Molecular Structures Involved in L-type Calcium Channel Inactivation
ROLE OF THE CARBOXYL-TERMINAL REGION ENCODED BY EXONS 40-42 IN alpha 1C SUBUNIT IN THE KINETICS AND Ca2+ DEPENDENCE OF INACTIVATION*

(Received for publication, September 10, 1996, and in revised form, November 14, 1996)

Nikolai M. Soldatov Dagger §, Roger D. Zühlke Dagger , Alexandre Bouron and Harald Reuter

From the Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The pore-forming alpha 1C subunit is the principal component of the voltage-sensitive L-type Ca2+ channel. It has a long cytoplasmic carboxyl-terminal tail playing a critical role in channel gating. The expression of alpha 1C subunits is characterized by alternative splicing, which generates its multiple isoforms. cDNA cloning points to a diversity of human hippocampus alpha 1C transcripts in the region of exons 40-43 that encode a part of the 662-amino acid carboxyl terminus. We compared electrophysiological properties of the well defined 2138-amino acid alpha 1C,77 channel isoform with two splice variants, alpha 1C,72 and alpha 1C,86. They contain alterations in the carboxyl terminus due to alternative splicing of exons 40-42. The 2157-amino acid alpha 1C,72 isoform contains an insertion of 19 amino acids at position 1575. The 2139-amino acid alpha 1C,86 has 80 amino acids replaced in positions 1572-1651 of alpha 1C,77 by a non-identical sequence of 81 amino acids. When expressed in Xenopus oocytes, all three splice variants retained high sensitivity toward dihydropyridine blockers but showed large differences in gating properties. Unlike alpha 1C,77 and alpha 1C,72, Ba2+ currents (IBa) through alpha 1C,86 inactivated 8-10 times faster at +20 mV, and its inactivation rate was strongly voltage-dependent. Compared to alpha 1C,77, the inactivation curves of IBa through alpha 1C,86 and alpha 1C,72 channels were shifted toward more negative voltages by 11 and 6 mV, respectively. Unlike alpha 1C,77 and alpha 1C,72, the alpha 1C,86 channel lacks a Ca2+-dependent component of inactivation. Thus the segment 1572-1651 of the cytoplasmic tail of alpha 1C is critical for the kinetics as well as for the Ca2+ and voltage dependence of L-type Ca2+ channel gating.


INTRODUCTION

DHP1-sensitive Ca2+ channels of class C (1) are voltage-gated channels, which start to open at membrane voltages more positive than -40 mV and slowly inactivate if Ba2+ is the charge carrier. Inactivation is usually greatly accelerated if Ba2+ is replaced by Ca2+ (2). The channels are also designated as L-type and are multisubunit proteins composed of the pore-forming alpha 1C subunit, which contains high affinity binding sites for DHPs (3-7), and of the auxiliary beta  and alpha 2delta subunits (8, 9). Analysis of the hydrophobicity profile of alpha 1C indicates four repetitive motifs of similarity (I-IV), each composed of six transmembrane segments (S1-S6) (10). Both, the short amino-terminal tail encoded by exons 1 and 2, and the long carboxyl-terminal tail encoded by exons 38-50 of the human alpha 1C gene (11) are located in the cytoplasm.

Expression of alpha 1C is regulated through alternative splicing (12), which has primarily been detected in the membrane-spanning regions of the molecule. However, there is evidence that the carboxyl-terminal tail is also affected by alternative splicing. Two partial transcripts have been identified in a cDNA library of human hippocampus (11, 13). They show that exons 40-43 encoding the second quarter of the putative cytoplasmic tail of the alpha 1C molecule are subject to alternative splicing and may give rise to new alpha 1C splice variants in the brain.

The functional role of the carboxyl-terminal tail attracts much attention because of its potential involvement in channel gating. Removal of approximately 70% of the tail causes an increase in the opening probability of the rabbit cardiac alpha 1C channel (14). A similar deletion mutant of the human cardiac alpha 1C showed faster inactivation of the channel as compared to the wild-type channel (15). It has been concluded that this tail part of alpha 1C may serve as a critical component of the gating structure that influences inactivation properties of the channel (15).

In this report we describe two recombinant plasmids, pHLCC72 and pHLCC86, which contain alternative exons encoding parts of the carboxyl-terminal tails that are found in human hippocampus transcripts. After expression in Xenopus oocytes, we have analyzed electrophysiological properties of alpha 1C,72 and alpha 1C,86 channels and compared them with the reference alpha 1C,77 channel (16). The results of our study show that amino acids encoded by exons 40-42 are important for the voltage dependence of activation and inactivation of the current through these channels, as well as for the kinetics and Ca2+ dependence of inactivation.


MATERIALS AND METHODS

Preparation of cDNAs Encoding alpha 1C Subunit Splice Variants

All splice variants were constructed within the frame of pHLCC77 (16) composed of exons 1-20, 22-30, 32-44, and 46-50 using the pBluescript SK(-) vector (Stratagene) flanked at the 5'-end with HindIII/BglII and at the 3'-end with BglII/BamHI fragments of the Xenopus beta -globin gene untranslated region sequences, respectively (17, 18). The recombinant plasmid pHLCC86 was prepared by replacing nucleotides 5104-5482 of pHLCC77, encoding exons 41 and 42, with the BsaI/BglII fragment of the coding frame of h54 cDNA, containing exons 40A and 40B and an upstream region of exon 43A (11). The recombinant plasmid pHLCC72 was constructed by substituting the SfuI (3726)/ScaI (5348) fragment of pHLCC77 with the respective fragment of h2.05 cDNA (11, 13), containing exon 41A with its 57-nt extension in the upstream direction. Nucleotide sequences of all obtained cDNAs were verified by the modified dideoxy termination method (19).

Template DNAs were linearized by digestion with BamHI (pHLCC77 and pHLCC86) or NotI (pHLCC72), and capped transcripts were synthesized in vitro with T7 RNA polymerase using the mRNA cap kit (Stratagene).

Expression of Ca2+ Channels in Xenopus Oocytes

Xenopus laevis oocytes were defolliculated 1 day before injection (20). cRNA samples were dissolved in 5 mM HEPES, pH 6.8, and oocytes were injected with 50-100 nl of a mixture containing cRNAs (0.5 µg/µl) for an alpha 1C splice variant, and for alpha 2delta (21) and beta 1 (22, 23) subunits in equimolar ratio. In some experiments beta 2A or beta 3 (24) subunits instead of beta 1 were used. Injected oocytes were stored for 5-8 days at 18 °C in sterile Barth's medium supplemented with 100 units of penicillin/ml and 100 µg of streptomycin/ml (Boehringer Mannheim, Rotkreuz, Switzerland). The medium was changed daily. Whole-cell Ba2+ currents (IBa) were recorded by a two-electrode voltage clamp method using an Axoclamp 2-A amplifier (Axon Instruments, Burlingame, CA) or a Warner Oocyte Clamp OC-725C (Warner Instrument Corp., Hamden, CT). Glass pipettes (Clark Electromedical Instruments, United Kingdom) were filled with 3 M CsCl and had resistances between 0.2 and 1 megohms. Throughout the experiments oocytes were continuously superfused at 5-15 ml/min. The Ba2+ bathing solution contained (in mM): Ba(OH)2 40, NaOH 50, KOH 1, HEPES 10 (pH 7.4 with methanesulfonic acid). Isradipine-containing solutions were prepared freshly from a stock solution.

In some experiments Ca2+ was used as charge carrier through the channels. One to 5 h prior to the recording of Ca2+ currents (ICa), oocytes were injected with 50 nl of a BAPTA solution containing 40 mM Na4-BAPTA and 10 mM HEPES (pH 7 with KOH). The bathing solution contained (in mM): Ca(NO3)2 40, NaOH 50, KOH 1, HEPES 10 (pH 7.4 with methanesulfonic acid).

Voltage-clamp commands, current recordings and leak current subtraction were performed by means of the EPC software (Cambridge Electronic Design, Cambridge, UK). The EPC software analysis module, the KaleidaGraph software (Abelbeck, Reading, CA), and the FigP software (Biosoft, Ferguson, MO) were used for the data analysis. Statistical values are given as means ± S.E. Membrane currents, filtered at 0.5-1 KHz and sampled at 2 KHz, were triggered by 0.25- or 1-s step depolarizations applied from Vh of -90 mV at a frequency of 0.033 Hz. All experiments were performed at room temperature (20-22 °C).

Inactivation characteristics of IBa through the three alpha 1C splice variants were measured with 2-s conditioning pre-pulses. An increase of the duration of conditioning pre-pulses from 2 s to 20 s produced, in all tested alpha 1C splice variants, an additional shift of the inactivation curves by 6 ± 2 mV toward more negative potentials without changes in their steepness, indicating that a steady state had not been reached with 2-s pre-pulses. However, the long pre-pulses were poorly tolerated by many oocytes; therefore, all inactivation curves reported in this paper have been obtained with a 2-s pre-pulse protocol and consequently are called "isochronic" inactivation curves.

To compare the sensitivities of alpha 1C splice variants toward DHPs, we have measured the fractional inhibition of IBa at Vh -90 mV by different concentrations of (+)-isradipine ranging from 10 nM to 1 µM. After application of isradipine, IBa was monitored at 30-s intervals until an equilibrium of the inhibition was reached. In these experiments endogenous, DHP-insensitive Ca2+ or Ba2+ currents (20) were not subtracted from recorded peak IBa amplitudes.


RESULTS

Structural Features of the Studied Splice Variants

We have compared electrophysiological properties of human alpha 1C splice variants: alpha 1C,77 and two of its homologues, alpha 1C,72 and alpha 1C,86. Both homologues contain substitutions in the region of exons 40-42 encoding the second quarter of the putative cytoplasmic tail of alpha 1C (Fig. 1, upper panel, see diagram). The recombinant plasmids for alpha 1C,72 (pHLCC72) and for alpha 1C,86 (pHLCC86) were prepared by incorporation of partial cDNA clones (h2.05 and h54) into the nucleotide sequence of pHLCC77. These partial clones have been isolated earlier from the human hippocampus cDNA library (11, 13) and proved to be products of alternative splicing of one and the same alpha 1C gene (11). The nucleotide sequence of exon 41 in pHLCC72 is extended by 57 nt in the upstream direction and thus produces an insertion of 19 residues into the amino acid sequence of the alpha 1C,72 channel at position 1575. In pHLCC86, 17 nt are deleted from the 3'-end of exon 40, the 102-nt exon 41 is replaced by the 118-nt exon 40B, and the 128-nt exon 42 is replaced by a 132-nt extension of exon 43 in the upward direction. Thus, 247 nt of the original pHLCC77 cDNA are replaced in pHLCC86 by 250 nt of a new coding sequence. At the amino acid level, this results in the replacement of 80 amino acid residues (1572-1651) of alpha 1C,77 with 81 essentially non-identical amino acids in alpha 1C,86 (Fig. 1). Amino acid alignments of the variable parts of the constructs have already been published (11). Because of this long stretch of non-identical amino acids, no mutagenesis has been attempted so far. All other parts of the recombinant channels studied in this work were unchanged.


Fig. 1. Electrophysiological properties and location of variable parts of three alpha 1C splice variants. Schematic diagrams (upper panel) show the arrangement of exons, numbered according to Ref. 10, in three alpha 1C splice variants. Exons incorporated into the coding sequences are shown in bold lines. Deleted exons are shown in light lines. Shaded boxes point to exons found in human hippocampus transcripts. The inset shows the test pulse protocol for the traces of IBa through alpha 1C,86 (A), alpha 1C,72 (B), and alpha 1C,77 (C). Averaged isochronic inactivation curves (12-16 experiments) of IBa through alpha 1C,86 (D), alpha 1C,72 (E), and alpha 1C,77 (F) were obtained with 2-s conditioning pre-pulses (protocol not shown). The equation for fitting the data is given in Table II. G-I, averaged current-voltage relationships (10-18 experiments) of IBa through alpha 1C,86, alpha 1C,72, and alpha 1C,77. Test pulses were applied at 30-s intervals. The equation for fitting the data is given in Table III. Vh = -90 mV. Pore-forming alpha 1C subunits were co-expressed with auxiliary beta 1 and alpha 2delta subunits at a 1:1:1 molar ratio.
[View Larger Version of this Image (32K GIF file)]


None of the studied splice variants has yet been shown to be expressed in the brain. The alpha 1C,86 channel is an "artificial" splice variant of the human alpha 1C. The partial cDNA clone h54 used to construct pHLCC86 shows further variability due to alternative splicing downstream of exon 43, which was not incorporated into the recombinant plasmid. A full-size transcript has not yet been cloned. Moreover, a fragment of an intron upstream of exon 40A indicates that h54 is not a part of a functional transcript but rather a product of post-transcriptional processing of the alpha 1C mRNA. However, both alpha 1C,72 and alpha 1C,86 showed a number of new characteristics pointing to an involvement of sequences encoded by exons 40-42 in important gating properties of the channel.

Differences between alpha 1C,77, alpha 1C,72, and alpha 1C,86 in Inactivation, Current-Voltage Relations, and Sensitivity to DHP Blockers

When cRNAs for alpha 1C,77, alpha 1C,72, or alpha 1C,86 were co-injected into Xenopus oocytes with cRNAs for auxiliary alpha 2delta (21) and beta 1 subunits (22, 23), they gave rise to functional Ca2+ channels with significantly different electrophysiological properties. Fig. 1 (A-C) show traces of IBa through splice variants of the pore-forming alpha 1C subunit recorded in response to depolarizing voltage clamp steps to +20 mV (1 s) from Vh = -90 mV. The inactivation kinetics of IBa through alpha 1C,86 was much faster than that through alpha 1C,77 and alpha 1C,72 channels. A direct comparison of time constants (tau ) of inactivation obtained from exponential fits of the current traces is shown in Table I. In the case of alpha 1C,77 and alpha 1C,72, the kinetics of the IBa decay was fitted best by a single-exponential function. For alpha 1C,86 an exponential fit indicated two time constants, where the slow time constant, tau s, was approximately 4 times that of the fast time constant, tau f (Table I). Subtraction of the DHP-insensitive, endogenous IBa did not change significantly the absolute tau  values. With beta 1 co-expressed, the fast component of the inactivation phase of IBa through alpha 1C,86 comprised 84.6 ± 1.3% (n = 12) of the total current recorded with a 1-s pulse, while the slow component was 15.4 ± 1.3% (n = 12) (Table I). The slow component of IBa through alpha 1C,86 was still significantly faster than that through alpha 1C,77 or alpha 1C,72 channels (Table I).

Table I.

Dependence of the kinetics of the IBa decay on the type of co-expressed alpha 1C and beta  subunits

Inactivation time constants, tau , of IBa were determined by test pulses to +20 mV from Vh = -90 mV. For alpha 1C,72 and alpha 1C,77 channels, tau  was obtained from a single-exponential equation: I(t) = Iinfinity  + I·exp(-t/tau ), where Iinfinity is the steady state amplitude of the current and I is the amplitude of the initial current. The best fit for the current through the alpha 1C,86 channel was obtained by a bi-exponential equation: I(t) = Iinfinity  + If·exp(-t/tau f) Is·exp(-t/tau s), where f and s stand for fast and slow components, respectively. IBa fractions refer to the respective contributions of fast and slow components to the total current through alpha 1C,86. In all cases the subunit composition of the analyzed channels was alpha 1C:beta :alpha 2delta (1:1:1, moles). n = number of tested oocytes. *, p < 0.05 compared to the respective alpha 1C,77 channel (one-way ANOVA and Tukey test).
 alpha 1C subunit (tau )  beta subunit  tau IBa fractions n

ms %
 alpha 1C,77  beta 1 484  ± 22 13
 beta 2A 590  ± 62 5
 beta 3 1,341  ± 144 2
 alpha 1C,72  beta 1 382  ± 13* 14
 beta 2A 721  ± 90 4
 alpha 1C,86 (fast component)  beta 1 47.5  ± 2.6* 84.6  ± 1.3 12
 beta 2A 71.3  ± 6.7* 70.2  ± 7.7 2
 beta 3 55.8  ± 3.9* 79.4  ± 2.6 2
 alpha 1C,86 (slow component)  beta 1 210.5  ± 10.2* 15.4  ± 1.3 12
 beta 2A 272.2  ± 6.8* 29.8  ± 7.7 2
 beta 3 202.1  ± 7.6* 20.6  ± 2.6 2

The time constants of inactivation of IBa through alpha 1C,77, alpha 1C,72 and alpha 1C,86 showed different voltage dependences (Fig. 2A). In the case of alpha 1C,77, the time constant of inactivation of IBa decreased only slightly from tau (0) = 455 ± 38 ms at 0 mV to tau (+40) = 347 ± 18 ms at +40 mV (n = 15), i.e. by a factor of 1.3 (Fig. 2, B and C). Similarly, only a small voltage dependence of inactivation time constants was observed for alpha 1C,72. In the alpha 1C,86 channel, however, a more than 3.5-fold decrease of the fast inactivation time constant, from tau (0) = 113 ± 14 ms (n = 10) to tau (+40) = 29 ± 1 ms (n = 14) was measured (Fig. 2, B and C).


Fig. 2. Dependence of IBa inactivation kinetics on membrane potential. A, traces of IBa recorded at 0, +20, and +40 mV. The voltage dependence of the inactivation time constant (tau ) was determined by fitting current traces of IBa in the range of 0 to +40 mV with exponential functions (B). Values of tau  for alpha 1C,72 (bullet , n = 10) and alpha 1C,77 (open circle , n = 15) were determined by mono-exponential fitting. A bi-exponential approximation was used to obtain tau  values for alpha 1C,86; only the fast component has been plotted in B and C (square , n = 10-14). To illustrate differences in the voltage dependence of tau  for the three alpha 1C splice variants, the values of tau  at each potential were normalized with respect to tau  at +40 mV (C). The subunit composition of the analyzed channels was alpha 1C:beta 1:alpha 2delta (1:1:1, mol).
[View Larger Version of this Image (18K GIF file)]


To characterize further the inactivation properties of the three splice variants of alpha 1C, we examined the rate of recovery of IBa from inactivation. Fig. 3 shows the ratio of maximum amplitudes of IBa elicited by two consecutive test pulses with different intervals. The duration of the first pulse was 0.4, 2, or 3 s for alpha 1C,86, alpha 1C,72, and alpha 1C,77, respectively, a time required to reach 80-90% of inactivation of the currents through these channels. The second pulse lasted 400 ms. In Fig. 3 the ratios of IBa at pulse 2 divided by IBa at pulse 1 are plotted as function of the time intervals between the two pulses. This represents the fractional recovery of IBa from inactivation. Only the initial phase of recovery of IBa from inactivation could be fitted with a single-exponential function. This phase had approximately the same time constant for all three alpha 1C splice variants (Fig. 3). However, IBa through the alpha 1C,86 channel reached full recovery much faster than IBa through alpha 1C,72 and alpha 1C,77. At the 0.15-s interval between pulses, when 93 ± 1% (n = 4) of IBa through alpha 1C,86 had recovered, only 57 ± 2% of IBa through alpha 1C,72 and 56 ± 2% for alpha 1C,77 were available. With 16-s intervals between pulses, all measured IBa had almost completely recovered from inactivation.


Fig. 3. Effect of alpha 1C subunit structures on the recovery of IBa from inactivation. Recovery of IBa through alpha 1C,86 (square ), alpha 1C,72 (open circle ), and alpha 1C,77 (bullet ) was measured with +20 mV pre-pulses of 0.4, 2, and 3 s in duration, respectively, and 0.4-s test pulses, both applied from Vh = -90 mV. The different pre-pulse durations for alpha 1C,86, alpha 1C,72, and alpha 1C,77 were necessary to achieve 80-90% inactivation of IBa through each channel. Pre-pulses and test pulses were separated by increasing intervals. Smooth lines represent fits of the mean data by single exponentials with time constants tau  = 27.2 ± 0.9 ms (alpha 1C,77), tau  = 30.0 ± 1.6 ms (alpha 1C,72), tau  = 24.0 ± 2.1 ms (alpha 1C,86) (n = 4). The pore-forming alpha 1C subunit was co-expressed with auxiliary beta 1 and alpha 2delta subunits at equimolar ratio.
[View Larger Version of this Image (16K GIF file)]


As reported previously (21, 24), auxiliary beta -subunits affect, among other properties, the kinetics of the Ca2+ channel current. We have found that beta 1, beta 2A or beta 3 subunits, when co-expressed with alpha 2delta and the splice variants of alpha 1C subunits, caused modulatory effects on the inactivation kinetics of IBa, which, however, were smaller than the differences between alpha 1C,86 and alpha 1C,77 or alpha 1C,72 (Table I).

Alternative splicing of exons 40-42 affects gating properties of the channel. Table II and Fig. 1 (D-F) show isochronic (2-s pre-pulses) inactivation characteristics of IBa through the three alpha 1C splice variants. Isochronic inactivation curves were shifted toward negative potentials by 5 mV (alpha 1C,72) and 11 mV (alpha 1C,86) with respect to that of alpha 1C,77 (Fig. 1, D-F; Table II, see V0.5 values). The slopes of isochronic inactivation curves were less steep for alpha 1C,86 and alpha 1C,72 channels than for alpha 1C,77 (Table II). Thus, cooperativity in the mechanism leading to inactivation of IBa may be different for alpha 1C,86 and alpha 1C,72 than for alpha 1C,77.

Table II.

Dependence of isochronic inactivation curves for IBa on alpha 1C and beta  subunits

Isochronic inactivation curves were measured using a two-step voltage clamp protocol. A 2-s conditioning pre-pulse was applied from Vh = -90 mV (10-mV increments up to +40 mV) followed by a 1-s test pulse to +20 mV. The intervals between each cycle were 30 s. Recorded peak current amplitudes were normalized to the maximum value determined in the range -60 to +20 mV. Isochronic inactivation curves were fitted by a Boltzmann function: IBa = 1/{1 + exp[(V - V0.5)/k]}, where V is the conditioning pre-pulse voltage, V0.5 is the voltage at half-maximum of inactivation, and k is a slope factor. n = number of tested oocytes. *, p < 0.05 compared to the respective alpha 1C,77 channel (one-way ANOVA and Tukey test).
 alpha 1C subunit  beta subunit V0.5 Slope n

mV
 alpha 1C,77  beta 1  -8.4  ± 1.1 5.7  ± 0.3 16
 beta 2A  -9.7  ± 1.4 7.3  ± 0.4 5
 beta 3  -9.1  ± 2.5 5.4  ± 0.1 3
 alpha 1C,72  beta 1  -13.6  ± 0.7* 8.6  ± 0.3* 12
 beta 2A  -11.8  ± 0.2 7.5  ± 0.3 2
 alpha 1C,86  beta 1  -19.9  ± 0.5* 7.8  ± 0.2* 13
 beta 2A  -21.4  ± 0.8* 7.7  ± 0.1 2
 beta 3  -16.8  ± 0.9 8.7  ± 0.1* 2

Current-voltage relationships also point to differences in the voltage dependence of IBa through alpha 1C,77 as compared to the other two splice variants (Table III, Fig. 1, G-I). In contrast to the negative shift of the inactivation curves of IBa through alpha 1C,72 and alpha 1C,86, their values for half-maximal activation were shifted toward more positive potentials by 6 mV and 11 mV, respectively (Fig. 1, G-I, and Table III). These data suggest that structural changes produced by alternative splicing of exons 40-42 in alpha 1C influence the voltage sensors of the channels for activation and inactivation in different ways. Since the reversal potentials of the current flowing through alpha 1C,77, alpha 1C,72, and alpha 1C,86 channels are not significantly different (Table III), the pore region determining the selectivity of the channel is probably the same in the studied splice variants (25).

Table III.

Dependence of parameters of current-voltage relationships on alpha 1C and beta  subunits

IBa was measured with 30 s intervals between 1-s test pulses in the range of -40 to +100 mV (10-mV increments) applied from Vh = -90 mV. In all cases the subunit composition of the analyzed channels was alpha 1C:beta :alpha 2delta (1:1:1, moles). I-V curves were fitted by IBa = Gmax (V - Erev)/{1 + exp[(V - V0.5)/kI-V]}, where Gmax = maximum conductance, Erev = reversal potential, V0.5 = voltage at 50% of IBa activation, and kI-V = slope factor. n = number of tested oocytes. Inhibition of IBa by isradipine (IC50) has been measured at a test potential of +20 mV from a Vh = -90 mV without correction for endogenous DHP-insensitive IBa. *, p < 0.05 compared to the respective alpha 1C,77 channel (one-way ANOVA and Tukey test).
Subunit
IBa(max) Erev V0.5 kI-V n IC50 for isradipine (n)
 alpha 1C  beta

µA mV mV nM
 alpha 1C,77  beta 1 1.47  ± 0.14 59.8  ± 0.9  -0.3  ± 1.1  -5.6  ± 0.3 18 146.2  ± 12.2 (34)
 beta 2A 0.58  ± 0.11 58.0  ± 0.7 2.2  ± 1.0  -7.0  ± 0.4 4
 beta 3 1.13  ± 0.02 59.3  ± 0.2 0.4  ± 1.1  -4.8  ± 0.2 2
 alpha 1C,72  beta 1 0.99  ± 0.12 60.3  ± 0.8 5.7  ± 2.0  -6.6  ± 0.3 12 39.8  ± 6.2 (10)*
 beta 2A 1.23  ± 0.26 63.7  ± 2.3 5.9  ± 5.7  -6.5  ± 0.6 3
 alpha 1C,86  beta 1 0.78  ± 0.15* 62.5  ± 1.2 11.5  ± 1.8*  -7.5  ± 0.3* 10 37.7  ± 5.1 (9)*
 beta 2A 1.04  ± 0.54 63.2  ± 4.9 8.0  ± 1.0  -7.1  ± 0.9 2
 beta 3 0.60  ± 0.03 63.0  ± 0.5 15.8  ± 0.7  -9.0  ± 0.1* 2

All three splice variants retain a high affinity for DHP blockers. When measured at Vh = -90 mV, the IC50 value for (+)-isradipine inhibition of IBa through alpha 1C,77 is about 3.5 times higher than those for the other splice variants (Table III).

Differences between Splice Variants in Ca2+-dependent Inactivation

Besides voltage-dependent inactivation, many L-type Ca2+ channels exhibit Ca2+-dependent inactivation (2). This latter mode of inactivation has also been shown for heterologously expressed L-type Ca2+ channels (26-28). In view of the marked kinetic differences in inactivation between alpha 1C,86, alpha 1C,77, and alpha 1C,72, we studied their respective Ca2+-dependent inactivation properties.

To buffer intracellular Ca2+ ions and to minimize contaminating Ca2+-dependent Cl- currents, 50 nl of 40 mM BAPTA solution were injected into the oocytes prior to the recordings. The BAPTA injection did not affect properties of IBa. However, it could have reduced the response to Ca2+ of Ca2+-dependent inactivation, although Neely et al. (26) have shown that the time course of Ca2+-dependent inactivation remains virtually unchanged over a 20-fold range of buffering capacity. In Fig. 4, representative current traces recorded from oocytes during superfusion with 40 mM Ba2+ solution and after switching to 40 mM Ca2+ solution were superimposed. Ca2+ current (ICa) amplitudes were much smaller in all three channels than IBa amplitudes. This is consistent with a lower conductance for Ca2+ than for Ba2+ ions of L-type calcium channels (29). The reduction was less pronounced in alpha 1C,86 compared to alpha 1C,77 and alpha 1C,72. The accelerated inactivation rate seen in alpha 1C,77 and alpha 1C,72, when Ca2+ was the charge carrier, was absent in alpha 1C,86. This is illustrated in Fig. 4B, where peak ICa has been scaled up to the level of peak IBa. The scaling factors for alpha 1C,77, alpha 1C,72, and alpha 1C,86 were 3.3, 2.9, and 1.8, respectively. In contrast to IBa inactivation kinetics of alpha 1C,77 and alpha 1C,72, ICa kinetics could not be fitted by a single exponential. With a bi-exponential fitting procedure, at +20 mV the fast time constants, tau f, of ICa inactivation were 27.7 ± 1.9 ms (n = 7) and 34.4 ± 5.5 ms (n = 4) for alpha 1C,77 and alpha 1C,72, respectively. The IBa inactivation time constants were 398.7 ± 39.6 ms (n = 7) and 348.1 ± 22.1 ms (n = 7) in these experiments. Thus, an acceleration of the inactivation kinetics by a factor of 13 and 10 was observed if Ca2+ ions were the charge carriers through alpha 1C,77 and alpha 1C,72. By contrast, inactivation kinetics of alpha 1C,86 were only slightly influenced by Ca2+ ions. The time constant, tau f, observed at +20 mV in Ca2+ containing solution was 78.2 ± 8.0 ms (n = 13) compared to 59.0 ± 3.0 ms (n = 10) in Ba2+. The apparent slowing of the inactivation kinetics of alpha 1C,86 by Ca2+ ions could be explained by a different surface potential with Ca2+ ions in the solution (30). This is also indicated by a slight shift toward more positive potentials of the current-voltage curve of all three calcium channel constructs when switching from Ba2+ to Ca2+ solution (Fig. 5, B-D, filled triangles).


Fig. 4. Ca2+-dependent inactivation occurs in alpha 1C,72 and alpha 1C,77 but not in alpha 1C,86. IBa and ICa through alpha 1C,77, alpha 1C,72, and alpha 1C,86 were recorded in Xenopus oocytes after injection of 50 nl of 40 mM BAPTA. Current traces were evoked by 400-ms depolarizing steps from Vh = -90 mV to +20 mV. The oocytes were superfused by a bath solution containing 40 mM Ba2+ or 40 mM Ca2+. A, IBa and ICa recorded from the same oocyte expressing either alpha 1C,77, alpha 1C,72, or alpha 1C,86. B, ICa traces from panel A normalized to peak IBa show that Ca2+-dependent inactivation is present in alpha 1C,77 and alpha 1C,72 but absent in alpha 1C,86. The individual alpha 1C subunits were co-expressed with auxiliary beta 1 and alpha 2delta subunits in an equimolar ratio.
[View Larger Version of this Image (15K GIF file)]



Fig. 5. Ca2+-dependent inactivation in alpha 1C,72 and alpha 1C,77 requires Ca2+ influx. IBa and ICa in BAPTA-injected oocytes were elicited by a double-pulse voltage protocol (A). 400-ms depolarizing pre-pulses, applied in 10-mV increments from -90 mV to voltages ranging from -40 mV to +80 mV were followed by a 400-ms test pulse to +20 mV after a 50-ms pulse interval at -90 mV. Representative current traces through alpha 1C,77 recorded from an oocyte superfused sequentially by 40 mM Ba2+ or 40 mM Ca2+ solutions are shown underneath the pulse protocol for pre-pulse potentials of -40, +20, and +80 mV. B-D, peak current amplitudes at pre-pulses (black-triangle) and test-pulses (triangle ) were normalized to the pre-pulse peak current at +20 mV and plotted versus the pre-pulse potential. The double-pulse protocol was applied to oocytes expressing alpha 1C,77 (B, n = 4), alpha 1C,72 (C, n = 4), and alpha 1C,86 (D, n = 7) with 40 mM Ba2+ (left panel) and 40 mM Ca2+ (right panel) as charge carrier. The individual alpha 1C subunits were co-expressed with auxiliary beta 1 and alpha 2delta subunits in an equimolar ratio.
[View Larger Version of this Image (34K GIF file)]


Ca2+-induced inactivation is dependent upon the size of Ca2+ influx through the channel pore (2). This can be studied by applying a double-pulse protocol as shown in Fig. 5A (upper panel). The duration of the pre-pulse was 400 ms. A pulse interval of 50 ms was chosen, which was long enough to minimize incomplete recovery from partial inactivation during the pulse intervals. The test pulse was always to +20 mV and also lasted 400 ms. The interval between cycles was 30 s. Representative current traces for alpha 1C,77 at the pre-pulse potentials -40 mV, +20 mV, and +80 mV for IBa (middle panel) and ICa (bottom panel) are shown in Fig. 5A. In Ba2+ solution, increasing pre-pulse potentials led to a persistent reduction (23.1%) of IBa through alpha 1C,77 at the test pulse (Fig. 5, A and B). This is due to incomplete recovery from partial inactivation under these experimental conditions (data not shown). By contrast, with Ca2+ as charge carrier, the test pulse current (ITP) exhibited a bell-shaped relation as a function of the pre-pulse potential (Fig. 5B). It was inversely related to the current amplitudes at the pre-pulse potentials (IPP). The maximal current reduction of ITP in Ca2+ solution was 53.5%, and became less with further depolarization during pre-pulses (Fig. 5, A and B). This is a strong indication for Ca2+-dependent inactivation triggered by Ca2+ influx through alpha 1C,77 channels. Application of the same protocol to alpha 1C,72 (Fig. 5C) resulted in a similar relationship between IPP and ITP as with alpha 1C,77. The maximal current reductions were 15.9% in Ba2+ solution and 48.9% in Ca2+ solution. However, for alpha 1C,86 (Fig. 5D) the relationships between IPP and ITP in Ba2+ and Ca2+ solutions (maximal current reductions were 22.8% and 24.3%, respectively) were almost identical and comparable to those obtained with alpha 1C,77 and alpha 1C,72 in Ba2+ solution. This provides further evidence that alpha 1C,86 lacks Ca2+-dependent inactivation.


DISCUSSION

Alternative splicing of the alpha 1 subunit of voltage-dependent Ca2+ channels contributes to the structural diversity of these ion channels, but only little is known about its functional importance. It has been shown that alternative splicing of the gene encoding the alpha 1C subunit of L-type Ca2+ channels contributes to differences in the voltage dependence of the sensitivity toward DHPs (16) and to the DHP tissue selectivity (31). In this study we have investigated electrophysiologically three putative splice variants of the human class C L-type Ca2+ channel.

We show that a segment of 80 amino acids replaced in alpha 1C,77 by a nonidentical sequence of 81 amino acids of alpha 1C,86 in the second quarter of the 662-amino acid carboxyl-terminal tail (1572-1651) caused a 10-fold increase in the rate of inactivation, an 11-mV hyperpolarizing shift in the voltage dependence of inactivation, and elimination of Ca2+-dependent inactivation, as well as an increase in the affinity of the channel to the DHP blocker (+)-isradipine. Some but not all of these effects were also partially visible in the alpha 1C,72 channel. It is structurally identical to the reference 2138-amino acid alpha 1C,77 channel, except for an insertion of 19 amino acids at position 1575 between sequences encoded by exons 40 and 41. There was a 5-mV hyperpolarizing shift of the voltage dependence of inactivation, the kinetics of inactivation of IBa through alpha 1C,72 was only 20% faster than that through alpha 1C,77 (Table I), and the DHP sensitivity of alpha 1C,72 was the same as that of alpha 1C,86 but about 4 times higher than that of alpha 1C,77 (Table III).

Over the last few years, a multitude of studies have shed light on the molecular basis of Ca2+ channel inactivation. It has been suggested that voltage- and Ca2+-dependent inactivation are regulated by distinct sites on the alpha 1C subunit (32). The structural determinants for voltage-dependent inactivation have been attributed to sequences near or in the S6 segments of domains I, III, and IV of the alpha 1 subunit (33, 34). Substituting as few as 9 amino acids from a rapidly inactivating class A Ca2+ channel near the transmembrane region IS6 for homologous residues in the alpha 1C subunit was sufficient to transform alpha 1C into a fast inactivating channel (33). More recently several studies have implicated carboxyl-terminal segments as being involved in voltage-dependent inactivation (15, 35).

The membrane-spanning regions, and consequently the voltage sensor in the S4 segments (36), are structurally identical in all three splice variants of alpha 1C studied in our work. The new amino acid sequences in the cytoplasmic carboxyl-terminal tail, encoded by alternative exons in alpha 1C,72 and alpha 1C,86, do not show hydrophobic stretches that would suggest their insertion into the plasma membrane. The differences in the voltage dependence of gating between alpha 1C,77, alpha 1C,72, and alpha 1C,86 may, therefore, be due to an altered interaction of cytoplasmic amino acid sequences with the intramembrane voltage sensor in the S4 segments of the alpha 1C protein. For example, a direct interaction of the amino acids encoded by exons 40-42 with the cytoplasmic ends of the charged transmembrane segments S4 may affect the mobility of the charged regions in response to a change in the transmembrane electric field (37). This could influence the transitions between open and closed states of the channel depending on the conformational flexibility of the cytoplasmic polypeptide chains, which may be highest for the alpha 1C,86 channel. However, we cannot rule out that the fast inactivation observed in alpha 1C,86 may be due to some modulatory effect on the interaction with auxiliary subunits, which are known to influence Ca2+ channel inactivation properties (38).

Ca2+-dependent inactivation seems to be mediated directly by binding of Ca2+ ions to the channel (39). Elimination of the Ca2+ selectivity by the E1145Q mutation in the pore region of repeat III of rabbit cardiac alpha 1C was associated with a loss of Ca2+-dependent inactivation (27). A Ca2+ binding site has also been implicated for a carboxyl-terminal segment near the transmembrane region IVS6 that includes a putative Ca2+ binding EF-hand motif (40-42). This motif is essential for Ca2+-dependent inactivation, although additional residues downstream to the EF-domain are required to exhibit the full effect (42-45). On the other hand, neither truncation of up to 70% of the carboxyl-terminal tail of cloned alpha 1C subunits (15, 28) nor cytoplasmic modification by trypsin of cloned cardiac Ca2+ channels in HEK 293 cells (15) and endogenous Ca2+ channels in ventricular myocytes (35) had any effect on Ca2+-dependent inactivation. However, in another study, Ca2+-dependent inactivation in ventricular myocytes was abolished by trypsin digestion (46), indicating that there is a limit to the extent by which the carboxyl terminus can be shortened before inactivation is impaired.

Our data show that substituting a stretch of 81 amino acids of alpha 1C,86 for a segment of 80 amino acids in alpha 1C,77 not only affects voltage-dependent inactivation, it also eliminates Ca2+-dependent inactivation. This substitution left the four putative transmembrane domains and the EF-hand motif intact. These structural regions are identical in both alpha 1C constructs. Thus, our study supports recent observations (43-45) suggesting that the EF-hand motif is not the only determinant of Ca2+-dependent inactivation (42). We could narrow down a carboxyl-terminal regulatory domain to a segment of maximally 81 amino acids.

Our studies have shown an alternatively spliced segment in the carboxyl terminus of the human alpha 1C subunit, which determines inactivation properties of the Ca2+ channel. It remains to be elucidated which parts and residues encoded by exons 40-42 are involved in voltage-dependent inactivation and whether these same sites are responsible for abolishing Ca2+-dependent inactivation of alpha 1C,86. Furthermore, it will be of great importance to clarify whether an alpha 1C,86-like splice variant is a functional class C Ca2+ channel in the brain.


FOOTNOTES

*   This work was supported by Grant 31-45093.95 from the Swiss National Science Foundation and a grant from the Sandoz Foundation. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Z74996[GenBank], Z34815[GenBank], and Z34817[GenBank].


Dagger    These authors have contributed equally to this study.
§   Present address: Dept. of Pharmacology, Georgetown Medical Center, Washington, DC 20007.
   To whom correspondence should be addressed: Dept. of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland. Tel.: 41-31-632-3281; Fax: 41-31-632-4992.
1    The abbreviations used are: DHP, dihydropyridine; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid Na4 salt; nt, nucleotide(s); Vh, holding potential.

Acknowledgments

We thank F. Hofmann (Munich) and V. Flockerzi (Heidelberg) for a gift of clones of beta  and alpha 2delta subunits, J. Tytgat (Leuven) for providing pGEMHE, H. Porzig and K. Baltensperger for reading the manuscript, and H. Van Hees for excellent technical assistance.


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