Amino Acids in Segment IVS6 and beta -Subunit Interaction Support Distinct Conformational Changes during Cav2.1 Inactivation*

Stanislav BerjukowDagger, Rainer MarksteinerDagger, Stanislav Sokolov, Regina G. Weiss, Eva Margreiter, and Steffen Hering§

Institut für Biochemische Pharmakologie, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria

Received for publication, November 20, 2000, and in revised form, March 7, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cav2.1 mediates voltage-gated Ca2+ entry into neurons and the release of neurotransmitters at synapses of the central nervous system. An inactivation process that is modulated by the auxiliary beta -subunits regulates Ca2+ entry through Cav2.1. However, the molecular mechanism of this alpha 1-beta -subunit interaction remains unknown. Herein we report the identification of new determinants within segment IVS6 of the alpha 12.1-subunit that markedly influence channel inactivation. Systematic substitution of residues within IVS6 with amino acids of different size, charge, and polarity resulted in mutant channels with rates of fast inactivation (kinact) ranging from a 1.5-fold slowing in V1818I (kinact = 0.98 ± 0.09 s-1 compared with wild type alpha 12.1/alpha 2-delta /beta 1a kinact = 1.35 ± 0.25 s-1) to a 75-fold acceleration in mutant M1811Q (kinact = 102 ± 3 s-1). Coexpression of mutant alpha 12.1-subunits with beta 2a resulted in two different phenotypes of current inactivation: 1) a pronounced reduction in the rate of channel inactivation or 2) an attenuation of a slow component in IBa inactivation. Simulations revealed that these two distinct inactivation phenotypes arise from a beta 2a-subunit-induced destabilization of the fast-inactivated state. The IVS6- and beta 2a-subunit-mediated effects on Cav2.1 inactivation are likely to occur via independent mechanisms.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Calcium (Ca2+) entry through Cav2.1, also known as class A or P/Q-type Ca2+ channels (1), plays a central role in triggering the release of neurotransmitters from presynaptic nerve terminals as well as influencing other critical neuronal functions (2). The pore-forming alpha 12.1-subunit is encoded by the CACNA1A gene, which is highly expressed in the central nervous system (3-6). Mutations and deletions in the alpha 12.1-subunit result in certain neurological disorders such as the familiar hemiplegic migraine, episodic ataxia type 2, and spinocerebellar ataxia type 6 (Ref. 5 and 7-11; see Ref. 12 for review). The importance of this channel type for neurological disorders is further emphasized by the recent finding that Cav2.1-deficient mice develop hallmark characteristics of severe ataxia and dystonia and subsequently die 3-4 weeks after birth (13).

The rate of voltage-dependent channel closure during depolarization, a process termed inactivation, is an important determinant of Ca2+ entry during a neuronal action potential. Three different types of Ca2+ channel inactivation processes have been identified in Cav2.1 channels: fast and slow voltage-dependent inactivation (14, 15) as well as a Ca2+-dependent inactivation mechanism (16). Cav2.1 inactivation is strongly influenced by alpha 12.1-beta -subunit interaction. For example, coexpression of beta 2a results in substantially slower IBa inactivation compared with channels composed of either beta 1a- or beta 3-subunits (14, 17, 18). Sequence stretches responsible for alpha 12.1 interaction with different beta -subunits have been identified on intracellular linkers between domains I-II (I-II linker) and the amino and carboxyl termini (19-21). Moreover, a soluble NSF (N-ethylmaleimide factor) attachment protein receptor (SNARE)-protein interaction domain within the II-III linker also influences inactivation gating of Cav2.1 (22-24).

The structural determinants of Cav2.1 inactivation are widely spread over the alpha 12.1-subunit (see Ref. 25 for review). The first molecular determinants involved in the process of Cav2.1 inactivation were disclosed by Zhang et al. (26). This study demonstrated a key role of segment IS6 and adjacent intra- and extracellular stretches in the rate of channel inactivation. An important role of segment IVS6 was subsequently demonstrated by Döring et al. (27). Mutations within IVS6 of Cav2.1 produce profound effects on the pharmacological properties of Cav2.1 as well as on inactivation (28). However, the precise relationship (e.g. common or independent mechanisms) between IVS6- and beta -subunit-mediated alterations in CaV2.1 inactivation remains to be elucidated.

Here we evaluate previously observed IVS6-mediated changes in Cav2.1 inactivation (27) at the level of single amino acids. The results identify a number of residues within IVS6 that represent critical determinants of voltage-dependent inactivation of CaV2.1. Systematic substitution of the important inactivation determinant Met-1811 by amino acid residues of different side chain length, charge, or polarity resulted in an array of channel constructs with inactivation properties that varied from being comparable with that of wild-type (e.g. M1811S) to an acceleration of inactivation by nearly 2 orders of magnitude (M1811Q). Thus, Met-1811 obviously imparts a strong influence on the rate of fast voltage-dependent inactivation of CaV2.1.

Coexpression of the alpha 12.1 mutants with either "fast-inactivating" (beta 1a, beta 3) or "slow-inactivating" (beta 2a) beta -subunits demonstrates that the beta 2a-subunit destabilizes the fast-inactivated channel conformation of CaV2.1. A model is proposed to describe the kinetic changes induced by beta 2a-subunit interaction with the different alpha 12.1 mutants.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Generation of alpha 12.1 Mutants-- The Cav2.1 mutants were constructed by introducing point mutations into alpha 12.1 cDNA (BI-2 accession number X57477; Ref. 3) by the "gene SOEing" technique (29). IVS6 segments of CaV2.1 and CaV1.1 differ by only eight amino acids. Thus, we systematically replaced each of these residues within Cav2.1 by their Cav1.1 counterparts (30) in order to generate the alpha 12.1 point mutants: Y1797V, V1801L, I1804Y, F1805M, S1808A, M1811I, L1812I, and V1818I. Residue Met-1811 (displaying the most dramatic changes in inactivation gating) was subsequently mutated to a number of alternative amino acids resulting in constructs M1811A, M1811S, M1811F, M1811I, M1811E, M1811N, M1811K, and M1811Q.

Amino acids in segment IVS6 that are conserved in all Ca2+ channels were substituted by alanine residues (except for the alanine at position 1817, which was replaced by a serine residue). Only 3 out of the 8 mutant cDNAs (N1813A, A1817S, and M1820A) resulted in functional channels when the cRNAs were injected into Xenopus oocytes. However, substitution of the other amino acids by a methionine residue resulted in functional Cav2.1 mutants: Y1799M, F1800M, S1802M, F1803M, F1809M. Ala-1817 was additionally mutated to a methionine, resulting in functional A1817M construct. All constructs were inserted into the polyadenylating transcription plasmids pNKS2 (a kind gift of Dr. O. Pongs) and verified by sequence analysis.

Electrophysiology-- Preparation of stage V-VI oocytes from Xenopus laevis, synthesis of capped off run-off poly(A+) cRNA transcripts from linearized cDNA templates, and injection of cRNA were performed as previously described in detail by Grabner et al. (31). Barium currents through calcium channels (IBa) were studied 2-7 days after microinjection of approximately equimolar cRNA mixtures of wild type alpha 12.1 IVS6 mutants (0.3 ng/50 nl) with beta 1a, beta 2a, or beta 3 (0.1 ng/50 nl) and alpha 2-delta (0.1 ng/50 nl) using the two microelectrode voltage clamp technique. The bath solution contained 40 mM Ba(OH)2, 50 mM NaOH, 5 mM HEPES, 2 mM CsOH adjusted to pH 7.4 with methanesulfonic acid as previously described (32). IBa of Cav3.1 (33) was measured using the same extracellular solution after injection of equimolar cRNA mixtures of alpha 13.1 (0.3 ng/50 nl) and alpha 2-delta (0.1 ng/50 nl) in to Xenopus oocytes. Endogenous chloride currents were suppressed by injecting 20-40 nl of a 0.1 M BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) solution 30-240 min before voltage clamp measurements. Voltage-recording and current-injecting microelectrodes were filled with 2.8 M CsCl, 0.2 M CsOH, 10 mM EGTA, 10 mM HEPES (pH 7.4) and had resistances of 0.3-2 megaohms. Only IBa with amplitudes ranging between 0.3 and 1.5 µA were analyzed.

The rate of inactivation (kdecay) was determined by fitting the initial phase of the current decay to IBa = C exp(-kdecayt). Voltage steps were applied from -80 mV to the peak current potential of the current voltage relationship (I-V) (i.e. voltage of the maximal inward current of the different alpha 12.1/alpha 2-delta /beta 1a constructs ranged from 7 ± 3 mV in L1812I to 20 ± 3 mV in mutant Y1797V). Coexpression of beta 2a did not significantly shift the peak of the I-V curve compared with other beta -subunit compositions.

The voltage of half-maximal inactivation (V0.5,inact) under quasi steady-state conditions was measured using a multi-step protocol. A control test pulse (50 ms to the peak potential of the I-V curve) was followed by a 1.5-s step to -100 mV followed by a 3-s conditioning step, a 4-ms step to -100 mV, and a subsequent test pulse to the peak potential. Inactivation during the 3-s conditioning pulse was calculated as IBa,inact = 1 - IBa test/IBa control.

The pulse sequence was applied every 3 min from a holding potential of -100 mV, and the estimated inactivation curves were fitted to a Boltzmann equation: IBa,inact = Iss + (1 - Iss)/(1 + exp[(V - V0.5,inact)/k]), where V is the membrane potential, V0.5,inact is the midpoint voltage of the inactivation curve, k is a slope factor, and Iss represents the fraction of a noninactivating current. The voltage of half-maximal activation (V0.5,act) was estimated from gpeak/gpeak,max = 0.5, where gpeak = Ipeak/(V - Erev), gpeak,max is the maximum value of gpeak measured at the descending part of the I-V curve, and Erev is the reversal potential.

Recovery from inactivation was studied using a conventional double-pulse protocol. After depolarizing the channels from a holding potential of -80 mV for 3 s to the peak current potential of the I-V curve, 30-ms test pulses were applied at various time intervals to the same voltage. Peak IBa values were normalized to the peak current measured during the pre-pulse, and the time course of IBa recovery from inactivation was fitted to a biexponential function (IBa,recovery = A exp(-t/tau fast) + B exp(-t/tau slow) +C).

The pClamp software package (Version 6.0 Axon Instruments, Inc.) was used for data acquisition and preliminary analysis. Microcal Origin 5.0 was employed for analysis and curve fitting. Data are given as the mean ± S.E. Statistical significance was calculated according to Student's unpaired t test (p < 0.05 for n >=  4).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

Role of Cav2.1-specific IVS6 Amino Acids in Inactivation Gating-- We have previously reported that replacing alpha 12.1 segment IVS6 by alpha 11.1 sequence results in a pronounced acceleration of the inactivation kinetics of the resulting Cav2.1/Cav1.1 chimera (AL23 in Ref. 27). Fig. 1A illustrates the putative folding structure of a Cav2.1 channel (segment IVS6 is highlighted). Of the 25 amino acids predicted to form transmembrane segment IVS6, 8 residues are different between alpha 12.1- and alpha 11.1-subunits (shaded residues shown in Fig. 1B). To assess their individual impact in inactivation gating, we substituted each of these residues by the corresponding alpha 11.1 amino acids. The resulting 8 mutant channels, Y1797V, V1801L, I1804Y, F1805M, S1808A, M1811I, L1812I, V1818L (Fig. 1C), were expressed together with alpha 2-delta and beta 1a auxiliary subunits in Xenopus oocytes, and their inactivation properties were analyzed using the two-microelectrode voltage clamp technique (see "Experimental Procedures").


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Fig. 1.   Segment IVS6 of Ca2+ channel alpha 1-subunits. A, putative transmembrane topology of a Ca2+ channel alpha 1-subunit. Each of the four domains consist of six transmembrane segments (segment IVS6 is shown in black). B, sequence alignment of IVS6 segments of various Ca2+ channel subtypes. Conserved amino acids are framed. Eight amino acids within the alpha 12.1 sequence different from alpha 11.1 are highlighted in black boxes. C, alpha -helical representation of the amino acid sequence of the segment IVS6 of the alpha 12.1-subunit. Sequence differences between CaV2.1 and CaV1.1 are illustrated by indicating the corresponding Cav1.1 amino acids.

Wild type alpha 12.1/alpha 2-delta /beta 1a channels inactivated at a rate (kinact) of 1.35 ± 0.25 s-1. Only two of the point mutations (M1811I and L1812I) induced a pronounced acceleration of inactivation kinetics. Mutants V1801L, F1805M, and S1808A exhibited a small but significant acceleration, whereas V1818I inactivated at a significantly slower rate compared with wild type Cav2.1 (Fig. 2A). An 8-fold acceleration in the rate of IBa inactivation observed for M1811I (kinact = 10.8 ± 1.5 s-1) was not significantly different from that of AL23 (kinact = 11 ± 0.8 s-1, see inset of Fig. 2A). The second fastest inactivation rate (kinact = 5.26 ± 0.39 s-1) was observed for mutation L1812I, located adjacent to M1811. Compared with wild type, the double mutant ML1811/1812II (ML/II)1 exhibited an accelerated rate of current inactivation (not significantly different from M1811I, p > 0.05, Fig. 2A). However, only ML/II, and not M1811I, exhibited a V0.5,inact value comparable with that of AL23 (Fig. 2, A and B). Thus, these data indicate that both Met-1811 and Leu-1812 significantly contribute to the AL23 inactivation phenotype.


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Fig. 2.   Substitutions of Cav2.1 IVS6 amino acids by Cav1.1 residues alter inactivation kinetics. A, rate of IBa decay in the wild-type CaV2.1 channel, IVS6 point mutants, double mutant ML1811/1812II (named here ML/II), and chimera AL23 consisting of Cav2.1 sequence with segment IVS6 substituted by the corresponding sequence of CaV1.1 (see Ref. 27). alpha 1-Subunits were coexpressed together with alpha 2-delta - and beta 1a-subunits. Significant differences (p < 0.05) compared with wild type are indicated by asterisks, n >=  4. Inset, scaled IBa of representative point mutants and chimera AL23, elicited by 300-ms steps from -80 mV to the peak current potential of the I-V curve. B, voltage dependence of half-maximal activation (V0.5,act, open circles) and half-maximal inactivation potential (V0.5,inact, filled circles), n >=  4.

The effects of the amino acid substitutions on the voltage dependence of channel activation and inactivation are illustrated in Fig. 2B. The mean voltages of half inactivation (V0.5,inact) ranged between -32 ± 2 mV in ML/II to -6 ± 3 mV in Y1797V. A comparison of the mean half-activation voltages revealed that only L1812I and V1801L induced a significant shift to more negative voltages (V0.5,act = -3 ± 2 mV (L1812I) and V0.5,act = -7 ± 2 mV (V1801L) compared with Cav2.1 V0.5,act = 2 ± 2, Fig. 2B).

Substitutions of Met-1811 by Charged and Polar Amino Acids Have Marked Effects on Cav2.1 Inactivation-- The data presented in Fig. 2 demonstrate that Met-1811 plays a pivotal role in the rate of voltage-dependent inactivation of Cav2.1. To more thoroughly evaluate the role of residue 1811, we systematically replaced this amino acid by residues of different size, polarity, and charge. Surprisingly, all substitutions of Met-1811 accelerated the time course of current inactivation. Replacements of Met-1811 by amino acids with side chains of different charge or polarity (Gln, Glu, Asn, Lys) induced a substantial acceleration of the IBa decay ranging from about 12-fold M1811K (kinact = 16.3 ± 1.3 s-1) to about 75-fold for M1811Q (kinact = 102 ± 3 s-1). Interestingly, M1811Q inactivated with similar kinetics as Cav3.1 (kinact = 104 ± 3 s-1), a channel known to display one of the fastest inactivation kinetics among all Ca2+ channel subtypes (Fig. 3). Substitutions by hydrophobic residues of different size resulted in less pronounced effects on IBa decay (range between 2-fold in M1811A (kinact = 2.23 ± 0.24 s-1) and 8-fold in M1811I (kinact = 10.8 ± 1.5 s-1); Fig. 3, A and B).


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Fig. 3.   Substitutions of Met-1811 reveal its key role in inactivation gating. A, mutations of a single amino acid in segment IVS6 of CaV2.1 to residues of different size, charge, and polarity accelerated the current decay to different extents (n = 3-7). The most dramatic acceleration was induced by mutation M1811Q with a rate of IBa decay (102 ± 3 s-1, n = 6). The rate of IBa inactivation of Cav3.1 (104 ± 3 s-1, gray column) estimated at the peak current potential of -20 mV is given for comparison (n = 4). Mutant alpha 12.1-subunits were coexpressed together with alpha 2-delta - and beta 1a-subunits. The alpha 13.1-subunit was coexpressed with the alpha 2-delta -subunit. Significant differences (p < 0.05) compared with alpha 12.1 are indicated by asterisks. Inset, scaled superimposed peak IBa during 100-ms depolarizations from -80 to -20 mV (Cav3.1) and to 20 mV (Cav2.1 and M1811Q). B, scaled superimposed IBa of the indicated Cav2.1 mutants (same voltage protocol as in Fig. 2). C, voltages of half-maximal activation (V0.5,act, open circles) and half-maximal inactivation potential (V0.5,inact, filled circles) of the Met-1811 mutants (n >=  3).

The effect of a given mutation on the time course of fast IBa inactivation usually correlated with the half-maximal voltage of Ca2+ channel inactivation (M1811K (V0.5,inact = -41 ± 1 mV), M1811E (V0.5,inact = -40 ± 2 mV), M1811Q (V0.5,inact = -32 ± 2 mV), and M1811N (V0.5,inact = -33 ± 2 mV), see Fig. 3C). The half-inactivation voltages of M1811F, M1811S, and M1811A were not significantly shifted compared with wild type alpha 12.1/alpha 2-delta /beta 1a (V0.5,inact = -8 ± 2 mV, Fig. 3C).

Half-maximal activation of the various Met-1811 mutants occurred between V0.5,act = 0 ± 2 mV (M1811S) and 10 ± 2 (M1811F). Small (<10 mV) but significant shifts in V0.5,act to more positive voltages were observed for M1811Q, M1811F, and M1811K (p < 0.05, Fig. 3C).

To investigate inactivation properties of methionine 1811 substitutions in more detail, we also analyzed the rate of recovery from inactivation. The rapidly inactivating construct M1811Q/alpha 2-delta /beta 1a recovered from fast inactivation with surprisingly similar kinetics as that of wild type alpha 12.1/alpha 2-delta /beta 1a channels (see Fig. 4A). Analogous observations were made for other Met-1811 mutants (Fig. 4B). Thus, these data suggest that despite pronounced acceleration in the rate of fast inactivation (ranging from about 12-fold in M1811K to almost 75-fold in M1811Q, Fig. 3A), these mutations facilitate entry into the fast-inactivated state without affecting stability of the inactivated state.


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Fig. 4.   Recovery from fast inactivation is unaffected by Met-1811 mutations. A: left panel, superimposed normalized IBa through M1811Q and Cav2.1; right panel, wild type alpha 12.1/alpha 2-delta /beta 1a channels recover from inactivation with biexponential kinetics (open circles, tau fast = 0.27 ± 0.03 s, tau slow = 4.85 ± 0.51 s, n = 4). Mutant M1811Q/alpha 2delta /beta 1a channels recovered from inactivation with similar kinetics as the wild type channel (tau fast = 0.29 ± 0.03 s, tau slow = 5.52 ± 0.48 s, n = 5). The voltage protocol is illustrated in the right panel. B, time constants of the fast recovery component (tau fast) of representative alpha 12.1(mutant)/alpha 2-delta /beta 1a channels are not different from wild-type (p > 0.05), n = 4-5.

Role of Conserved IVS6 Amino Acids in Fast Voltage-dependent Inactivation-- As illustrated in Fig. 1B, eight amino acids in segment IVS6 are perfectly conserved in all Ca2+ channel subtypes. To analyze the impact of these residues on inactivation gating, we mutated each conserved amino acid to an alanine (alanine at position 1817 to serine). Only three of these mutants (N1813A, A1817S, and M1820A) formed functional Ca2+ channels in Xenopus oocytes after injection of the cRNAs together with alpha 2-delta  - and beta 3-subunits. The remaining five amino acids (and additionally A1817) were subsequently mutated to methionine. Each methionine mutant (Y1899M, F1800M, S1802M, F1803M, F1809M, and A1817M) formed a functional alpha 12.1(mutant)/alpha 2-delta /beta 3 (kinact = 3.15 ± 0.25 s-1) channel. The strongest effects on IBa decay were observed for mutants A1817M (3.6-fold, kinact = 11.6 ± 0.9 s-1), N1813A (2.7-fold, kinact = 8.03 ± 0.6 s-1), and S1802M (1.5-fold with kinact = 5.57 ± 0.27 s-1, see Fig. 5A for other mutants). The same trend was observed for mutants N1813A and A1817M coexpressed with the beta 1a-subunit (data not shown). Significant (p < 0.01) shifts (compared with -19 ± 2 mV in alpha 12.1/alpha 2-delta /beta 3) in the half-maximal voltage of the inactivation curves were observed for mutants A1817M (-40 ± 2 mV), A1817S (-35 ± 3 mV), N1813A (-24 ± 2 mV), and M1820A (-24 ± 1 mV). The voltages of half-maximal activation and inactivation of the different channel constructs are summarized in Fig. 5C.


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Fig. 5.   Substitution of conserved IVS6 amino acids by alanine or methionine. A, two of the point mutants exhibit a pronounced acceleration of IBa inactivation compared with wild type (N1813A 8.03 ± 0.61 s-1; A1817M 11.5 ± 0.9 s-1). The alpha 12.1(mutant)-subunits were coexpressed with alpha 2-delta - and beta 3-subunits. Significant differences (p < 0.05) to wild type are indicated by asterisks, n = 3-5. B, scaled superimposed IBa of A1817M and N1813A (same voltage protocol as in Fig. 2) illustrate faster inactivation compared with wild type alpha 12.1/ alpha 2-delta /beta 3 channels. C, the voltages of half-maximal activation (V0.5,act, open circles) and half-maximal inactivation (V0.5,inact, filled circles) of the mutant channels (n = 3-5).

IVS6 Mutations Produce Different Kinetic Phenotypes of beta 2a-Subunit Modulation-- It is well established that different beta -subunits differentially modulate the inactivation kinetics of Cav2.1 (14, 15, 17, 18, 34). To elucidate the role of structural changes in different parts of segment IVS6 on beta -subunit modulation, we systematically analyzed IBa inactivation kinetics of our Cav2.1 mutants when expressed in combination with either beta 1a-subunit or beta 2a-subunits.

As illustrated in Fig. 6, we observed two principal patterns of beta 2a-subunit modulation. In most of the IVS6 mutants, coexpression of the beta 2a-subunit dramatically slowed the fast component in IBa decay (called herein type I modulation, Fig. 6, A and B, left panels). This pattern of beta 2a modulation was previously documented for wild type alpha 12.1 (14, 15, 17, 18). A different type of modulation was observed for M1811Q, M1811E, M1811N, and M1811K. In those fast-inactivating alpha 12.1(mutant)/alpha 2-delta /beta 1a constructs, coexpression of beta 2a had much less effect on the transient current decay but, instead, induced a slowly inactivating IBa component (type II modulation, Fig. 6, A and B, right panels). Thus, our data clearly demonstrate that depending on the initial rate of IBa inactivation, coexpression of the beta 2a-subunit results in distinct inactivation phenotypes.


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Fig. 6.   beta 2a modulation of IVS6 Cav2.1 mutants. A, the rates of IBa decay of the mutant Cav2.1 channels are shown in a semilogarithmic scale. Black bars correspond to alpha 12.1 mutants coexpressed with alpha 2-delta - and beta 2a-subunits (n = 3-6, significant differences (p < 0.05) compared with alpha 12.1 are indicated by asterisks), and open circles represent the corresponding inactivation rates of alpha 12.1(mutant)/alpha 2-delta /beta 1a channels (n = 3-7). B, IBa of CaV2.1, M1811I (type I, left panels) and M1811Q, M1811N (type II, right panels) illustrate the two patterns of beta 2a-subunit modulation. Type I (evident as a significant decrease in the rate of fast inactivation kinact) was found to be characteristic for channel mutants with only moderately changed inactivation kinetics. Type II modulation barely affected the transient current component but, instead, attenuated a slow phase in IBa inactivation. Type II modulation was distinctive for most rapidly inactivating mutants. Note the different time scales in left and right panels.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Structural determinants of Cav2.1 inactivation are located in different parts of Ca2+ channel alpha 1-subunits, including pore-forming transmembrane segments and loops, intracellular domain linkers, and the carboxyl terminus (Refs. 35-39, see Ref. 25 for review). Fast and slow voltage-dependent inactivation of this channel type are regulated by auxiliary beta -subunits and other intracellular regulator proteins (14, 15, 17, 18, 22-24).

The molecular mechanism of Ca2+ channel inactivation and its modulation by various beta -subunits are still incompletely understood. In particular, the question of whether beta -subunit-induced changes in Ca2+ channel gating are dependent on inactivation determinants in segment IVS6 remains unanswered.

In the present study we created 25 alpha 12.1 point mutants with substantially different inactivation properties by replacing residues in segment IVS6 that are conserved in all other Ca2+ channel classes by either alanine or methionine residues. In addition, we also mutated specific alpha 12.1 IVS6 residues to the corresponding residues found in alpha 11.2. The impact of inactivation determinants in segment IVS6 in beta -subunit modulation was subsequently analyzed by expressing the mutant alpha 12.1 with either fast-inactivating beta 1a- and beta 3-subunits or the slow-inactivating beta 2a-subunit.

Hot Spots of Inactivation Determinants in Segment IVS6-- The individual replacement of eight non-conserved IVS6 amino acids in alpha 12.1 by the corresponding alpha 11.1 residues enabled the identification of two amino acids that strongly influence Cav2.1 inactivation. Our data demonstrate that mutations of M1811I result in an acceleration in the time course of IBa decay to nearly the same extent as the replacement of the entire IVS6 segment by alpha 11.1 sequence (Fig. 2A). The second strongest effect was observed for substitution L1812I located adjacent to Met-1811 (Fig. 1A). Simultaneous replacement of both alpha 12.1 residues by their alpha 11.1 counterparts almost perfectly reproduced the phenotype of chimera AL23 (27), a chimera in which the entire IVS6 segment is of alpha 11.1 sequence. The similarity between ML/II and AL23 is emphasized by the fact that these two constructs exhibit not only similar rates of IBa inactivation (Fig. 2A) but also similar midpoint voltages of activation and steady-state inactivation (Fig. 2B). Significant but less pronounced effects on channel inactivation were also observed for mutations V1801L, F1805M, and S1808A (Fig. 2A). Other amino acid substitutions had minor effects on the time course of IBa inactivation. However, their contribution to Cav2.1 inactivation is illustrated by the significant shifts of the availability curve (Fig. 2B).

Most dramatic changes in Cav2.1 inactivation occurred upon substitution of Met-1811 by glutamine or other charged/polar amino acids (M1811Q > M1811N > M1811E > M1811K). For example, M1811Q induced a 75-fold acceleration in the initial rate of IBa inactivation compared with wild type, resulting in a Cav2.1 mutant inactivating with similar kinetics as Cav3.1 (Fig. 3).

We mutated each of the amino acids that are conserved between all Ca2+ channel alpha 1-subunits in order to probe the role of these residues in Ca2+ channel inactivation. Out of the eight alanine substitution mutants, only N1813A, M1820A, and A1817S formed functional Ca2+ channels. It is unclear if the cRNA of the non-expressing mutants is translated and the resulting alpha 1-subunits represent non-conducting channels or if expression is stopped on the level of translation. However, substitution of the residues concerned by methionine (Y1899M, F1800M, S1802M, F1803M, F1809M, and A1817M) resulted in functional alpha 12.1 mutants. Of these mutants, the most significant acceleration in channel inactivation was observed for mutations N1813A and A1817M. However, compared with up to a 75-fold acceleration in IBa inactivation observed upon mutation of Met-1811, substitutions of the non-conserved IVS6 residues clearly exhibit a less significant influence on the rate of Cav2.1 inactivation.

Thus, we hypothesize that Met-1811 along with the closely located Leu-1812, Asn-1813, and Ala-1817 residues plays a critical role in helix packing within this putative bundle-crossing region of Ca2+ channel alpha 1-subunits (in analogy to the orientation of S6 segments in KcsA channels (40); see Ref. 41 for review). Consistent with this notion, strong effects of amino acid substitutions within the inner pore region of Ca2+ channel alpha 1-subunits have previously been reported for segment IVS6 of alpha 11.2 (42) and segment IIIS6 of alpha 12.1 (15).

The Role of IVS6 Mutations in beta 2a-Subunit Modulation of Cav2.1 Inactivation-- Multiple beta -subunits appear to be associated to various extents in different parts of the mammalian brain with the alpha 12.1-subunit (43-45). Therefore, inactivation of Cav2.1 is expected to be modulated by tissue-specific beta -subunit expression. To gain a deeper understanding of the molecular mechanism of beta -subunit modulation of alpha 12.1, we investigated the role of inactivation determinants within segment IVS6 on Cav2.1 inactivation observed in the presence of either beta 1a- or beta 2a-subunits. Thus, we compared the kinetic properties of fast-inactivating alpha 12.1(mutant)/alpha 2-delta /beta 1a channels with the corresponding channel construct formed using beta 2a-subunits (alpha 12.1(mutant)/alpha 2-delta /beta 2a). As illustrated in Fig. 6, A and B, we observed two principle phenotypes of beta 2a modulation. One pattern of modulation (type I, Fig. 6A) was exhibited by wild type Cav2.1 channels and all IVS6 mutations that induced only moderate changes in the rate of fast inactivation (Figs. 2A and 3A). Coexpressing these constructs with beta 2a resulted in a dramatic decrease of fast inactivation (illustrated in the left panels of Fig. 6B for wild type alpha 12.1/alpha 2-delta /beta 2a and M1811I/alpha 2-delta /beta 2a channels). Type II modulation was found only in mutants that exhibited very rapid inactivation kinetics (such as M1811Q/alpha 12.1/alpha 2-delta /beta 1a and M1811N/alpha 12.1/alpha 2-delta /beta 1a, Fig. 6, A and B, right panels). For these mutants, coexpression of beta 2a did not markedly slow the transient component in IBa inactivation but, instead, attenuated a slow component of current decay.

One hypothesis that can be put forward to explain the different kinetic phenotypes is that structural changes in segment IVS6 affects the interaction of beta 2a with alpha 12.1(mutant)-subunits. Under this scenario, IVS6 residues would form part of a receptor for a beta 2a-modulated inactivation gate or lid interacting from the intracellular side of the channel pore (see Ref. 46 for a proposed hinged lid mechanism of Cav2.3 involving the I-II linker and segments IIS6 and IIIS6; see also Ref. 47). In the frame of such a hypothesis, structural changes in segment IVS6 would alter inactivation by changing the affinity of such a receptor site.

Alternatively, the alpha 12.1-beta interaction might be unaffected by structural changes in segment IVS6, and type I and type II modulation could simply result from an interplay of changes in microscopic inactivation rate constants induced by the different constructs. We have, therefore, analyzed the beta 2a-induced changes in inactivation gating of alpha 12.1 point mutants with type I and type II modulation in terms of a simple Cav2.1 inactivation model that accounts for state transitions between open (O), fast-inactivated (Fast-I), and slow-inactivated states (Slow-I) during a membrane depolarization (Fig. 7A; see also Ref. 15). Each of the alpha 12.1 point mutations caused individual effects on the microscopic rate constants of fast (alpha , beta ) and slow inactivation (gamma , delta ). Fig. 7B illustrates four typical examples for type I or type II modulation simulated by means of domestically written software: wild type alpha 12.1/alpha 2-delta /beta 1a channels, mutation M1811I/alpha 2-delta /beta 1a inducing an 8-fold acceleration of IBa decay, and two mutants that were typical for type II modulation (M1811Q/alpha 2-delta /beta 1a with 75- and M1811N/alpha 2-delta /beta 1a with about 20-fold faster IBa decay than wild type).


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Fig. 7.   Simulation of type I and type II modulation of mutant Cav2.1 inactivation. A, kinetic scheme of CaV2.1 inactivation with microscopic rates of fast (Fast-I) (alpha , beta ) and slow (Slow-I) (gamma , delta ) inactivation (see also Ref. 15). B, left column: IBa of the mutants from Fig. 6B are superimposed by simulated inactivation kinetics (dashed lines). Right column: alpha , beta , gamma , and delta  values used to simulate inactivation kinetics by means of the model shown in A. The beta 2a-induced changes in inactivation gating of all channel constructs are reproduced by a similar (~1000-fold) acceleration of the backward transition rate from the fast-inactivated to the open state. In wild type (alpha 12.1/alpha 2-delta /beta 1a) and other channel constructs with a comparable rate of fast inactivation, the impact of the beta 2a-subunit-induced increase in the backward rate constant beta  (100 s-1) is much larger than the impact of the forward rate alpha  (1-6 s-1). Consequently, only a negligible amount of channels reside in Fast-I, and the kinetics of IBa decay of alpha 12.1/alpha 2-delta /beta 2a channels are mainly determined by the rates of slow inactivation (gamma  and delta ). In faster-inactivating mutants, the forward rate alpha  (23 s-1 in M1811N and 90 s-1 in M1811Q) is comparable with the backward rate beta  (about 30 s-1), resulting in biphasic inactivation kinetics with a pronounced transient component (determined by the rates alpha  and beta ) and a slowly decaying second phase in IBa inactivation (rates gamma  and delta ).

An ~1000-fold acceleration of a single microscopic transition rate (beta ) from the fast-inactivated to the open state reproduced the beta 2a-subunit-induced changes in inactivation in all cases. A similar acceleration in the rate constant beta  reproduced type I and type II modulation of other alpha 12.1 mutants illustrated in Fig. 6A (data not shown).

Taken together, the simulations suggest that the beta 2a-subunit destabilizes the fast-inactivated channel conformation similarly for each of the alpha 12.1 mutants. Therefore, the different inactivation patterns of wild type Cav2.1 and alpha 12.1(mutant)/alpha 2-delta /beta 2a constructs (type I and type II) are likely to arise from the interplay of microscopic transition rates between the open and fast-inactivated states (see the legend of Fig. 7).

In summary, we identified new hot spots of determinants of Ca2+ channel inactivation within the IVS6 segment of alpha 12.1. Substitution of two Cav2.1 amino acids by their Cav1.1 counterparts (ML1811/1812II) reproduced the inactivation properties (i.e. rate of inactivation, V0.5,act, and V0.5,inact) of a previously studied chimeric channel (AL23, Ref. 27), suggesting that these residues markedly contribute to voltage-dependent inactivation of Cav2.1. Nevertheless, the structural details of how the IVS6 segment within Cav2.1 influences channel inactivation remain to be clarified. Noncovalent interactions of specific IVS6 residues with neighboring transmembrane segments (i.e. IS6 or IVS5) are feasible. It will, therefore, be interesting to study if similar changes in Cav2.1 inactivation occur upon amino acid substitutions on other S6 segments by charged or polar residues.

The most intriguing result of this study, perhaps is that IVS6- and beta 2a-subunit-mediated conformational changes during Cav2.1 inactivation apparently occur in an independent manner (Fig. 7). Hence, type I and type II kinetics of the different alpha 12.1 IVS6 mutants can be simulated by assuming a uniform beta 2a-mediated destabilization of the fast-inactivated channel state (Fig. 7).

    ACKNOWLEDGEMENTS

We thank Prof. H. Glossmann for continuous support, Dr. Perez-Reyes for the cDNA of the alpha 13.1-subunit, Dr. E. N. Timin for the computer simulation software, and Dr. R.T. Dirksen for comments on the manuscript.

    FOOTNOTES

* This work was supported by Fonas zur Förderung der Wissenscheflichen Forschung Grants P12649-MED and P12828-MED, a grant from the Else Kröner-Fresenius-Stiftung, and a grant from the Austrian National Bank (to S. H.).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 Contributed equally to this work.

§ To whom correspondence should be addressed. Tel.: 43-512-507-3154; Fax: 43-512-588627; E-mail: Steffen.Hering@uibk.ac.at.

Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M010491200

    ABBREVIATIONS

The abbreviation used is: ML/II, ML1811/1812II..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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