regulated presynaptic Ca2+ concentration under its resting level is likely to depress the G-protein-dependent tonic or phasic inhibition of exocytotic N- and P/Q-type Ca2+ channels. The fact that PIP2 not only regulates basal current rundown (17) but also interferes with the G-protein-dependent regulation of Ca2+ channels, as already described for G-protein gated K+ channels, extends the regulatory role of phosphoinositides on Ca2+ channels and opens new perspectives to the understanding of the molecular mechanisms of these regulations. Such a pathway may indeed play an important role in the adjustment of synaptic efficacy.


   FOOTNOTES
 
* This work was supported by Association Française contre les Myopathies, Association pour la Recherche contre le Cancer, and the Fondation Simone Cino Del Duca. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains supplemental material. Back

§ Supported by the Fondation pour la Recherche Médicale and French Ministry of Education. Back

** To whom correspondence should be addressed. E-mail: charnet{at}crbm.cnrs-mop.fr.

1 The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol; BAPTA, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate; GDP{beta}S, guanyl-5'-yl thiophosphate; DRG, dorsal root ganglia; DAMGO, [D-Ala, N-Me-Phe,Gly-ol]-enkephalin; PTX, pertussis toxin; PH domain, pleckstrin homology domain. Back

2 M. Rousset, T. Cens, and P. Charnet, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Cesar Labarca, Jean Philippe Pin, Laurent Prézeau, and Vincent Homburger for the gift of various cDNAs, Drs. C. Doucet, I. Lefèvre, D. T. Yue, P. F. Mery, A. Lacampagne, and P. Jeanneau for fruitful discussions, and Alain Bernet and Jean Marc Donnay for preparation of oocytes.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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L. Kreiner and A. Lee
Endogenous and Exogenous Ca2+ Buffers Differentially Modulate Ca2+-dependent Inactivation of CaV2.1 Ca2+ Channels
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