Molecular Determinants of L-type Ca2+ Channel Inactivation
SEGMENT EXCHANGE ANALYSIS OF THE CARBOXYL-TERMINAL CYTOPLASMIC MOTIF ENCODED BY EXONS 40-42 OF THE HUMAN alpha 1C SUBUNIT GENE*

Nikolai M. SoldatovDagger , Murat Oz, Kathleen A. O'Brien, Darrell R. Abernethy, and Martin Morad

From the Georgetown University Medical Center, Department of Pharmacology, Washington, D. C. 20007

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Recently we have described a splice variant of the L-type Ca2+ channel (alpha 1C,86) in which 80 amino acids (1572-1651) of the conventional alpha 1C,77 were substituted by another 81 amino acids due to alternative splicing of exons 40-42. Ba2+ current (IBa) through alpha 1C,86 exhibited faster inactivation kinetics, was strongly voltage-dependent, and had no Ca2+-dependent inactivation. An oligonucleotide-directed segment substitution and expression of the mutated channels in Xenopus oocytes were used to study the molecular determinants for gating of the channel within the 80-amino acid domain. Replacement of segments 1572-1598 or 1595-1652 of the "slow" alpha 1C,77 channel with the respective segments of the "fast" alpha 1C,86 gave rise to rapidly inactivating alpha 1C,86-like channel isoforms. We found that replacement of either motifs 1572IKTEG1576 or 1600LLDQV1604 of alpha 1C,77 with the respective sequences of alpha 1C,86 caused strong but partial acceleration of IBa inactivation. Replacement of both sequences produced an alpha 1C,86-like fast channel which had no Ca2+-dependent inactivation. These results support the hypothesis that motifs 1572-1576 and 1600-1604 of alpha 1C,77 contribute cooperatively to inactivation kinetics of alpha 1C and are critical for Ca2+-dependent inactivation of the channel.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The voltage-gated class C ("cardiac") L-type Ca2+ channel is composed of the pore-forming alpha 1C subunit, containing the high affinity binding sites for dihydropyridines and other organic Ca2+ channel blockers, and the auxiliary beta - and alpha 2delta -subunits (1, 2). Unlike dihydropyridine-insensitive Ca2+ channels, which exhibit voltage-dependent inactivation of Ca2+ current (ICa), L-type Ca2+-channels are in addition inactivated by Ca2+, but not Ba2+ ions permeating through the channel (3). The Ca2+-binding site responsible for the Ca2+-induced inactivation of the class C Ca2+ channel is thought to be located near the inner mouth, but outside the electric field of the pore (4). Substituting a small 142-amino acid segment of the Ca2+-insensitive alpha 1E channel with a homologous segment of the cytoplasmic tail of alpha 1C, confers Ca2+ sensitivity on the alpha 1E channel (5).

Alternative splicing of the human alpha 1C subunit generates multiple isoforms of the channel (6), including those with structurally altered carboxyl-terminal tail (7). Recently two splice variants of the principal 2138-amino acids pore-forming alpha 1C subunit, alpha 1C,86 and alpha 1C,77, exhibiting strong differences in their gating properties were described (8). The alpha 1C,86 channel has 80 amino acid residues (1572-1651) in the second quarter of the 662-amino acid cytoplasmic tail of the conventional channel (alpha 1C,77), replaced with 81 non-identical amino acids (Fig. 1) due to alternative splicing of exons 40-42 (7). Both splice variants retained high sensitivity toward dihydropyridine blockers, but IBa through alpha 1C,86 inactivated 10 times faster than alpha 1C,77 at +20 mV. The inactivation rate of alpha 1C,86 was strongly voltage-dependent but essentially Ca2+-independent suggesting that the segment 1572-1651 of the carboxyl-terminal tail of alpha 1C is critical for the kinetics as well as for voltage and Ca2+ dependence of inactivation of alpha 1C channel. Extended segment-substitution studies, reported here, show that amino acid residues 1572-1576 and 1600-1604 of alpha 1C,77 contribute in a cooperative manner to the Ca2+-binding motif(s) responsible for the feedback inhibition of alpha 1C channel by Ca2+ and the kinetics of decay of IBa in the absence of Ca2+.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Preparation of alpha 1C,77 Mutants-- All mutations were incorporated into the nt1 sequence of pHLCC77 cDNA encoding human Ca2+ channel alpha 1C,77 subunit (9) subcloned into pAlter-1 vector (Promega) or Bluescript SK(-) vector (Stratagene) and composed of exons 1-20, 22-30, 32-44, and 46-50 (EMBL Data Bank accession number Z34815). To improve expression in Xenopus oocytes, pHLCC77 was supplemented at the 5'-end with HindIII/BglII and at the 3'-end with BglII/BamHI fragments of untranslated region sequences of the Xenopus beta -globin gene (10), respectively. Mutations were introduced through segment exchange using specifically designed mutation primers and the Altered Sites® II in vitro Mutagenesis System (Promega) according to the manufacturer's manual. Table I shows mutation primers that have been synthesized (Genosys Biotechnologies, Inc.) and used for segment exchange with pHLCC77 in pAlter-1 vector as a template. Double mutant alpha 1C,77M1,3-encoding construct was prepared using the M1 primer and the mutated recombinant plasmid pHLCC77M3 as a template. Mutated plasmid pHLCC77L, encoding alpha 1C,77L, was constructed by co-ligating the SfuI (3342)/SacI (4787) 1.4-kilobase fragment of the recombinant plasmid pHLCC86, encoding alpha 1C,86 (8), and 0.7-kilobase SacI (4787)/AatII (5495) fragment of the mutated plasmid pHLCC77M2 with the AatII (5498)/SfuI (3342) 6.7-kilobase fragment of pHLCC86 containing pBluescript vector. Similarly, the mutated plasmid pHLCC77K encoding alpha 1C,77K was prepared by co-ligating the AatII (5498)/SfuI (3342) fragment of pHLCC86 with the SfuI (3342)/SacI (4787) fragment of the mutated plasmid pHLCC77M2 and SacI (4787)/AatII (5498) fragment of the recombinant plasmid pHLCC86. Nucleotide sequences of the obtained cDNAs were verified using the ABI PRISMTM Dye Terminator Cycle Sequencing Kit with AmpliTaq DNA Polymerase (Perkin-Elmer). All template DNAs were linearized by digestion with BamHI. Capped transcripts were synthesized in vitro with T7 (pHLCC77L and pHLCC77K) or SP6 (all other mutants) RNA polymerases using the mRNA cap kit (Stratagene) and stored as suspensions in ethyl alcohol at -70 °C. Before injection, mRNA samples were dried and dissolved in water (0.5 µg/µl).

                              
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Table I
Nucleotide sequences and positions of oligonucleotide mutation primers
All primers were designed as antisense oligonucleotides. Here and throughout the paper, nt positions are given relative to the first coding triplet. Mismatches are presented in lower case, while nt shown in upper case are identical to pHLCC77.

Functional Expression of Ca2+ Channels in Xenopus Oocytes-- Xenopus laevis oocytes were defolliculated 6-12 h prior to injection with 50-100 nl of a mixture containing mRNAs for alpha 1C-, beta 1- (11), and alpha 2delta -subunits (12) in equimolar ratio. Injected oocytes were incubated at 18 °C in sterile Barth's medium supplemented with 10,000 units/liter penicillin, 6 mg/liter streptomycin, 50 mg/liter gentamicin, and 90.1 mg/liter theophilline. Membrane currents were recorded at room temperature (20-22 °C) by a two-electrode voltage clamp method using a GeneClamp 500 amplifier (Axon Instruments). Electrodes were filled with 3 M CsCl and had resistance between 0.2 and 1 megohms. The Ba2+ and Ca2+ extracellular (bath) solutions contained 50 mM NaOH, 1 mM KOH, 10 mM HEPES, and 40 mM Ba(OH)2 or 40 mM Ca(NO3)2, respectively (pH adjusted to 7.4 with methanesulfonic acid). In some experiments, about 1 h prior to recording, Ca2+ current (ICa)-expressing, oocytes were injected with 50 nl of 94 mM Cs4-BAPTA (pH 7.4). Currents were filtered at 1 kHz. Data were acquired using pClamp 5.5 software (Axon Instruments), corrected for leakage using an on-line P/4 subtraction paradigm and analyzed with KaleidaGraph software. Results are shown as mean ± S.E. "Endogenous" current, determined in the presence of 5 µM (±)-PN200-110 to block the L-type current, did not exceed 3% of the total current.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Strategy of Segment Exchange Analysis-- Since it was unclear whether the altered properties of the alpha 1C,86 channel were due to the lost determinants normally present in alpha 1C,77 and/or were due to those imposed by the new 81-amino acid motif, we chose a well defined slow L-type Ca2+ channel isoform (alpha 1C,77 channel (9)) as a primary target for the mutation studies. A series of segment exchange experiments were performed on pHLCC77 plasmid encoding alpha 1C,77 to map the molecular determinants for the alpha 1C inactivation kinetics as well as its voltage and Ca2+ dependence within the experimentally targeted 80-amino acid (1572-1651) sequence of the carboxyl-terminal tail. To narrow the search, we substituted initially two large fragments of this sequence in alpha 1C,77 with respective sequences from alpha 1C,86 (Fig. 1, mutants 77L and 77K) by introducing an HLCC86-specific restriction site into the alpha 1C,77-encoding DNA sequence (mutant 77M2, Fig. 1). The mutated channels were then expressed in Xenopus oocytes by coinjecting an equimolar mixture of cRNAs encoding the mutated alpha 1C-, beta 1- (11), and alpha 2delta -subunits (12). Because both mutants showed accelerated inactivation kinetics of IBa and loss of Ca2+-dependent inactivation, shorter segments of 5-7 amino acids in this region of slowly inactivating alpha 1C,77 were replaced by residues from the equivalent positions of rapidly inactivating alpha 1C,86 using oligonucleotide-directed segment exchange technique (mutants 77M1, 77M3, and 77M5 shown in Fig. 1). As we shall show below, it was the double mutant 77M1,3 (Fig. 1) that best mimicked the properties of the alpha 1C,86 channel.


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Fig. 1.   Scheme illustrating segment exchange analysis of the specific carboxyl-terminal motif (1572-1651) of alpha 1C,77. Corresponding sequence of alpha 1C,86 (1572-1652) is shown on the top. Indicated amino acids of alpha 1C,86 replace the respective residues in the amino acid sequence of alpha 1C,77 thus forming mutants in which all other amino acids remain identical to alpha 1C,77. Positions of amino acids are shown on the right. Amino acids sharing identical positions in both alpha 1C,77 and alpha 1C,86 are shown in bold caps. Vertical arrow marks a position of 19-amino acid insertion in alpha 1C,72 (8).

Introduction of HLCC86-specific SacI Restriction Site within the Segment (1595-1598)-coding Sequence Does Not Change Properties of Mutated alpha 1C,77M2-- The nt sequence of the alpha 1C,86-encoding plasmid contains a convenient SacI restriction site which is absent from pHLCC77. This site, when introduced into the alpha 1C,77-coding DNA sequence, allows the transfer of large segments of the 81-amino acid motif of the "fast" alpha 1C,86 channel (e.g. 77L and 77K, Fig. 1) into alpha 1C,77.

We introduced this SacI restriction site into pHLCC77 by replacing the segment (nt 4783-4794), which encodes the amino acid residues 1595-1598 of the "slow" alpha 1C,77 channel, with the respective segment of pHLCC86 coding for SSHP (mutant 77M2, Fig. 1). The resulting alpha 1C,77M2 channel showed no significant electrophysiological differences compared with alpha 1C,77 (Table II, III, and IV; Fig. 2, A-C). Similar to alpha 1C,77, the 77M2 mutant showed marked acceleration of inactivation rate when Ca2+ was the charge carrier through the channel (Fig. 2A). Consistent with previous observations (13), ICa was 4.0 ± 0.3 (n = 6) times smaller than IBa. tau f had a U-shaped voltage dependence (Fig. 2C) for the Ca2+-transporting channel consistent with previous observations (4).

                              
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Table II
Kinetics of IBa decay in segmental mutants of alpha 1C,77
Inactivation time constants (tau ) of IBa were determined from double exponential fitting of current traces elicited by 1-s test pulses to +20 mV from Vh = -90 mV. The fit was obtained by equation: 1(t) = Iinfinity  + If · exp(-t/tau f) Is · exp(-t/tau s), where Iinfinity is the steady state amplitude of the current, I is the amplitude of the initial current, f and s stand for fast and slow components, respectively.


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Fig. 2.   Electrophysiological properties of alpha 1C,77M2 channel recorded in bath solutions containing either 40 mM Ba2+ or 40 mM Ca2+. A, current traces measured in an oocyte injected with Cs4-BAPTA. The rapid component of inactivation of ICa was approx 2.8 times faster (tau f(20) = 34.5 ms) than the rapid component of IBa, and comprised 89.2% of the total current, while the slow component (tau s(20) = 295 ms) represented only a minor fraction of the current. B, averaged I-V curve (bullet , n = 11) and voltage dependence of tau f (open circle , n = 7) for IBa through alpha 1C,77M2 channel. tau f at 0 mV was 82.3 ± 15.7 ms (n = 6) compared with 113.5 ± 13.8 ms (n = 7) at +40 mV; tau s(0) = 582.6 ± 101.4 ms compared with tau s(40) = 484.1 ± 93.1 ms. C, Current-voltage relation (bullet ) and voltage dependence of tau f (open circle ) for ICa through alpha 1C,77M2 channel in Cs4-BAPTA-injected oocytes (n = 6).

Large Segments Exchange Between alpha 1C,86 and alpha 1C,77-- Using the pHLCC86-specific SacI restriction site in alpha 1C,77M2-coding nt sequence, alpha 1C,77L and alpha 1C,77K mutants were constructed by substituting segments 1572-1598 and 1595-1651 of alpha 1C,77M2 with the corresponding 27- and 58-amino acid segments of the fast alpha 1C,86 channel (Fig. 1). Electrophysiological analysis of these mutants indicated that both alpha 1C,77L and alpha 1C,77K have properties very similar to those of the fast alpha 1C,86 channel. Indeed, superimposed normalized traces of IBa at +20 mV through alpha 1C,77L, alpha 1C,77K, and alpha 1C,86 channels (Fig. 3A) show that about 90% of IBa decayed rapidly in all these channels (Table II), and their voltage dependence were almost identical (Table III, Fig. 3B). Comparison of the steady-state inactivation curves for alpha 1C,77L, alpha 1C,77K, and alpha 1C,86 using 2-s conditioning prepulses, however, showed that the steady-state inactivation curve for alpha 1C,77L was shifted by about -20 mV with respect to that of alpha 1C,86 and by -30 mV relative to alpha 1C,77 (Table IV). Thus, structural changes produced by the substitution of the 27-amino acid segment alone altered the inactivation properties of the alpha 1C,77L mutant more effectively than the exchange of the 81-amino acid segment of alpha 1C,86, containing the 27-amino acid motif.


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Fig. 3.   Comparison of electrophysiological properties of alpha 1C,77L and alpha 1C,77K channels with alpha 1C,86. A, representative traces of IBa scaled to the same amplitude. B, averaged I-V curves for IBa through alpha 1C,77L (bullet , n = 7), alpha 1C,77K (black-square, n = 3), and alpha 1C,86 (open circle , n = 4) channels. C, averaged voltage dependence of tau f for IBa through alpha 1C,77L (bullet , n = 7), alpha 1C,77K (black-square, n = 3), and alpha 1C,86 (open circle , n = 10) channels. In alpha 1C,77L channel, tau f decreased from 56.1 ± 9.6 ms (74.6 ± 3.6% of IBa) at 0 mV to 24.3 ± 0.9 (94.4 ± 0.7% of IBa) at +40 mV. In alpha 1C,77K channel, tau f decreased from 81.3 ± 14.6 ms (62.3 ± 7.0% of IBa) at 0 mV to 27.9 ± 2.9 ms (94.8 ± 2.6% of IBa) at +40 mV. In alpha 1C,86 channel, tau f decreased from 62.3 ± 10.9 ms (56.6 ± 6.1% of IBa) at 0 mV to 33.6 ± 1.9 ms (94.8 ± 0.8% of IBa) at +40 mV. D, averaged I-V curves (open circle , bullet ) and voltage dependence of tau f (square , black-square) for ICa through alpha 1C,77L (open circle , square ; n = 3) and alpha 1C,77K (bullet , black-square; n = 3) channels.

                              
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Table III
Parameters of current-voltage relationships for segmental mutants of alpha 1C,77
Ba2+ currents were measured in response to 1-s test pulses to +20 mV from Vh = -90 mV applied at 30-s intervals in the range of -40 to +60 mV with 10-mV increments. The fit was obtained by equation: IBa = Gmax (V - Erev)/{1 + exp[(V - V0.5)/kI-V]}, where Erev is reversal potential, V0.5 - voltage at 50% of IBa activation, and kI-V - slope factor. Gmax, maximum conductance (not shown); instead are presented maximum amplitudes of IBa (IBa(max)) measured at voltage for the peak current.

                              
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Table IV
Influence of segmental mutations of alpha 1C,77 on steady-state inactivation
Steady-state inactivation curves were measured using a 2-step voltage clamp protocol. A 2-s conditioning pulses were applied at 30-s intervals with 10-mV increments up to +20 mV from Vh -90 m V followed by a 250-ms test pulse to +20 m V. Peak current amplitudes were normalized to maximum value. The curves were fitted by a Boltzmann function: IBa = 1{1 + exp[(V - V0.5)/k]}, where V is the conditioning pulse voltage; V0.5 is the voltage at half-maximum of inactivation, and k is a slope factor.

The voltage dependence of tau f for IBa through alpha 1C,77L and alpha 1C,77K channels was similar to that of alpha 1C,86 (Fig. 3C). There was approximately a 2.5-3-fold decrease of tau f at +40 mV compared with tau f at 0 mV. In both channels the fraction of IBa following the fast decay accounted for approximately 95% of the total current at +40 mV. However, a larger fraction of the current through 77L and 77K mutants continued to inactivate rapidly at 0 mV. Thus, voltage dependence of inactivation of IBa through alpha 1C,77L was stronger than in alpha 1C,86.

Similar to alpha 1C,86 (8), alpha 1C,77L and alpha 1C,77K channels did not exhibit Ca2+-dependent inactivation of ICa (Fig. 3D) as indicated by the absence of characteristic U-shaped voltage dependence of tau f (e.g. see for example, alpha 1C,77M2, Fig. 2C). Therefore, alpha 1C,77L and alpha 1C,77K channels appear to have electrophysiological properties quite similar to those of alpha 1C,86 channel. This data suggests that there may be at least two molecular determinants for the gating kinetics and Ca2+ dependence of inactivation of the channel, one located possibly within the 22-amino acid segment 1572-1593 (see Fig. 1), and the other within the 54-amino acid motif (1599-1652).

Motif M1 Determines the Fractional Ratio of Fast to Slow Inactivation-- To further narrow the 22-amino acid motif (1572-1593) that confers the inactivation properties of alpha 1C,86 to alpha 1C,77L channel, a number of alpha 1C,77 mutants containing shorter segments of this motif were prepared. The carboxyl-terminal part (1588-1592; mutant M5 in Fig. 1) of the 77L motif did not cause appreciable changes in the properties of the mutated alpha 1C,77M5 channel as compared with alpha 1C,77 (Fig. 4A, Tables II and III). Within the remaining 16-amino acid sequence (1572-1587), we identified a 5-amino acid segment (1572-1576, mutant 77M1 in Fig. 1) that was critical for the faster inactivation. Electrophysiological properties of the alpha 1C,77M1 channel are presented in Fig. 4. The inactivation kinetics of IBa through alpha 1C,77M1 was much faster than through the alpha 1C,77 channel (Fig. 4A). This may have resulted from about 2-fold increase in the fraction of IBa that inactivates rapidly (Table II). Interestingly, the time constant of inactivation of the fast current component remained virtually unchanged at +20 mV as compared with that of the alpha 1C,77 channel. Furthermore, the voltage dependence of IBa through alpha 1C,77M1 channel (Fig. 4B) was not significantly different from that of the alpha 1C,77 channel (Table III). However, the steady-state inactivation of IBa through alpha 1C,77M1 shifted by approximately -20 mV with respect to the alpha 1C,77 channel (Table IV). At the same time, the voltage dependence of time constants of inactivation of IBa through alpha 1C,77M1 (Fig. 4B) was steeper than in alpha 1C,77 but did not reach values seen with the alpha 1C,77L channel (Fig. 3C). In contrast to the other rapidly inactivating alpha 1C,77 mutants, tau f for ICa through alpha 1C,77M1 channel exhibited weak U-shaped voltage dependence (Fig. 4C) consistent with the idea that alpha 1C,77M1 remains somewhat Ca2+ sensitive. Thus, amino acids in positions 1572-1576 may be important for the fractional ratio of fast to slow inactivation of IBa.


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Fig. 4.   Electrophysiological properties of alpha 1C,77M1 channel. A, representative traces of IBa through alpha 1C,77M1, alpha 1C,77M5, alpha 1C,77L, and alpha 1C,77 channels scaled to the same amplitude. B, averaged I-V curve (bullet , n = 10) and voltage dependence of tau f (open circle , n = 13) for IBa through alpha 1C,77M1 channel. The time constant of inactivation decreased from tau f(0) = 84.9 ± 6.4 ms (61.1 ± 3.6% of IBa) to tau f(40) = 56.7 ± 4.7 ms (75.7 ± 5.3% of IBa). C, I-V curve (bullet ) and voltage dependence of tau f (open circle ) for ICa through alpha 1C,77M1 channel (n = 3).

A Second 5-Amino Acid Motif Critical for the Faster Kinetics of Inactivation of IBa and Its Ca2+ and Voltage Dependence-- Segment exchange analysis also revealed a second 5-amino acid motif 1600-1604 (mutant 77M3 in Fig. 1) involved in the gating of the channel. Inactivation of IBa through alpha 1C,77M3 occurred with time constants similar to those of the fast alpha 1C,86 (Fig. 5A, Table II). However, the fraction of the slow component of IBa was approximately 3 times greater at +20 mV in the alpha 1C,77M3 channel, causing considerable retardation of the inactivation compared with alpha 1C,86. Both channels exhibited approximately the same strong voltage dependence of tau f for IBa (compare Figs. 3C and 5B). When tau f for the inactivation of ICa through the alpha 1C,77M3 channel was plotted versus membrane potential, neither strong nor U-shape voltage dependence were observed (Fig. 5D). Thus, alpha 1C,77M3 channel lacks the Ca2+-dependent inactivation.


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Fig. 5.   Electrophysiological properties of alpha 1C,77M3 channel and double-mutated alpha 1C,77M1,3 channel. A, representative traces of IBa through alpha 1C,86, alpha 1C,77M1, alpha 1C,77M3, and double mutated alpha 1C,77M1,3 channels scaled to the same amplitude. Averaged I-V curves (bullet ) and voltage dependence of tau f (open circle ) for IBa through alpha 1C,77M3 (B, n = 5) and alpha 1C,77M1,3 (C, n = 5) channels. Time constant of inactivation of alpha 1C,77M3 decreased from tau f(0) = 57.1 ± 6.3 ms (60.6 ± 3.8% of IBa) to tau f(40) = 40.6 ± 7.5 ms (80.8 ± 4.5% of IBa). Time constant of inactivation of alpha 1C,77M1,3 decreased from tau f(0) = 66.0 ± 19.3 ms (73.8 ± 15.0% of IBa) to tau f(40) = 29.6 ± 1.7 ms (93.6 ± 1.7% of IBa). Averaged I-V curves (bullet ) and voltage dependence of tau f (open circle ) for ICa through alpha 1C,77M3 (D, n = 4) and alpha 1C,77M1,3 (E, n = 4) channels.

Acceleration of inactivation kinetics and disruption of Ca2+ sensitivity of inactivation produced by M3 mutation in alpha 1C,77 channel were not accompanied by changes in the properties of the voltage sensor observed in alpha 1C,86 (8). The voltage dependence of IBa through alpha 1C,77M3 (Fig. 5B, Table III) and its steady-state inactivation (Table IV) remained similar to those of alpha 1C,77 (Tables III and IV). Therefore, the 5-amino acid motif (1600-1604) responsible for the faster kinetics, and Ca2+ and voltage dependence of inactivation is distinct from the molecular structures that cause voltage shifts of activation and inactivation in alpha 1C,86 (8).

We extended the M3 mutation of the alpha 1C,77 channel and found that amino acids immediately preceding the M3 motif may cause changes in voltage dependence of inactivation. The "extended" mutant alpha 1C,77M3m contained uncharged hydrophobic Val replacing the positively charged Lys-1599, common for both the slow (alpha 1C,77) and fast (alpha 1C,86) channels (Fig. 1), and preceded by bulky Phe residue. IBa through the alpha 1C,77M3m channel exhibited a strong shift of steady-state inactivation curve toward negative potentials (Table IV) with opposite but much smaller shift of the current-voltage relation (Table III). Similar strong shift of the steady-state inactivation was displayed by the alpha 1C,77L mutant, which had no overlapping mutated structure (Fig. 1). Therefore, within the cytoplasmic motif (1572-1651) of alpha 1C,77, there are at least two non-overlapping regions that may strongly affect intramembrane voltage sensors of the channel responsible for inactivation.

Double Mutant M1 + M3 Shows the Best Fit to the alpha 1C,86 Channel-- When M1 mutation was combined with the M3 mutation, the resulting alpha 1C,77M1,3 channel exhibited all the properties of the alpha 1C,86 channel. Fig. 5A shows representative trace of IBa through alpha 1C,77M1,3 elicited by step depolarization to +20 mV from Vh -90 mV. Approximately 90% of the current inactivated with tau f of 40 to 45 ms, a distinct characteristic of alpha 1C,86 channel (Table II). Furthermore, the time constant of inactivation of IBa through alpha 1C,77M1,3 was strongly voltage-dependent (Fig. 5C). As compared with alpha 1C,77M1 and alpha 1C,77M3, the voltage dependence of IBa through the double mutated channel was shifted by about +15 mV (Fig. 5C) but all other parameters were essentially identical to those of alpha 1C,86 channel (Table III).

Double-mutated alpha 1C,77M1,3 channel did not show Ca2+-dependent inactivation as evidenced by the absence of characteristic U-shaped voltage dependence of tau f for ICa (Fig. 5E). Unlike alpha 1C,77M3 channel (Fig. 5D), inactivation kinetics of ICa through alpha 1C,77M1,3 was strongly voltage dependent (Fig. 5E). Taken together, these data suggest that motifs M1 and M3 complement each other in disrupting the functional site of Ca2+-dependent inactivation. Thus, two 5-amino acid motifs located at positions 1572-1576 and 1600-1604 of alpha 1C,77 cooperatively participate in the gating function of the channel.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Unlike Ba2+ current, the magnitude of inactivation of ICa depends on the size of ICa so that the time constant of inactivation exhibits a U-shaped voltage dependence. Ca2+ accelerates inactivation of the L-type Ca2+ channel by reducing its open probability by interacting with the alpha 1C subunit (4, 14) in equimolar ratio (15). The Ca2+-binding site for Ca2+-dependent inactivation has been postulated to be very close to the internal opening of the pore (4) but outside of the conduction pathway (16). The Ca2+-dependent inactivation was thought to be linked (17) to a 29-amino acid domain (1506-1534) with homology to Ca2+-binding EF-hand motif (18). Recent experiments using mutation analysis (5), however, have excluded amino acids 1506-1534 from this function. Other observations (5, 8) suggest that the location of the site for Ca2+-dependent inactivation may be at least 40 amino acids toward the carboxyl terminus of the polypeptide chain.

None of the mutations described in this paper have caused appreciable changes in the kinetics of ICa inactivation. However, the kinetics of IBa decay was changed in a gradual manner (Table II). Since tau f decreases in the following order among the mutants: alpha 1C,77 approx  alpha 1C,77M2 approx  alpha 1C,77M5 > alpha 1C,77M1 > alpha 1C,77M3 alpha 1C,77K approx  alpha 1C,77L approx  alpha 1C,77M1,3 approx  alpha 1C,86, it is likely that retardation of inactivation can be eased through specific structural changes not requiring Ca2+. The values of tau f for the decay of IBa through the fast mutants are almost identical to those for ICa through the slow channel, suggesting that both modes of inactivation may be thermodynamically similar.

It would be reasonable to assume that mutations leading to faster decay rates of IBa disrupt intraprotein interactions responsible for retardation of inactivation, which otherwise may be achieved through interaction with Ca2+. First, inactivation time constants of IBa through fast channels (alpha 1C,77K, alpha 1C,77L, alpha 1C,77M1,3, and alpha 1C,86) do not exhibit a U-shaped voltage dependence, and therefore Ba2+ does not influence inactivation in a current-dependent manner. Second, all fast channels are deprived of Ca2+-dependent inactivation.

At least two partially overlapping sequences, 1572-1598 in alpha 1C,77L and 1572-1576 plus 1600-1604 in alpha 1C,77M1,3, can transform a conventional slow alpha 1C,77 channel into the fast (alpha 1C,86-like) one. The motif in the 77L mutation can apparently be narrowed to 1572-1587 because its partial mutants, alpha 1C,77M2 and alpha 1C,77M5, exhibit properties of the slow channel.

It is the 77M1 motif (1572-1576, Fig. 1) that is common to both fast mutants. However, when each motif is introduced alone, it causes only partial disruption of Ca2+ sensitivity and acceleration of IBa. Therefore, for the full effect to occur, M1 must be supplemented with another determinant. The latter may be mobilized either from the adjacent sequence (1577-1587) of the 77L motif or from a distant 77M3 motif. These segments in alpha 1C,77 channel are responsible for Ca2+-dependent inactivation and, in the absence of Ca2+, for the accelerated kinetics of IBa inactivation. Thus, these motifs are involved in the gating function of the channel possibly through a direct interaction with the pore.

It is possible that 8 out of 11 amino acids (1574-1584, Fig. 1) of alpha 1C,77 represent residues that may form coordination bonds with Ca2+. However, disruption of this motif by insertion of 19 additional amino acids at position 1576 neither changed the kinetics of IBa decay, nor the Ca2+-dependent inhibition of otherwise invariant channel isoform, alpha 1C,72 (8). Moreover, alpha 1C,77M1 mutant retained some Ca2+-induced inactivation property. Since segment 1577-1584 in both the slow alpha 1C,72 and the fast alpha 1C,77M1,3 channels is intact (Fig. 1), the remaining segment (1572-1575) is apparently critical for gating. Coordination of Ca2+ in Ca2+-binding sites involves seven bonds that are spatially distributed as an octahedron (19) and can be separated into two vertices. Our data suggest that either of the two motifs, 1577-1587 or 1600-1604, may serve as the second critical element for the channel gating. Additional point mutation analysis is needed to make a final judgment about critical amino acids in the proposed Ca2+-coordination motif responsible for the channel gating.

Our data point to the complexity of molecular determinants for Ca2+ dependence of inactivation. In addition to motifs discussed above, there may be other sequences which determine fast inactivation kinetics and loss of Ca2+-induced inactivation of alpha 1C,77K channel. Investigation of these motifs is in progress.

    ACKNOWLEDGEMENTS

We are grateful to F. Hofmann and V. Flockerzi for a gift of clones of beta 1- and alpha 2delta -subunits.

    FOOTNOTES

* This work was supported in parts by grant-in-aid of the American Heart Association, Nation's capital affiliate (to N. M. S.), National Institutes of Health Grants HL16152 (to M. M.), and AG08226 and GM08386 (to D. R. A.).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 and reprint requests should be addressed: Georgetown University Medical Center, Dept. of Pharmacology, 3900 Reservoir Rd., NW, Washington, D. C. 20007. Tel.: 202-687-8450; Fax: 202-687-8458.

1 The abbreviations used are: nt, nucleotide(s); BAPTA, 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetate; Vh, holding potential.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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