Promotion and Inhibition of L-type Ca2+ Channel Facilitation by Distinct Domains of the beta  Subunit*

Thierry CensDagger , Sophie RestituitoDagger , Alice Vallentin, and Pierre Charnet§

From the Centre de Recherche de Biochimie Macromoléculaire, CNRS Unité Propre de Recherche 1086, 1919 Route de Mende, BP 5051, F34033 Montpellier, France

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Ca2+ current potentiation by conditioning depolarization is a general mechanism by which excitable cells can control the level of Ca2+ entry during repetitive depolarizations. Several types of Ca2+ channels are sensitive to conditioning depolarization, however, using clearly distinguishable mechanisms. In the case of L-type Ca2+ channels, prepulse-induced current facilitation can only be recorded when the pore-forming alpha 1C subunit is coexpressed with the auxiliary beta 1, beta 3, or beta 4, but not beta 2, subunit. These four beta  subunits are composed of two conserved domains surrounded by central, N-terminal, and C-terminal variable regions. Using different deleted and chimeric forms of the beta 1 and beta 2 subunits, we have mapped essential sequences for L-type Ca2+ channel facilitation. A first sequence, located in the second conserved domain of all beta  subunits, is responsible for the promotion of current facilitation by the beta  subunit. A second sequence of 16 amino acids, located on the N-terminal tail of the beta 2 subunit, induces a transferable block of L-type current facilitation. Site-specific mutations reveal the essential inhibitory role played by three positive charges on this segment. The lack of prepulse-induced current facilitation recorded with some truncated forms of the beta 2 subunit suggests the existence of an additional inhibitory sequence in the beta 2 subunit.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Beyond their role in membrane excitability, voltage-gated Ca2+ channels are of particular interest due to the fundamental importance of the biological processes regulated by incoming Ca2+ ions (for review, see Ref. 1). Several different types of Ca2+ channel have been identified in neurons, where they are crucial for secretion, synaptic transmission, or gene expression (2-4). Ca2+ entry through L-, N-, T-, or P-type channels is primarily regulated by the level of membrane depolarization. However, the frequency of these depolarizations is also an important regulatory factor that has been implicated in the regulation of synaptic transmission and that may be of importance for long-term potentiation. Accordingly, repetitive stimuli or strong pre-depolarization can up-regulate the activity of different types of Ca2+ channel, giving rise to the so-called Ca2+ current facilitation (for review, see Ref. 5). Although a similar increase is recorded for the various Ca2+ channels, different mechanisms, however, have been proposed. Experimental evidence firmly correlates a given mechanism with a particular Ca2+ channel type, as is the case for N-type Ca2+ channels, which are sensitive to a direct, voltage-dependent G-protein block.

L-type Ca2+ channel facilitation has been described using various preparations, and different mechanisms leading to current potentiation have been proposed. Strong pre-depolarizations can induce a change in gating mode in cardiac L-type Ca2+ channels, whereas the same protocol appears to lead, in skeletal and vascular muscle, to a voltage-dependent phosphorylation of the channel by protein kinase A and calmodulin kinase II, respectively (6-8). At the molecular level, these effects seem to be related to different isoforms of the L-type Ca2+ channel (alpha 1C, alpha 1D, or alpha 1S), different splice variants, and/or different cellular preparations. Current facilitation of the neuronal L-type Ca2+ channel (9) has been recorded, using the appropriate protocol, in isolated neurons (10, 11) or after expression of the alpha 1C subunit in Xenopus oocytes (12, 13). This type of facilitation can be distinguished from cardiac, skeletal, or vascular L-type Ca2+ channel facilitation (6-8, 14-18) by its insensitivity to phosphatase inhibitors and permeating ions (12), suggesting a specific mechanism for neuronal facilitation. As opposed to N-type Ca2+ channels, coexpression of specific beta  subunits (beta 1, beta 3, or beta 4) is absolutely necessary for the development of alpha 1C subunit facilitation (12, 19). The inhibitory effect of the beta 2 subunit (13) and the insensitivity to G-protein stimulation or inhibition (12) are also distinctive features. Altogether, these data suggest that the molecular mechanisms of L- and N-type Ca2+ channel facilitation are different processes. In this work, using deleted and chimeric subunits constructed from permissive and nonpermissive beta  subunits, we demonstrate the existence of a conserved sequence in the beta  subunits necessary for L-type Ca2+ channel facilitation. We also identify a short autoinhibitory segment on the N-terminal tail of the beta 2 subunit partly responsible for the insensitivity of the alpha 1C + beta 2 subunit combination to prepulse facilitation. Expression of individual beta  subunits could therefore reveal latent properties that are central for neuronal plasticity.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Preparation of Truncated, Chimeric, and Mutated Forms of the beta  Subunits and Xenopus Oocyte Injection-- The following calcium channel subunits were used: alpha 1C and alpha 2-delta (9), beta 1b (20), and beta 2a (21). All these subunit cDNAs were inserted into the pMT2 expression vector under the control of an SV40 promoter (9).

Deleted Mutants of the beta  Subunit-- beta 1-TF1-4 and beta 2-TF1-4 were constructed by PCR.1 The sense primer was engineered to possess an EcoRI site and a start codon (boldface) when necessary. In the reverse primer, an XbaI site (preceded by a stop codon in the case of the TF4 subunits) was added at the 5'-end. Numbers in parentheses correspond to the position of the primer in the sequence. GenBankTM accession numbers were X61394 for the beta 1b subunit and M80545 for the beta 2a subunit. The primers used were as follows: beta 1-TF1: sense, EcoRI-ATG-(235-257 beta 1b), and antisense, XbaI-(1837-1857 beta 1b); beta 1-TF2: sense, EcoRI-ATG-(559-579 beta 1b), and antisense, identical to beta 1-TF1; beta 1-TF3: sense, EcoRI-ATG-(706-726 beta 1b), and antisense, identical to beta 1-TF1; beta 1-TF4: sense, EcoRI-(64-84 beta 1b), and antisense, XbaI-TCA-(1298-1317 beta 1b); beta 2-TF1: sense, EcoRI-ATG-(424-444 beta 2a), and antisense, XbaI-TCA-(2168-2190 beta 2a); beta 2-TF2: sense, EcoRI-ATG-(748-769 beta 2a), and antisense, identical to beta 2-TF1; beta 2-TF3: sense, EcoRI-ATG-(1009-1031 beta 2a), and antisense, identical to beta 2-TF1; and beta 2-TF4: sense, EcoRI-(115-133 beta 2a), and antisense, XbaI-TCA-(1600-1623 beta 2a). The PCR product was checked and purified on agarose gel.

beta Subunit Chimeras-- The chimeras were obtained by a PCR strategy as described (22) using the Expand High Fidelity PCR system (Boehringer Mannheim). The N-terminal fragments were amplified using the following primers: beta CH1: sense, identical to beta 2-TF4, and antisense, (559-576 beta 1b + 731-747 beta 2a); beta CH2: sense, identical to beta 2-TF4, and antisense, (706-723 beta 1b + 878-894 beta 2a); beta CH3: sense, identical to beta 2-TF4, and antisense, (706-724 beta 1b + 993-1008 beta 2a); and beta CH4: sense, identical to beta 2-TF4; and antisense, (235-249 beta 1b + 409-423 beta 2a). The C-terminal fragments were amplified using the following primers: beta CH1: sense, (731-747 beta 2a + 559-576 beta 1b), and antisense, identical to beta 1-TF1; beta CH2: sense, (878-894 beta 2a + 706-723 beta 1b), and antisense, identical to beta 1-TF1; beta CH3: sense, (993-1008 beta 2a + 706-724 beta 1b), and antisense, identical to beta 1-TF1; beta CH4: sense, (407-423 beta 2a + 235-249 beta 1b), and antisense, identical to beta 1-TF1.

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. beta -Subunit truncated forms and chimeras were finally digested using EcoRI and XbaI, subcloned into pBluescript (Stratagene) for sequencing (DiDeoxy Terminator technology, Applied Biosystems) and in vitro translation (TNT® coupled reticulocyte lysate systems, Promega), and subsequently into pMT2 for injection and expression.

beta 2M123 and beta 2/beta 1-CH4M123 Constructions-- The mutants beta 2M123 and beta 1/beta 2-CH4M123 were obtained by a PCR strategy as described (22) using the Expand High Fidelity PCR system. The N-terminal fragments were amplified using the following primers: beta 2M123 and beta 2/beta 1-CH4M123: sense, identical to beta 2-TF4, and antisense, 5'-GGACACCCGTACTGCCGCGGCATGTACCAGCCCG-3' ((388-420 beta 2a), excepted the mutated sequence is underlined), using beta 2 as a matrix. The C-terminal fragments were amplified using the following primers: beta 2M123: sense, 5'-GGGCTGGTACATGCCGCGGCAGTACGGGTGTCC-3' (388-420 beta 2a, excepted mutated sequence underlined), and antisense, identical to beta 2-TF1, using beta 2 as a matrix; and beta 2/beta 1-CH4M123: sense, identical to beta 2M123, and antisense, identical to beta 1-TF1, using beta 2/beta 1-CH4 as a matrix. The complete sequence was obtained as described for chimeras.

All the constructions of the beta  subunits used in this paper were checked for correct translation and molecular weight by in vitro translation using the T7 or T3 TNT coupled reticulocyte lysate system (Promega). All these constructions were able to increase Ba2+ current amplitude when co-injected with the alpha 1C subunit, demonstrating association with the alpha 1C subunit (respective current amplitudes for alpha 1C alone, -368 ± 48 nA (n = 5); for beta 1, beta 1-TF1, beta 1-TF2, beta 1-TF3, and beta 1-TF4, -813 ± 306 nA (n = 20), -702 ± 235 nA (n = 6), -687 ± 273 nA (n = 14), -757 ± 414 nA (n = 10), and -411 ± 100 nA (n = 5), respectively; for beta 2, beta 2-TF1, beta 2-TF2, beta 2-TF3, and beta 2-TF4, -815 ± 403 nA (n = 9), -1220 ± 440 nA (n = 11), -1035 ± 399 nA (n = 17), -527 ± 200 nA (n = 5), and -1447 ± 678 nA (n = 6), respectively; and for beta 2/beta 1-CH1, beta 2/beta 1-CH2, beta 2/beta 1-CH3, and beta 2/beta 1-CH4, -930 ± 424 nA (n = 8), -1200 ± 540 nA (n = 9), -1633 ± 823 nA (n = 3), and -1650 ± 644 nA (n = 18), respectively).

Deleted Mutants of the alpha 1C Subunit-- alpha 1CDelta N and alpha 1CDelta C were generated by PCR using the Expand Long Template PCR system (Boehringer Mannheim). An N-terminal fragment was generated to introduce the untranslated avian myeloblastosis virus sequence just before the sequence of the alpha 1C subunit (the antisense primers used were 5'-CCTCGTGTTTTCATTGACCATGGTGGAAGTATTTGAAAGAAA-3' for alpha 1CDelta C (nucleotides 290-310; GenBankTM accession number M67515), and 5'-GCAGCTGCATTGGCATTCATGGTGGAAGTATTTGAAAGAAA-3' for alpha 1CDelta N (nucleotides 377-396)). This fragment was then used as a megaprimer to generate the final mutated forms of alpha 1C with an antisense primer (5'-GTTTTCCTTTTGCGGCCGCGTACAGGCCTCCAGCCCTCCTGAAGATGTC-3' for alpha 1CDelta C (NotI, stop codon in boldface, nucleotides 5378-5404) and 5'-ATAGTTTAGCGGCCGCCTACAGGTTGCTGACATAGGACC-3' for alpha 1CDelta N (NotI, nucleotides 6699-6721)). The mutated forms were subcloned into the pMT2 expression vector.

Xenopus oocyte preparation and injection (5-10 nl of alpha 1 + alpha 2-delta  + beta  subunit cDNAs at approx 0.3 ng/nl) were performed as described elsewhere (13). Oocytes were then incubated for 2-7 days at 19 °C under gentle agitation before recording.

Electrophysiological Recordings-- Whole cell Ba2+ currents were recorded under two-electrode voltage clamp using the GeneClamp 500 amplifier (Axon Instruments, Inc., Foster City, CA). Current and voltage electrodes (<1 megaohms) were filled with 2.8 M CsCl and 10 mM BAPTA, pH 7.2, with CsOH. Ba2+ current recordings were performed after injection of BAPTA (one or two 40-70-ms injection at 1 bar of 100 mM BAPTA-free acid (Sigma), 10 mM CsOH, and 10 mM HEPES, pH 7.2). The recording solution had the following composition: 10 mM BaOH, 20 mM tetraethyl ammonium hydroxide, 50 mM N-methyl-D-glucamine, 2 mM CsOH, and 10 mM HEPES, pH 7.2, with methanesulfonic acid. Only oocytes expressing Ba2+ currents with amplitudes in the range of 0.5-5 µA were analyzed to ensure sufficient resolution and to avoid voltage-clamp problems. Currents were filtered and digitized using a DMA-Tecmar Labmaster and subsequently stored on an IPC 486 personal computer using pClamp software (Version 6.02, Axon Instruments, Inc.). Ba2+ currents were recorded during a typical test pulse from -80 to +10 mV of 0.4-s duration. Current facilitation was elicited by applying a 200- or 400-ms depolarization (pre-depolarization) to +60 to +140 mV, 50 ms prior the test pulse. Current amplitudes were measured using Clampfit (pClamp Version 6.02, Axon Instruments, Inc.). Facilitation was quantified by dividing the current amplitude recorded after pre-depolarization by the control current amplitude (without pre-depolarization). All values are presented as means ± S.D. The significance of the difference between two means was tested using Student's t test (p < 0.05).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We have previously shown that the activity of the alpha 1C subunit L-type Ca2+ channel, recorded during a single voltage step at +10 mV, could be up-regulated by applying strong positive pre-depolarizations (12). This type of current facilitation was sensitive to the expression of a beta  subunit (12), but was not affected by the omission of the alpha 2-delta subunit. We have further identified the existence of permissive and nonpermissive beta  subunits for this facilitation (13).

Fig. 1 shows that application of a +120-mV pre-depolarization to an oocyte expressing the alpha 1C and beta 1 subunits (see protocol shown on top of the figure) increases the Ba2+ current recorded during a subsequent depolarization (in the range of -20 to +30 mV). The same pre-depolarization was without effect when applied to oocytes expressing the alpha 1C and beta 2 subunits (Fig. 1A). This lack of facilitation was recorded independently of the amplitude of the test pulse, as exemplified for voltage steps from -20 to +30 mV (Fig. 1, A and B). It was not due to a beta 2 subunit-induced shift in the voltage dependence of facilitation since stronger (or longer) pre-depolarizations were also inefficient in producing alpha 1C + beta 2 subunit current potentiation (13). Given that the alpha 1C subunit, when expressed alone, was not sensitive to pre-depolarization, we concluded that the beta 1, beta 3, or beta 4 subunit carried specific sequences able to produce novel properties when expressed with the alpha 1C subunit. We used the beta 2 subunit sequence as a matrix for chimeric beta  subunits to identify these sequences.


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Fig. 1.   Regulation of L-type Ca2+ channel facilitation by different beta  subunits. A, Ba2+ currents recorded from oocytes injected with cDNA coding for the alpha 1C and beta 1 or beta 2 subunits. Current facilitation (open circle ) was recorded during a typical 400-ms-long depolarizing pulse to (from top to bottom) -20, -10, 0, +10, +20, or +30 mV by applying a conditioning depolarization to +120 mV during 200 ms. Note the lack of facilitation when the beta 2 subunit was expressed. B, current voltage curves obtained from the oocytes shown in A and recorded during test pulses preceded, or not, by a 400-ms-long conditioning depolarization to +120 mV. Note that expression of the beta 2 subunit prevented facilitation for all these pulses.

The four beta  subunit genes identified so far are organized in two conserved domains (C1 and C2, 60-80% homology; white boxes in Fig. 2A), surrounded by variable regions where homology is lower (V1-3; black boxes) (see also Ref. 23). The sequence responsible for the interaction with the alpha 1 subunit (the beta  subunit interaction domain) has been localized to the second conserved domain (C2; see arrow in Fig. 2A), and recent studies suggest that important deletions outside this region have only slight effects on the capacity of this subunit to affect calcium channel function (24). Keeping these results in mind, we constructed and expressed a series of N- and C-terminal deletions of the beta 1 and beta 2 subunits in which this beta  subunit interaction domain was preserved and four chimeric beta  subunits, between the permissive beta 1 and nonpermissive beta 2 subunits, in which each of the five domains was exchanged (Fig. 2B).


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Fig. 2.   Schematic drawing of the different beta  subunits used in this study. A: amino acid similarity among the four beta  subunits. The white boxes represent the two conserved regions where homology is high (>90% between the beta 1 and beta 2 subunits). B: left panel, representation of the different truncated and chimeric forms of the beta 1 and beta 2 subunits. The precise location of the deletion is depicted on top of each schematic. Note that for each truncated beta  subunit, the second conserved region, which includes the alpha 1 subunit-binding site (24), is preserved to allow association with the pore-forming subunit (see "Materials and Methods"). All chimeras are composed of a C-terminal half from the beta 1 subunit and an N-terminal half from the beta 2 subunit. In beta 2/beta 1-CH2, a central insertion (black boxes in B and C) present in the beta 2 subunit (amino acids 170-203) and in beta 2/beta 1-CH3 has been removed. In beta 2/beta 1-CH4, the first 57 amino acids of the beta 1 subunit were replaced by the first 16 amino acids of the beta 2 subunit. Right panel: autoradiograms of SDS-polyacrylamide gels of in vitro translated, [35S]methionine-labeled, truncated and chimeric beta  subunits. Films were exposed overnight.

Fig. 3 shows the effects of N- and C-terminal deletions of the beta 1 subunit on current facilitation. Prepulse facilitation was elicited using a 200-ms-long prepulse at +100 mV. Under these conditions, the average current potentiation recorded by expressing the full-length beta 1 subunit with the alpha 1C subunit was 47 ± 18% (n = 17) larger than the control current, recorded without pre-depolarization. Deletion of the first variable region (V1, first 58 amino acids, beta 1-TF1) of the beta 1 subunit did not affect its capacity to induce facilitation since the increase in current recorded after the pre-depolarization was 54 ± 16% (n = 5) (Fig. 3). Subsequent deletions of the N-terminal region of the beta 1 subunit including the first conserved domain and the second variable region (first 166 and 215 amino acids, respectively; beta 1-TF2 and beta 1-TF3) were also without effect on facilitation (respective increases of 66 ± 6% (n = 7) and 48 ± 9% (n = 9)) (Fig. 3). In all cases, the increase in current following pre-depolarization was accompanied by an acceleration of inactivation, as already noted for class C L-type Ca2+ channel facilitation (12). Similarly, the C-terminal deletion of the last variable domain (amino acids 418-597) (Fig. 2) produced a truncated beta 1 subunit that was able, when expressed with the alpha 1C subunit, to generate Ca2+ channels sensitive to pre-depolarization (43 ± 13% (n = 5) of current increase; see trace labeled beta 1-TF4 in Fig. 3). Altogether, these data suggested that the sequences necessary to induce L-type current facilitation are located in the second conserved domain of the beta 1 subunit (C2, amino acids 215-418). Since this domain is highly conserved between the beta 1 and beta 2 subunits (92% homology), the beta 2 subunit should also be able to produce facilitation. Under these conditions, the lack of facilitation recorded with the beta 2 subunit may be due to some inhibitory processes instead of a missing facilitatory sequence.


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Fig. 3.   L-type current facilitation recorded with truncated forms of the beta 1 subunit. Ba2+ currents were recorded during a depolarization to +10 mV from a holding potential of -80 mV in Xenopus oocytes injected with the alpha 1C subunit and different truncated forms of the beta 1 subunit. Current facilitation was recorded, without significant differences upon coexpression of each of these truncated forms, when the appropriate conditioning depolarization was applied (200 ms in duration from -80 to +100 mV; traces labeled *). Left panel, constructions used; middle panel, current traces (scale bars = 1 µA, except for beta 1-TF4, 200 nA); right panel, averaged facilitation, quantified as the ratio of the current amplitude after pre-depolarization divided by the control current amplitude.

We have tested this idea by constructing a series of four truncated beta 2 subunits. In beta 2-TF1, the first 16 amino acids of the beta 2 subunit were removed. As shown in Fig. 4, Ba2+ currents recorded from oocytes expressing this subunit with the alpha 1C subunit were not sensitive to pre-depolarization since their amplitude was not increased by the conditioning voltage step (5 ± 2%, n = 11). The subsequent deletion of the first conserved domain (C1) gave rise to beta 2-TF2 (Fig. 4). Surprisingly, application of pre-depolarization to oocytes injected with this subunit, in conjunction with the alpha 1C subunit, significantly increased the Ba2+ current recorded during a subsequent test pulse to +10 mV (38 ± 6% (n = 8) compared with 5 ± 6% (n = 9) for the full-length beta 2 subunit). The facilitated current displayed the typical inactivating phase usually recorded with this paradigm (compare traces labeled beta 2-TF2 in Fig. 4 and beta 1 in Fig. 1). A larger deletion including the central variable region V3 (amino acids 1-212, beta 2-TF3 in Fig. 4) had the same effect and transformed this subunit into a permissive subunit for facilitation (average increase of 42 ± 13%, n = 3). Deletion of V3, the C-terminal variable sequence of the beta 2 subunit (amino acids 416-604, beta 2-TF4 in Fig. 4), however, had no effect, and currents recorded with or without pre-depolarization were almost indistinguishable (7 ± 3%, n = 6).


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Fig. 4.   L-type current facilitation recorded with truncated forms of the beta 2 subunit. The same recording conditions were used as those described in the legend to Fig. 3, but using truncated beta 2 subunits. Left panel, constructions used; middle panel, current traces (scale bars = 1 µA, except for beta 2-TF3, 500 nA); right panel, averaged facilitation, quantified as the ratio of the current amplitude with pre-depolarization divided by the control current amplitude. #, significantly different from the beta 2 subunit.

The voltage dependence of facilitation of these truncated subunits was characterized by using depolarization in the range of +60 to +140 mV. Such depolarizations induced an increase in Ba2+ currents recorded during a subsequent test pulse for alpha 1C subunit Ca2+ channels containing beta 1, beta 1-TF1, beta 1-TF3, or beta 2-TF2. As shown in Fig. 5 (A and B) for typical recordings, this increase rose with the amplitude of the conditioning depolarization, reaching a plateau for amplitude greater than +100 mV. The voltage dependence of this increase was similar for all these constructions (e fold changes for beta 1, beta 1-TF1, beta 1-TF3, and beta 2-TF2 were 45 ± 12 mV (n = 3), 23 ± 15 mV (n = 4), 52 ± 26 mV (n = 5), and 47 ± 27 (n = 3), respectively). Depolarization of the same amplitude was, however, without effect when the alpha 1C subunit was expressed with either beta 2 or beta 2-TF4 (Fig. 5B). The induction of facilitation by deletions of the N-terminal tail (V1 + C1 domains) of the beta 2 subunit strongly suggests (i) the existence of a conserved facilitatory sequence in the C2 domain of the beta  subunit, able to induce facilitation by both the beta 1 and beta 2 subunits and (ii) the presence, in the full-length beta 2 subunit, of an inhibitory sequence located in the first 128 amino acids. To test this hypothesis more directly, we constructed four chimeras based on the N-terminal truncated beta 1 subunits beta 1-TF1, beta 1-TF2, and beta 1-TF3, which were all able to induce facilitation (Fig. 3). The results are shown in Fig. 6. The average facilitation, recorded using the standard protocol, was 47 and 5% for the beta 1 and beta 2 subunits, respectively (Figs. 3, 4, and 6). In the first construct, the missing sequence of beta 1-TF3 (V1 + C1 + V2 domains) was replaced with the corresponding sequence of the beta 2 subunit (amino acids 1-212) (Fig. 2). Expression of this beta 2/beta 1-CH3 chimera with the alpha 1C subunit prevented the promotion of facilitation normally recorded with beta 1-TF3 (9 ± 1%, n = 3) (Fig. 6). The lack of facilitation recorded with the beta 2 subunit can therefore be transferred to the beta 1 subunit by addition of these amino acids, which confirmed the existence of an inhibitory sequence. In beta 2/beta 1-CH2, beta 2/beta 1-CH1, and beta 2/beta 1-CH4, the contribution of the beta 2 subunit to the total sequence of the chimera was further reduced. beta 2/beta 1-CH2 was deleted of an insert specifically found in the V2 domain of the beta 2 subunit (amino acids 170-212, black box in Fig. 6). beta 2/beta 1-CH1 and beta 2/beta 1-CH4 kept only the V1 + C1 and V1 sequences, respectively, from the beta 2 subunit, with the remaining sequence coming from the beta 1 subunit (Fig. 2). As shown in Fig. 6, none of these constructs was able to promote current facilitation in response to pre-depolarization. The extreme case is the beta 2/beta 1-CH4 chimera, where addition of the first 16 amino acids of beta 2 to beta 1 was able to completely block current facilitation (4 ± 5%, n = 10; not significantly different from the beta 2 subunit; see bottom of Fig. 6), suggesting that an inhibitory sequence was indeed present in these few amino acids.


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Fig. 5.   Voltage dependence of alpha 1C subunit current facilitation recorded with truncated beta 1 and beta 2 subunits. Current facilitation, quantified as described in the legend to Fig. 3, was recorded with prepulse (PP) amplitude varying from +60 to +140 mV (the protocol is shown on top of the figure). A, oocytes were injected with the alpha 1C and alpha 2-delta subunits and beta 1, beta 1-TF1, or beta 1-TF3. B, oocytes were injected with the alpha 1C and alpha 2-delta subunits and beta 2, beta 2-TF2, or beta 2-TF4. Note that facilitation of increasing amplitude was recorded with expression of beta 1, beta 1-TF1, beta 1-TF3, or beta 2-TF2, whereas prepulses of various amplitude were without effect on Ba2+ currents recorded upon coexpression of alpha 1C, alpha 2-delta , and beta 2 or beta 2-TF4.


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Fig. 6.   L-type current facilitation recorded with chimeras between the beta 1 and beta 2 subunits. The same recording conditions were used as those described in the legend to Fig. 3, but using chimeras between beta  subunits. Left panel, chimeric beta  subunits used; middle panel, current traces (scale bars = 1 µA); right panel, averaged facilitation, quantified as the ratio of the current amplitude with pre-depolarization divided by the control current amplitude. Facilitation was not observed when the first 16 amino acids of the beta 2 subunit were present (beta 2/beta 1-CH4). #, significantly different from the beta 1 subunit.

The primary sequence of this inhibitory segment of the beta 2 subunit is shown in Fig. 7 (top). The sequence is characterized by the presence of a short stretch of positively charged amino acids (boldface letters). To test the possible involvement of these charges in the inhibition of facilitation, we mutated these three arginines of the beta 2/beta 1-CH4 chimera to alanines. The mutated subunit, beta 2/beta 1-CH4M123, was then coexpressed with the alpha 1C subunit. As shown in Fig. 7, Ba2+ currents recorded from oocytes expressing this combination of mutated subunits could be increased by the application of conditioning depolarization (28 ± 5%, n = 7), reversing the effect of the insertion of the N-terminal tail of the beta 2 subunit (compare traces recorded with the non-mutated and mutated beta 2/beta 1-CH4 chimeras in Fig. 7; difference was statistically significant).


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Fig. 7.   Involvement of positive charges of beta 2/beta 1-CH4 in inhibition of current facilitation. Top, schematic showing the N-terminal sequences of the beta 2 subunit incorporated in the beta 2/beta 1-CH4 chimera. The three arginines that are mutated to alanine in beta 2/beta 1-CH4M123 are in boldface. Middle, current traces recorded when the non-mutated (left) and mutated (right) beta 2/beta 1-CH4 subunits were coexpressed with the alpha 1C subunit. Note that the replacement of the arginines allowed prepulse-induced facilitation. Facilitation is normally not observed with this chimera (scale bars = 1 µA). Bottom, average current facilitation recorded with beta 2/beta 1-CH4 and beta 2/beta 1-CH4M123. The prepulse lasted 200 ms and had an amplitude of +100 mV (test pulse of 400 ms at +10 mV). #, significantly different from beta 2/beta 1-CH4.

Although facilitation was a property specifically carried by the beta  subunit, participation of the alpha 1 subunit appeared also to be essential since this type of current potentiation was recorded only with the alpha 1C pore-forming subunit. In a first attempt to identify critical amino acids of the alpha 1C subunit involved in this regulation, we constructed two deletions in the alpha 1C subunit that have been reported to increase Ca2+ current amplitude (25, 26). alpha 1CDelta N had its first 29 amino acids removed, corresponding to the deletion made in the rabbit alpha 1C subunit (first 60 amino acids) (26). Similarly, alpha 1CDelta C had a deletion of amino acids 1706-2143, corresponding to deletion of the homologous residues 1733-2171 of the rabbit subunit (25). Our prediction was that these sequences could be involved in a tonic block of the Ca2+ channel activity that could be relieved either by deletion (25, 26) or by voltage-dependent interactions with beta  subunits, thus inducing current facilitation. We thus expressed these two truncated forms of the alpha 1C subunit with either the beta 1 or beta 2 subunit and tested their capacity to respond to conditioning pre-depolarization (Fig. 8). As expected, the amplitudes of the Ba2+ currents recorded with both alpha 1CDelta N and alpha 1CDelta C were systematically larger than those recorded with the full-length alpha 1C subunit (data not shown). However, deletion in neither the N-terminal nor C-terminal tail affected the response of these subunits to pre-depolarizations with the beta 1 or beta 2 subunit. Respective increases in Ba2+ current recorded with the beta 1 and beta 2 subunits were 80 ± 30% (n = 9) and 11 ± 11% (n = 11) for alpha 1CDelta N and 63 ± 36% (n = 3) and 11 ± 3% (n = 2) (9 and 14%) for alpha 1CDelta C. We therefore conclude that these two sequences, despite their role in modulating current amplitude, were not directly involved in the regulation of alpha 1C subunit voltage-dependent facilitation.


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Fig. 8.   N- and C-terminal deletions of the alpha 1C subunit have no effect on current facilitation. The same recording conditions were used as those described in the legend to Fig. 3, but using truncated alpha 1C subunits. Left panel: constructions used; middle panel, current traces (scale bars = 1 µA); right panel, averaged facilitation recorded with these deleted forms coexpressed with the beta 1 or beta 2 subunit. alpha 1CDelta N was made by removing the first 29 amino acids, whereas alpha 1CDelta C had the last 437 amino acids removed (amino acids 1706-2143).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Functional Diversity of Facilitation-- Our results show that neuronal L-type Ca2+ channel facilitation can be promoted or blocked by distinct short sequences of the ancillary beta  subunit. The lack of effect of acute injection of okadaic acid, the insensitivity to incoming Ca2+ ions (12), and the requirement for permissive beta 1 beta 3, and beta 4 subunits (13) clearly distinguished this type of facilitation from L-type facilitations previously characterized in cardiac, skeletal, and vascular cells (6-8,14-18) and suggest that different underlying mechanisms are involved. The presence or absence of these different types of facilitation with related L-type Ca2+ channels, such as, for example, the cardiac L-type Ca2+ channel, may depend not only on the type of auxiliary subunit associated with the alpha 1 subunit in a particular cell type, but also on specific kinase activities (6, 8, 17, 27) or expression of other associated proteins such as ryanodine receptors (for the L-type Ca2+ channel) (28, 29) or syntaxine (for the N-type Ca2+ channel) (30). All of these elements have been shown to be crucial for the normal development of specific forms of facilitation. Our results, however, emphasize the importance of the auxiliary beta  subunits for neuronal L-type facilitation and can explain the diversity of the response of the same alpha 1C subunit to conditioning depolarizations when recorded in cardiac and neuronal cells. Differentially spliced variants of the beta 2 subunit have been described in rat, rabbit, and human (23). At least three display important variations in their N-terminal V1 domains, suggesting that functional differences regarding L-type Ca2+ channel facilitation may exist. One consequence of the differential expression of these splicing variants could therefore be to finely tune the level of Ca2+ entry during sustained or repetitive depolarizations.

Molecular Mechanism of Neuronal alpha 1C Subunit Facilitation-- Although we have previously noted the importance of normal protein kinase A activity for facilitation (12), a mechanism involving voltage-dependent phosphorylation of the channel as a key step for the promotion of facilitation can be disregarded for the following reasons. First, we have demonstrated that ATPgamma S, AMP-PCP, and okadaic acid cannot stabilize or prevent L-type Ca2+ channel facilitation (12). Second, facilitation can be recorded on expression of the beta 1 subunit, but not the beta 2/beta 1-CH4 chimera, which retains the putative protein kinase A and C phosphorylation sites present in the beta 1 subunit (and of course in the alpha 1C subunit). Third, cardiac L-type Ca2+ channels, which are highly sensitive to protein kinase A phosphorylation (31), are completely resistant to this type of voltage-dependent facilitation (13). Altogether, these data suggest that facilitation requires a phosphorylated alpha 1C subunit, but do not support the existence of a voltage-dependent phosphorylation step, as described in skeletal muscle (7), in the pathway leading to current potentiation.

We have mapped two distinct regions in the sequence of the Ca2+ channel beta  subunit as critical for the promotion and inhibition of L-type Ca2+ channel facilitation (Fig. 9). The promoting sequence, localized in the C2 domain of beta 1, appears to be conserved on the four beta subunits, as shown by the capability of beta 3 and beta 4 (12, 13) and beta 2-TF2 (this work) to promote facilitation. This domain contains the beta  subunit interaction domain identified by De Waard et al. (32) and shown to be responsible for the beta  subunit-induced regulation of Ca2+ channel properties. Facilitation is, however, the first property demonstrated to be carried out by the beta  subunit, although it appears to be specific to the alpha 1C subunit. This suggests that the molecular consequences of beta  subunit binding are specific among the alpha 1 subunits. Among possible molecular mechanisms of facilitation, we have excluded the involvement of a voltage-dependent block of the channel by the amino- and carboxyl-terminal tails of the alpha 1C subunit, although their deletions have been shown to increase the L-type current amplitude (25). Another mechanism could be a prepulse-induced reactivation of inactivated channels since the same sequence that is shown here to block facilitation has already been shown to block voltage-dependent inactivation (33). Whether or not the three arginine residues that we have mutated and the chimeras used in this work also affect inactivation remains to be determined. Further experiments to test the effects of these on channel inactivation will be useful in clarifying this issue.


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Fig. 9.   Schematic drawing summarizing the results presented in this study. Shown is a schematic representation of the putative secondary structure of the alpha 1C and beta  subunits and their possible implication in L-type Ca2+ channel facilitation. Deletions of the N- and C-terminal tails of alpha 1C subunits have no effect on current facilitation. However, whereas the second conserved segment of the beta  subunit is necessary for facilitation (marked as Excitatory), the N-terminal tail (V1; see sequence on the figure) of the beta 2 subunit has a unique role in preventing current potentiation. The sequence of the V1 domain, with charged amino acids in boldface, is shown on the bottom of the figure.

The inhibitory sequence is found only in the beta 2 subunit. The lack of current facilitation produced by coexpression of the beta 2/beta 1-CH4 chimera suggests that specific interactions with the first few amino acids of the beta 2 subunit are important for the mechanism by which facilitation is blocked. Furthermore, the abolition of the effect by the mutation of the three arginines located in this short sequence suggests that electrostatic interactions may be of importance for this inhibition. The absence of facilitation recorded with beta 2-TF1 is worth noting and could also suggest the existence of another inhibitory sequence within the C1 domain of the beta 2 subunit. At this point, however, we are not able to precisely locate the inhibitory binding site(s) of these sequences on the alpha 1C or beta 2 subunit. On-going experiments, using chimeras between alpha 1 subunits permissive and nonpermissive for facilitation, should allow us to further define this binding site and to precisely determine the molecular mechanism of this form of neuronal L-type current facilitation. Whether the same sequences of the beta 2 subunit are also important for other types of Ca2+ channel facilitation, such as G-protein-dependent N-type Ca2+ channel facilitation, remains to be determined.

    ACKNOWLEDGEMENTS

We thank Drs. T. Snutch and E. Perez Reyes for kindly providing calcium channel alpha 1C and beta  subunit cDNAs; Drs. D. Fisher, P. F. Mery, and J. P. Pin for helpful comments on the manuscript; and Drs. S. Galas, J. C. Labbe, T. Lorca, and N. Morin for technical expertise in in vitro translation.

    FOOTNOTES

* This work was supported in part by CNRS, the Association Française contre les Myopathies, Association pour la Recherche contre le Cancer Grant ARC 9115, the Ligue Nationale contre le Cancer, and the Fondation pour la Recherche Médicale.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 Supported by the Ministère Education Nationale Recherche Enseignement Supérieur, the Association Française contre les Myopathies, and the Association pour la Recherche contre le Cancer.

§ To whom correspondence should be addressed. Tel.: 33-67-61-33-52; Fax: 33-67-52-15-59; E-mail: charnet{at}xerxes.crbm.cnrs-mop.fr.

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

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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