Voltage and Calcium Use the Same Molecular Determinants to Inactivate Calcium Channels*

Thierry Cens, Sophie Restituito, Simon Galas, and Pierre CharnetDagger

From the Centre de Recherches de Biochimie Macromoléculaire, CNRS UPR 1086, 1919 Route de Mende, F34293 Montpellier, France

    ABSTRACT
Top
Abstract
Introduction
References

During sustained depolarization, voltage-gated Ca2+ channels progressively undergo a transition to a nonconducting, inactivated state, preventing Ca2+ overload of the cell. This transition can be triggered either by the membrane potential (voltage-dependent inactivation) or by the consecutive entry of Ca2+ (Ca2+-dependent inactivation), depending on the type of Ca2+ channel. These two types of inactivation are suspected to arise from distinct underlying mechanisms, relying on specific molecular sequences of the different pore-forming Ca2+ channel subunits. Here we report that the voltage-dependent inactivation (of the alpha 1A Ca2+ channel) and the Ca2+-dependent inactivation (of the alpha 1C Ca2+ channel) are similarly influenced by Ca2+ channel beta  subunits. The same molecular determinants of the beta  subunit, and therefore the same subunit interactions, influence both types of inactivation. These results strongly suggest that the voltage and the Ca2+-dependent transitions leading to channel inactivation use homologous structures of the different alpha 1 subunits and occur through the same molecular process. A model of inactivation taking into account these new data is presented.

    INTRODUCTION
Top
Abstract
Introduction
References

Calcium channels formed integral membrane proteins through which Ca2+ ions can flow into the cells during membrane depolarization, thereby activating many fundamental physiological functions such as contraction, synaptic transmission, or gene activation (1, 2). According to their biophysical and pharmacological properties, they have been classified into L, N, P, Q, R, and T type Ca2+ channels (2-6). All these types are able to respond to membrane depolarization by opening a pore, selective for Ca2+ and other divalent cations, and thus generating an inward current into the cells (2, 7). During sustained depolarizations, all of these Ca2+ currents progressively undergo a voltage-dependent inactivation with specific kinetics, primarily regulated by the membrane potential (2, 3, 8, 9). Of particular interest in the case of the L-type voltage-gated Ca2+ channels is the existence of a second type of inactivation driven by the intracellular Ca2+ concentration (10-12). In this case, Ca2+ ions entering into the cell through calcium channels can bind to a specific site located close to the inner mouth of the channel and promote the so-called Ca2+-dependent inactivation (12-15). This type of inactivation, as opposed to the voltage-dependent inactivation, is not recorded when Ba2+ or Sr2+ permeate the channel (2, 4, 11, 12). While voltage-dependent inactivation has been found in all types of Ca2+ channels, the Ca2+-dependent inactivation seems to be specific to the L-type, dihydropyridine-sensitive, Ca2+ channels and has been suggested to arise from a completely different mechanism (5, 12, 15-19).

It is strongly believed that all types of functionally characterized Ca2+ channels possess at least three different subunits: alpha 1, the pore-forming subunit, and alpha 2-delta and beta , two regulatory subunits (20-26). The biophysical and pharmacological properties of a Ca2+ channel are primarily driven by the alpha 1 subunit (13, 27-31), for which 10 genes (named class A, B, C, D, E, F, G, H, I, and S) have been identified (23, 25, 26, 32-34). The beta  subunit (four genes identified: beta 1-4) and, to a lesser extent, the alpha 2-delta subunit (only one gene) seemed to have only a regulatory role on these properties (8, 35-42). The alpha 1 subunit is an integral membrane protein organized in four homologous domains, each containing six alpha -helical membrane-spanning segments (23, 25, 26). It has been shown that the differences in the kinetics of the voltage-dependent inactivation between two distinct alpha 1 subunits (encoding class E and A calcium channels, respectively) were due to the segment S6 of the first domain (I-S6) (30), suggesting a role for important and delimited molecular structures in the process of inactivation as was the case for K+ channels (43). On the other hand, a typical Ca2+ binding site (44) and other regions involved in the Ca2+-dependent inactivation (45, 46) have recently been located in the carboxyl-terminal tail of the alpha 1 subunit. The identification of two different structures regulating voltage and Ca2+-dependent inactivation supports the hypothesis of different underlying mechanisms for these two types of inactivation. However, donation of this Ca2+ binding site (EF-hand motif) to the alpha 1E subunit gave chimerical Ca2+ channels that did support Ca2+-dependent inactivation, whereas the parental alpha 1E subunit did not. The fact that Ca2+-dependent inactivation can be transplanted by the sole donation of a Ca2+ binding site presupposes that the molecular backbone responsible for the closure of the channel during sustained depolarization may be shared by voltage- and Ca2+-dependent inactivation and would therefore be present on all types of Ca2+ channel.

To test this hypothesis, we have compared the regulation of inactivation of the class A and class C Ca2+ channels by different beta  subunits in conditions where pure voltage and Ca2+-dependent mechanisms drove inactivation, respectively. We found that voltage and Ca2+-dependent inactivations were similarly regulated by the four beta  subunits. Moreover, deleted or chimerical forms of the beta  subunit that altered the regulation of the voltage-dependent inactivation also modified the Ca2+-dependent inactivation. We proposed a scheme for the inactivation mechanism and suggested that common distal steps shared by Ca2+- and voltage-dependent inactivations use homologous molecular structures. This scheme may be useful in elaborating new experiments designed to understand the molecular mechanisms underlying voltage and Ca2+-dependent inactivations.

    EXPERIMENTAL PROCEDURES

Preparation of Truncated and Chimerical Forms of the beta  Subunits-- The following calcium channel subunits were used: alpha 1A (47), alpha 1B (48), alpha 1E (49), alpha 1C (50), beta 1b (40), beta 2a (38), beta 3 (37), and beta 4 (36). All of these subunit cDNAs were inserted into the pMT2 expression vector (51). Ndel-beta 2 was obtained by PCR.1 The sense primer was engineered to possess an EcoRI site (italic type), a start codon (boldface type), and the 748-769 sequence (underlined, accession number M80545) of the beta 2a subunit (5'-GGAATTCATGGAGAACATGAGGCTACAGCAGCATG-3'). In the reverse primer, an XbaI site was added at the 5'-end of the 2190-2168 sequence of beta 2a (5'-GCTCTAGATCATTGGCGGATGTATACATCCC-3'). The PCR product was checked and purified on agarose gel. The chimera were obtained by a PCR strategy as described (52). The N-terminal fragments were amplified using the following primers: for beta ch1, sense primer, 5'-GGAATTCAGCCCCCTGAAAGGAGATC-3' (representing EcoRI plus positions 114-133 of beta 2a) and reverse primer, 5'-CAGCAGGCGAAGGCTGTCTAGTTTGACCGGGCTTG-3' (representing positions 576-559 of beta 1b, accession number X61394, plus positions 747-731 of beta 2a); for beta ch4, sense primer same as beta ch1 sense primer and reverse primer, 5'-GTAGGACTCTGCTGAGCCATAGGACACCCGTACTC-3' (representing positions 235-249 of beta 1b + positions 407-423 of beta 2a); for beta ch5, sense primer, 5'-GGAATTCATGGTCCAGAAGAGCGGCATG-3' (representing EcoRI plus positions 67-84 of beta 1b) and reverse primer, 5'-TAGGAGTCTGCCGAACCCTGACGGACAAAGCTGTT-3' (representing positions 424-440 of beta 2a plus positions 217-234 of beta 1b). The C-terminal fragments were amplified using the following primers: for beta ch1, sense primer, 5'-CAAGCCCGGTCAAACTAGACAGCCTTCGCCTGCTG-3' (representing positions 731-747 of beta 2a plus positions 559-576 of beta 1b) and reverse primer, 5'-GCTCTAGATCAGCGGATGTAGACGCCTTG-3' (representing XbaI plus positions 1857-1837 of beta 1b); for beta ch4, sense primer, 5'-GAGTACGGGTGTCCTATGGCTCAGCAGAGTCCTAC-3' (representing positions 407-423 of beta 2a plus positions 235-249 of beta 1b) and reverse primer same as the beta ch1 reverse primer; for beta ch5, sense primer, 5'-AACAGCTTTGTCCGTCAGGGTTCGGCAGACTCCTC-3' (representing positions 217-234 of beta 1b plus positions 424-440 of beta 2a) and reverse primer, 5'-GCTCTAGATCATTGGCGGATGTATACATCCC-3' (representing XbaI plus positions 2168-2190 of beta 2a).

All PCR products were separated on 1% agarose gel, cut out, and purified. A second PCR was performed using these products to produce the final chimeras. Ndel-beta 2 and -beta chimera were finally digested using EcoRI and XbaI, subcloned into pBluescript (Stratagene) for sequencing (DiDeoxy Terminator technology; Applied Biosystems), and subsequently subcloned into pMT2 for injection and expression.

I-II and III-IV loops of the alpha 1A and alpha 1C subunits were produced by PCR, subcloned into pBluescript, and in vitro transcribed (T7 m-Message m-Machine; Ambion) for oocyte injection (at an mRNA concentration of 1 µg/µl). Correct translation and molecular weight of these loops were checked by in vitro translation (T7-TNT coupled reticulocyte lysate system from Promega).

Xenopus oocyte preparation and injection (5-10 nl of alpha 1, alpha 1 plus beta , or alpha 1 plus alpha 2-delta plus beta  cDNAs at ~0.3 ng/nl) were performed as described elsewhere (42, 53). Oocytes were then incubated for 2-7 days at 19 °C under gentle agitation before recording.

Electrophysiological Recordings-- Whole cell Ba2+ and Ca2+ currents were recorded under two-electrode voltage clamp using the GeneClamp 500 amplifier (Axon Instruments, Burlingame, CA). Current and voltage electrodes (less than 1 megaohm) were filled with 2.8 M CsCl, 10 mM BAPTA, pH 7.2, with CsOH. Ca2+ and Ba2+ current recordings were performed after injection of BAPTA (one or two 40-70-ms injections at 1 bar of 100 mM BAPTA free acid (Sigma), 10 mM CsOH, 10 mM HEPES, pH 7.2, using solutions of the following composition: 10 mM BaOH/CaOH, 20 mM tetraethylammonium hydroxide, 50 mM N-methyl-D-glucamine, 2 mM CsOH, 10 mM HEPES, pH 7.2, with methanesulfonic acid. Ca2+ and Ba2+ current amplitudes were usually in the range of 1-5 µA, except for the alpha 1C subunit expressed alone (70-250 nA) as reported by others (35). Expression of the alpha 2delta subunit was sometimes performed but did not significantly modify either the kinetics of the two types of inactivation or the effects of overexpressing intracellular I-II and III-IV loops. All of the results mentioned here were obtained without alpha 2delta .

Currents were filtered and digitized using a DMA-Tecmar labmaster and subsequently stored on an IPC 486 personal computer by using version 6.02 of the pClamp software (Axon Instruments). Ba2+ or Ca2+ currents recorded during a typical test pulse from -80 mV to +10 mV of 2.5-s duration were well fitted using a biexponential function: i(t) = (A1* exp(-(t - K)/Tau1) + A2 * exp(-(t - K)/Tau2)) C, where t is the time; K is the zero time; A1, A2, Tau1, and Tau2 are the amplitudes and time constants of the two exponential components; and C is the fraction of noninactivating current.

Current amplitudes and inactivation time constants were measured using Clampfit (pClamp version 6.02, Axon Instruments). Pseudo-steady state inactivation (2.5 s of conditioning depolarization) and current-voltage curves were fitted using the equations I/Imax R + (1 - R)/(1 + exp((V - V0.5)/k)) (for the inactivation curve) and I/Imax = g * (V - Erev)/(1 + exp((V - V0.5)/k)) (for the current-voltage curve), where g is a normalized conductance, Erev is the extrapolated reversal potential for barium, k is the slope factor, V0.5 is the potential for half-inactivation or activation, V is the conditioning depolarization (inactivation curve) or the membrane potential used to record current (current-voltage curve), and R is the proportion of noninactivating current. All values are presented as mean ± S.D. Student's t test was used at the 0.05 confidence level to test the significance of the difference between two means.

    RESULTS AND DISCUSSION

When expressed in Xenopus oocytes, each class of Ca2+ channels displays only one type of inactivation, which can be driven either by membrane potential or change in intracellular Ca2+ concentration (Fig. 1, A and B). Current kinetics of the alpha 1A, alpha 1B and alpha 1E Ca2+ channel, co-expressed with the ancillary Ca2+ channel beta 1 or beta 2 subunits, were identical using either Ca2+ or Ba2+ as charge carriers, as shown by the superimposed scaled traces seen in Fig. 1, A and B. These kinetics were only modulated by the amplitude of the depolarization, as expected for a voltage-dependent inactivation (not shown) (41, 54, 55). In contrast, Ca2+ currents recorded from oocytes injected with the alpha 1C subunit inactivated markedly faster than the corresponding Ba2+ currents (Fig. 1A). This behavior, typical of the so-called Ca2+-dependent mechanism of inactivation (13, 56-58), was present whether the beta 1 or the beta 2 subunit was co-expressed. Despite differences in their initiating events, voltage- and Ca2+-dependent inactivations were nevertheless both sensitive to the co-expression of the ancillary beta  subunits. Co-expression of the alpha 1A subunit with the beta 1 or beta 2 subunit, for example, led to fast or slowly decaying Ba2+ currents, respectively (Fig. 1C, left). These differences in current kinetics were similar, in the case of the alpha 1A subunit (but also alpha 1B and alpha 1E), using either 10 mM Ca2+ or Ba2+ as the charge carrier (see Fig. 1, A and B). Fast and slowly inactivating Ca2+ currents were also recorded upon co-expression of the beta 1 or beta 2 subunit with the alpha 1C subunit (Fig. 1C, right). In this case, however, the use of 10 mM Ba2+ as the charge carrier completely blocked current inactivation for these two subunit combinations, further demonstrating the absolute requirement of Ca2+ for alpha 1C inactivation (Fig. 1, A and B). To understand the molecular mechanisms underlying these two different types of inactivation, the alpha 1A and alpha 1C subunits were chosen as prototype Ca2+ channels with pure voltage and Ca2+-dependent inactivations, respectively. Averaged data using long depolarizations and extended to the co-expression of the four known beta  subunits with these two alpha 1 subunits are shown in Fig. 2. When compared with the corresponding alpha 1 subunit expressed alone, voltage- and Ca2+-dependent inactivations were significantly accelerated upon co-expression of the beta 1 subunits (Fig. 2, alpha 1A (top left) and alpha 1C (top right); p < 0.05). The same effect was found upon co-expression of the beta 3 or beta 4 subunit, without significant difference when compared with alpha 1A plus beta 1. Co-expression of the beta 2 subunit, however, slowed both the voltage and the Ca2+-dependent inactivation when expressed with the alpha 1A or the alpha 1C subunit, respectively (significantly different from alpha 1A plus beta 1). A kinetic analysis of these current decays confirmed and extended this observation. Voltage- as well as Ca2+-dependent inactivation could be well approximated by a biexponential decay. The fast time constant (Tau1, Fig. 2) of inactivation of the alpha 1A or alpha 1C subunits was always accelerated upon co-expression of each of the beta  subunits (no statistical differences between the four beta  subunits). The slow time constant (Tau2, Fig. 2) was either decreased (beta 1, beta 3, and beta 4) or increased (beta 2), for these two alpha 1 subunits, after co-expression of a beta  subunit, and it was thus responsible for the marked slowing of inactivation recorded with the beta 2 subunit (together with an increase in the relative amplitude of the slow component in both cases; not shown). Taken together, these data demonstrate that beta  subunits modified the same kinetic components of the voltage- and Ca2+-dependent inactivations and thus suggest that they affect the same molecular processes on the two alpha 1 subunits. Because protein-protein interactions between the alpha 1 and the beta  subunits are the basis of these regulations (8, 41, 42, 51, 59-63) and because alpha 1 subunits alone are able to undergo both voltage-dependent (alpha 1A) and Ca2+-dependent (alpha 1C) inactivation (28-30, 41, 44, 51), we hypothesized that homologous molecular determinants on these different pore-forming subunits are responsible for these distinct modes of inactivation. One immediate prediction is that structural modifications of the beta  subunit, modifying the voltage-dependent inactivation, should also change, in parallel, the Ca2+-dependent inactivation. We have tested this hypothesis using four different constructions of the beta  subunit (Fig. 3, top). A large deletion in the amino terminus of the beta 2 subunit (125 amino acids) strongly accelerated the slow voltage-dependent inactivation of the alpha 1A subunit, usually recorded with the full-length beta 2 subunit (Fig. 3, Ndel-beta 2, left). This deleted beta 2 subunit had exactly the same effect on the alpha 1C Ca2+-dependent inactivation, which became as fast as beta 1 (see Fig. 3, Ndel-beta 2, right). When this deleted sequence was inserted at a homologous position in the beta 1 subunit (beta ch1; see Fig. 3, top, for construction and traces), Ba2+ currents recorded from oocytes expressing alpha 1A plus beta ch1 subunits displayed the slow inactivation kinetics typically recorded with the beta 2 subunit. A similar slowing of the Ca2+-dependent inactivation was recorded when this chimera was expressed with the alpha 1C subunit (Fig. 3, beta ch1, right). These data suggest that the presence of the amino-terminal end of the beta 2 subunit is responsible for the slow current decays in both voltage and Ca2+-dependent inactivation. This was further confirmed by the use of chimera beta ch4 and beta ch5 (see Fig. 3, top, for constructions). Co-expression of beta ch4 (where the beta 1 subunit had its first variable domain (64), corresponding to amino acids 0-58, replaced by the homologous amino acids of the beta 2 subunit) decreased the voltage-dependent inactivation (alpha 1A) as well as the Ca2+-dependent inactivation (alpha 1C). In both cases, inactivation became as slow as in the case of co-expression of the full-length beta 2 subunit (Fig. 3, beta ch4 traces). The reverse chimera beta ch5 (where the beta 2 subunit has its first domain, corresponding to amino acids 1-16, replaced by the homologous domain of beta 1) had opposite effects and induced fast-inactivating currents comparable with those recorded with the beta 1 subunit (or Ndel-beta 2), irrespective of the alpha 1 subunit and therefore of the mode of inactivation. All of these data, summarized at the bottom of Fig. 3, clearly show that any change in the structure of the beta  subunit affecting the voltage-dependent inactivation also modified the Ca2+-dependent inactivation and suggest that the same mechanism underlies these two distinct phenomena.


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Fig. 1.   Regulation of voltage- and Ca2+-dependent inactivation by beta  subunits. A and B, Ba2+ and Ca2+ currents recorded from oocytes injected with the alpha 1A, alpha 1B, alpha 1C, or alpha 1E Ca2+ channel subunits together with the auxiliary beta 1 (A) or beta 2 (B) subunit. Inactivation (I2/I1) was quantified by dividing the current at the end of a 400-ms-long depolarization (I2) by the peak current (I1) and is displayed as a bar graph for these different subunit combinations in the presence of external Ba2+ and Ca2+ (10 mM). Corresponding scaled traces are displayed at the bottom of each combination. Currents were recorded during 400-ms-long depolarizations to +10 mV from a holding potential of -100 mV: alpha 1A plus beta 1 (n = 6); alpha 1A plus beta 2 (n = 7); alpha 1B plus beta 1 (n = 5); alpha 1B plus beta 2 (n = 5); alpha 1C plus beta 1 (n = 11); alpha 1C plus beta 2 (n = 10); alpha 1E plus beta 1 (n = 6); alpha 1B + beta 2 (n = 6). For a given combination of subunits, an asterisk indicates when inactivation of Ca2+ current was significantly faster (p < 0.05) than that of the Ba2+ current. C, Ba2+ and Ca2+ current traces recorded during long depolarization of oocytes expressing the alpha 1A or the alpha 1C subunits, respectively, with either the beta 1 or the beta 2 subunit. Current traces were scaled at the same size and superimposed for comparison (depolarization of 2.5 s to +10 mV from a holding potential of -100 mV).


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Fig. 2.   Effect of the beta  subunits on the kinetics of inactivation of the alpha 1A and alpha 1C Ca2+ channels. Top, inactivation was quantified as the ratio of the noninactivating current at the end of the pulse over peak amplitude (I2/I1). Depolarizations of 2.5 s were used to maximize measurements of current inactivation. Dashed lines, superimposed fit of the two exponential components Tau1 and Tau2 necessary to describe current inactivation kinetics. I2/I1, Noninactivating currents calculated for different subunit combinations having either a voltage-dependent inactivation (alpha 1A, barium) or a Ca2+-dependent inactivation (alpha 1C, calcium). Note that the beta 2 subunit decreased both the voltage-dependent (alpha 1A subunit recorded in the presence of Ba2+) and the Ca2+-dependent (alpha 1C subunit recorded in the presence of Ca2+) inactivation (n = 8, 10, 6, 7, and 7 for alpha 1A, alpha 1A plus beta 1, alpha 1A plus beta 2, alpha 1A plus beta 3, and alpha 1A plus beta 4, respectively; n = 6, 9, 7, 2, and 4 for alpha 1C, alpha 1C plus beta 1, alpha 1C plus beta 2, alpha 1C plus beta 3, and alpha 1C plus beta 4, respectively). *, alpha 1 plus beta 1 significantly different from alpha 1 alone; #, alpha 1 plus beta 2 significantly different from alpha 1 plus beta 1 (see "Results and Discussion"). Tau1, fast time constants of voltage-dependent inactivation (alpha 1A; recorded using Ba2+) and Ca2+-dependent inactivation (alpha 1C; recorded using Ca2+). Note that, in both cases, the fast time constant was accelerated by expression of the different beta  subunits. Tau2, only the beta 2 subunit increased the slow time constant of both the voltage-dependent (alpha 1A) and the Ca2+-dependent (alpha 1A) inactivations. Currents were recorded during depolarization at +10 mV of 2.5 s (n = 7, 13, 4, 8, and 7 for alpha 1A, alpha 1A plus beta 1, alpha 1A plus beta 2, alpha 1A plus beta 3, and alpha 1A plus beta 4, respectively; n = 3, 9, 8, 3, and 3 for alpha 1C, alpha 1C plus beta 1, alpha 1C plus beta 2, alpha 1C plus beta 3, and alpha 1C plus beta 4, respectively.


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Fig. 3.   Mutations on the beta  subunit have the same effects on the two types of inactivation. A, schematic representation of the truncated and chimeric forms of the beta  subunit used to study the regulation of inactivation. B, effect of these constructions on voltage and Ca2+-dependent inactivation. Oocytes were injected with the alpha 1A or the alpha 1C subunits in combination with the beta 1 (n = 12 and 7, respectively), beta 2 (n = 6 and 7), Ndel-beta 2 (n = 4 and 11), beta ch1 (n = 3 and 9), beta ch4 (n = 6 and 3), and beta ch5 (n = 5 and 4) subunits. Currents were recorded during depolarization at +10 mV during 2.5 s, and traces were scaled. Note that the presence of the N-terminal sequence of the beta 2 subunit governed the kinetics of both types of inactivation. C, bar graph, averaged ratio I2/I1 of the noninactivating alpha 1A and alpha 1C currents for the six combinations of beta  subunit tested. beta 1 and beta 2 were the same values as in Fig. 2. Each subunit has the same effect on the two types of inactivation. An asterisk represents a statistical difference between alpha 1A plus beta 2 and the constructs.

Voltage- as well as Ca2+-dependent inactivations are known to be intrinsic properties of the alpha 1 subunit. However, until now, only voltage-dependent inactivation was known to be regulated by auxiliary beta  subunits (8, 41, 42, 51, 65). We show here the presence of two kinetic components of inactivation (characterized by Tau1 and Tau2) in both alpha 1A- and alpha 1C-directed currents. These two components are both either voltage-dependent (alpha 1A inactivation) or Ca2+-dependent (alpha 1C inactivation; see Fig. 2), suggesting that the two types of inactivation each have two different underlying mechanisms. Our results emphasize the fact that a given beta  subunit regulates, in the same way, each of these two components, independently of the mode of inactivation (voltage- or Ca2+-dependent), and therefore the type of alpha 1 subunit. To take into account these new data, we propose a new scheme of inactivation where voltage and Ca2+ inactivate these two different channels by using a "ball and chain" mechanism, with blocking particles and binding sites encoded by homologous sequences on the alpha 1A and alpha 1C subunits and therefore sensitive to the same molecular interactions with the beta  subunit (see Fig. 4). Binding of the particle to its binding site would ensure channel inactivation, as in the case of potassium channels (43). In the case of voltage-dependent inactivation, this binding is not ion-sensitive and can occur with either Ba2+ or Ca2+ as charge carriers, voltage-dependence being due to state-dependent changes in the mobility of the particle and/or the accessibility of the binding site. The Ca2+-dependent mechanism of inactivation is essentially the same, except that accessibility to, or functionality of, the binding site needs the fixation of a Ca2+ ion to a site located near the inner mouth of the channel (44) (Fig. 4, right). In our scheme, this fixation would produce a conformational modification of the binding site into a high affinity state, therefore allowing binding of the ball and inactivation of the channel. The sensitivity of this newly formed inactivated state to the continuous presence of a bound Ca2+ ion on the alpha 1 subunit might explain the differences in the immobilization of channel gating charges between voltage- and Ca2+-dependent inactivation (56). In this scheme, regulation of inactivation by beta  subunits occurs mainly through their N-terminal tail, where essential palmitoylation sites have just been identified (66-68), and would be due to modifications in the mobility of the inactivating particle by bound auxiliary subunits (Fig. 4). Accordingly, this region of the beta 2 subunit has also been shown to influence voltage-dependent inactivation of the alpha 1A Ca2+ channel (68-70). Therefore, the I-II loop, connecting domains I and II of the pore-forming alpha 1 subunit represents an attractive candidate for the blocking particle, since it possesses the alpha 1 interaction domain sequence, responsible for the interaction with the beta  subunit (60), and multiple mutations in this domain affect inactivation (71, 72). Accordingly, co-expression of an excess of free alpha 1A I-II loop with the slowly inactivating alpha 1A plus beta 2 Ca2+ channels significantly accelerated voltage-dependent inactivation (alpha 1A(I-II); Fig. 5, A and B). Similar effects on the voltage-dependent inactivation of the alpha 1A plus beta 2 Ca2+ channels were also recorded after co-expression of the I-II loop from alpha 1C (alpha 1C(I-II); Fig. 5, A and B) but not with the III-IV loop from alpha 1A (alpha 1A(III-IV)), suggesting that the alpha 1C I-II loop can promote voltage-dependent inactivation of the alpha 1A Ca2+ channel. Indeed, similar changes were obtained when steady state inactivation was studied, with smaller noninactivating current and hyperpolarizing shift of the half-inactivation potentials obtained with I-II loops but not the III-IV loop (Fig. 5C; mean values for V0.5 and R as follows: H2O, -12 ± 7 mV and 0.57 ± 0.08 (n = 12); alpha 1A(III-IV), -13 ± 7 mV and 0.61 ± 0.13 (n = 15); alpha 1A(I-II), -22 ± 5 mV and 0.35 ± 0.13 (n = 11); alpha 1C(I-II), -16 ± 7 mV and 0.41 ± 0.09 (n = 7)). Co-expression of these loops did not affect significantly (or slightly hyperpolarized; see Fig. 5C) the voltage dependence of activation and the average current amplitude (not shown). These latter data confirmed a direct effect on inactivation rather than a displacement of the beta 2 subunit from alpha 1A by an excess of free alpha 1 interaction domain, which would be expected to depolarize the current-voltage curve (mean activation potentials were as follows: H2O, -8 ± 3 mV (n = 12); alpha 1A(III-IV), -11 ± 6 mV (n = 15); alpha 1A(I-II), -15 ± 6 mV (n = 11); alpha 1C(I-II), -7 ± 12 mV (n = 7)) and depress current amplitude (62). Possible molecular determinants for the binding site include the carboxyl end of the alpha 1 subunit, where an EF-hand Ca2+ binding motif and other sequences, important for Ca2+ dependent inactivation, have been identified (44-46, 72, 73). Supporting our scheme, transfer of this domain to the alpha 1E subunit confers Ca2+-dependent inactivation (44), while alpha 1C chimera subunits harboring a carboxyl-terminal tail originating from a non-Ca2+-sensitive alpha 1 subunit lost their Ca2+-sensitive inactivation while preserving voltage-dependent inactivation (72).


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Fig. 4.   Proposed mechanism underlying voltage- and Ca2+-dependent inactivation. The schematic drawing represents the hypothetical mechanism underlying voltage- and Ca2+-dependent inactivation. The blocking site of the inactivating particle is always present in alpha 1 subunits displaying voltage-dependent inactivation (top). In the case of Ca2+-dependent inactivation (bottom), this site needs the binding of Ca2+ to be functional. The inactivating particle is encoded by a homologous sequence in both types of Ca2+ channel alpha 1 subunit. beta  subunits regulate Ca2+ channel inactivation by modifying the mobility of the blocking particle (see "Results and Discussion").


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Fig. 5.   Overexpression of the alpha 1 subunit I-II loop speeds up inactivation. A, left, schematic representation of the two-dimensional structure of the alpha 1A subunit. Loop I-II and loop III-IV represent connecting loops between domains I-II and domains III-IV of the alpha 1 subunit. Loop I-II and loop III-IV of the alpha 1A and alpha 1C subunits (alpha 1A(I-II), alpha 1A(III-IV), and alpha 1C(I-II), respectively) were subcloned into pBluescript and in vitro transcribed for oocyte injection. Right, autoradiogram of SDS-polyacrylamide gel of in vitro translated [35S]methionine-labeled alpha 1A(I-II), alpha 1A(III-IV), and alpha 1C(I-II) loops showing the correct production of these loops from the corresponding synthetic RNA. The film was exposed overnight. B, left, typical current traces recorded from oocytes injected with cDNA coding for the alpha 1A and the beta 2 subunits co-injected with H2O, alpha 1A I-II loop (alpha 1A(I-II)), alpha 1C I-II loop (alpha 1C(I-II)), or alpha 1A III-IV loop (alpha 1A(III-IV)) RNAs. Currents were recorded during 2.5-s-long depolarizations to +10 mV from a holding potential of -100 mV. Right, averaged current inactivation (I2/I1; see Fig. 1) calculated from different oocytes: H2O (n = 15); alpha 1A(III-IV) (n = 15); alpha 1A(I-II) (n = 11); and alpha 1C(I-II) (n = 7). Asterisk, significantly different from control (H2O). C, steady state (left) and current-voltage (right) curves recorded from the same oocytes as in C, co-injected with cDNA coding for the alpha 1A and the beta 2 subunits and with H2O (square), alpha 1A I-II loop (alpha 1A(I-II); circle), alpha 1C I-II loop (alpha 1C(I-II); triangle), or alpha 1A III-IV loop (alpha 1A(III-IV), inverted triangle) RNAs. The holding potential was -100 mV. Smooth lines represent the best fit using equations described under "Experimental Procedures." Calculated values for half-inactivation potentials and R for these particular oocytes were as follows: H2O, -11 mV and 0.65; alpha 1A(III-IV), -10 mV and 0.66; alpha 1A(I-II), -17 mV and 0.31; alpha 1C(I-II), -11 mV and 0.51. Half-activation potentials were as follows: H2O, -8 mV; alpha 1A(III-IV), -7 mV; alpha 1A(I-II), -7 mV; alpha 1C(I-II), -5 mV (see "Results and Discussion" for averaged values).

The presence of the two kinetic components (Tau1 and Tau2) can be best explained by the existence of an additional blocking particle instead of multiple binding sites, since (i) fast and slow inactivation have distinct regulation by beta  subunits and (ii) the two components are either voltage- (alpha 1A) or Ca2+- (alpha 1C) dependent, as expected for a unique binding site. Differences in the proportion of each component between voltage- and Ca2+-dependent inactivation may be due to sequence variation between the alpha 1 subunits. This scheme also disregards any participation of phosphatases in the process of Ca2+-dependent inactivation, since the intracellular Ca2+ concentration was sufficiently buffered to inhibit Ca2+-dependent enzyme, and ATPgamma S, as well as okadaic acid, had no effect either on Ca2+ current amplitude or on kinetics (74, 75). In conclusion, we show that voltage and Ca2+ use homologous structure of related alpha 1 subunits to inactivate Ca2+ channels, and we provide evidence for a direct channel block by the intracellular I-II loop of the alpha 1 subunit. The use of similar molecular determinants for the two types of inactivation suggests that voltage-dependent inactivation has been modified during evolution to provide the structural basis for the Ca2+-mediated inactivation, therefore preventing cellular Ca2+ overload.

    ACKNOWLEDGEMENTS

We thank Dr. T. Snutch and E. Perez-Reyes for kindly providing calcium channel cDNAs; Dr. T. Lorca, J. C. Labbe, and N. Morin for invaluable technical help during the course of these experiments; and Drs. D. Pietrobon, D. Yue, P. F. Mery, M. Moris, and D. Fisher for helpful comments on the manuscript.

    FOOTNOTES

* This work was supported by CNRS, INSERM, MRES, the Association Française contre les Myopathies, the Association pour la Recherche contre le Cancer, the Fondation pour la Recherche Médicale, the Ligue nationale contre le cancer, and NATO.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 33-467-61-33-52; Fax: 33-467-52-15-59; E-mail: charnet{at}crbm.cnrs-mop.fr.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; ATPgamma S, adenosine 5'-O-(thiotriphosphate); BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid.

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Abstract
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