Fast inactivation of voltage-dependent calcium channels. A hinged-lid mechanism?

We recently described domains II and III as important determinants of fast, voltage-dependent inactivation of R-type calcium channels (Spaetgens, R. L., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 22428-22438). Here we examine in greater detail the structural determinants of inactivation using a series of chimeras comprising various regions of wild type alpha(1C) and alpha(1E) calcium channels. Substitution of the II S6 and/or III S6 segments of alpha(1E) into the alpha(1C) backbone resulted in rapid inactivation rates that closely approximated those of wild type alpha(1E) channels. However, neither individual or combined substitution of the II S6 and III S6 segments could account for the 60 mV more negative half-inactivation potential seen with wild type alpha(1E) channels, indicating that the S6 regions contribute only partially to the voltage dependence of inactivation. Interestingly, the converse replacement of alpha(1E) S6 segments of domains II, III, or II+III with those of alpha(1C) was insufficient to significantly slow inactivation rates. Only when the I-II linker region and the domain II and III S6 regions of alpha(1E) were concomitantly replaced with alpha(1C) sequence could inactivation be abolished. Conversely, introduction of the alpha(1E) domain I-II linker sequence into alpha(1C) conferred alpha(1E)-like inactivation rates, indicating that the domain I-II linker is a key contributor to calcium channel inactivation. Overall, our data are consistent with a mechanism in which inactivation of voltage-dependent calcium channels may occur via docking of the I-II linker region to a site comprising, at least in part, the domain II and III S6 segments.     

Voltage and calcium use the same molecular determinants to inactivate calcium channels

During sustained depolarization, voltage-gated Ca2+ channels progressively undergo a transition to a nonconducting, inactivated state, preventing Ca2+ overload of the cell. This transition can be triggered either by the membrane potential (voltage-dependent inactivation) or by the consecutive entry of Ca2+ (Ca2+-dependent inactivation), depending on the type of Ca2+ channel. These two types of inactivation are suspected to arise from distinct underlying mechanisms, relying on specific molecular sequences of the different pore-forming Ca2+ channel subunits. Here we report that the voltage-dependent inactivation (of the alpha1A Ca2+ channel) and the Ca2+-dependent inactivation (of the alpha1C Ca2+ channel) are similarly influenced by Ca2+ channel beta subunits. The same molecular determinants of the beta subunit, and therefore the same subunit interactions, influence both types of inactivation. These results strongly suggest that the voltage and the Ca2+-dependent transitions leading to channel inactivation use homologous structures of the different alpha1 subunits and occur through the same molecular process. A model of inactivation taking into account these new data is presented.     

Interaction of SNX482 with domains III and IV inhibits activation gating of alpha(1E) (Ca(V)2.3) calcium channels.

We have investigated the action of SNX482, a toxin isolated from the venom of the tarantula Hysterocrates gigas, on voltage-dependent calcium channels expressed in tsa-201 cells. Upon application of 200 nM SNX482, R-type alpha(1E) calcium channels underwent rapid and complete inhibition, which was only poorly reversible upon washout. However, upon application of strong membrane depolarizations, rapid and complete recovery from inhibition was obtained. Tail current analysis revealed that SNX482 mediated an approximately 70 mV depolarizing shift in half-activation potential, suggesting that the toxin inhibits alpha(1E) calcium channels by preventing their activation. Experiments involving chimeric channels combining structural features of alpha(1E) and alpha(1C) subunits indicated that the presence of the domain III and IV of alpha(1E) is a prerequisite for a strong gating inhibition. In contrast, L-type alpha(1C) channels underwent incomplete inhibition at saturating concentrations of SNX482 that was paralleled by a small shift in half-activation potential and which could be rapidly reversed, suggesting a less pronounced effect of the toxin on L-type calcium channel gating. We conclude that SNX482 does not exhibit unequivocal specificity for R-type channels, but highly effectively antagonizes their activation.     

Identification of inactivation determinants in the domain IIS6 region of high voltage-activated calcium channels

We have recently reported that transfer of the domain IIS6 region from rapidly inactivating R-type (alpha(1E)) calcium channels to slowly inactivating L-type (alpha(1C)) calcium channel confers rapid inactivation (Stotz, S. C., Hamid, J., Spaetgens, R. L., Jarvis, S. E., and Zamponi, G. W. (2000) J. Biol. Chem. 275, 24575-24582). Here we have identified individual amino acid residues in the IIS6 regions that are responsible for these effects. In this region, alpha(1C) and alpha(1E) channels differ in seven residues, and exchanging five of those residues individually or in combination did not significantly affect inactivation kinetics. By contrast, replacement of residues Phe-823 or Ile-829 of alpha(1C) with the corresponding alpha(1E) residues significantly accelerated inactivation rates and, when substituted concomitantly, approached the rapid inactivation kinetics of R-type channels. A systematic substitution of these residues with a series of other amino acids revealed that decreasing side chain size at position 823 accelerates inactivation, whereas a dependence of the inactivation kinetics on the degree of hydrophobicity could be observed at position 829. Although these point mutations facilitated rapid entry into the inactivated state of the channel, they had little to no effect on the rate of recovery from inactivation. This suggests that the development of and recovery from inactivation are governed by separate structural determinants. Finally, the effects of mutations that accelerated alpha(1C) inactivation could still be antagonized following coexpression of the rat beta(2a) subunit or by domain I-II linker substitutions that produce ultra slow inactivation of wild type channels, indicating that the inactivation kinetics seen with the mutants remain subject to regulation by the domain I-II linker. Overall, our results provide novel insights into a complex process underlying calcium channel inactivation.     

Functional roles of the extracellular segments of the sodium channel alpha subunit in voltage-dependent gating and modulation by beta1 subunits.

Voltage-gated sodium channels consist of a pore-forming alpha subunit associated with beta1 subunits and, for brain sodium channels, beta2 subunits. Although much is known about the structure and function of the alpha subunit, there is little information on the functional role of the 16 extracellular loops. To search for potential functional activities of these extracellular segments, chimeras were studied in which an individual extracellular loop of the rat heart (rH1) alpha subunit was substituted for the corresponding segment of the rat brain type IIA (rIIA) alpha subunit. In comparison with rH1, wild-type rIIA alpha subunits are characterized by more positive voltage-dependent activation and inactivation, a more prominent slow gating mode, and a more substantial shift to the fast gating mode upon coexpression of beta1 subunits in Xenopus oocytes. When alpha subunits were expressed alone, chimeras with substitutions from rH1 in five extracellular loops (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had negatively shifted activation, and chimeras with substitutions in three of these (IISS2-S6, IIIS1-S2, and IVS3-S4) also had negatively shifted steady-state inactivation. rIIA alpha subunit chimeras with substitutions from rH1 in five extracellular loops (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) favored the fast gating mode. Like wild-type rIIA alpha subunits, all of the chimeric rIIA alpha subunits except chimera IVSS2-S6 were shifted almost entirely to the fast gating mode when coexpressed with beta1 subunits. In contrast, substitution of extracellular loop IVSS2-S6 substantially reduced the effectiveness of beta1 subunits in shifting rIIA alpha subunits to the fast gating mode. Our results show that multiple extracellular loops influence voltage-dependent activation and inactivation and gating mode of sodium channels, whereas segment IVSS2-S6 plays a dominant role in modulation of gating by beta1 subunits. Evidently, several extracellular loops are important determinants of sodium channel gating and modulation.     

Structure and function of voltage-dependent ion channel regulatory beta subunits

Voltage-dependent K(+), Ca(2+), and Na(+) channels play vital roles in basic physiological processes, including management of the action potential, signal transduction, and secretion. They share the common function of passively transporting ions across cell membranes; thus, it would not be surprising if they should exhibit similarities of both structure and mechanism. Indeed, the principal pore-forming (alpha) subunits of each show either exact or approximate 4-fold symmetry and share a similar transmembrane topology, and all are gated by changes in membrane potential. Furthermore, these channels all possess an auxiliary polypeptide, designated the beta subunit, which plays an important role in their regulation. Despite considerable functional semblences and abilities to interact with structurally similar alpha subunits, however, there is considerable structural diversity among the beta subunits. In this review, we discuss the similarities and differences in the structures and functions of the beta subunits of the voltage-dependent K(+), Ca(2+), and Na(+) channels.     

A novel Ca(V)1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits

Voltage-dependent L-type Ca(2+) (Ca(V)1.2) channels are the principal Ca(2+) entry pathway in arterial myocytes. Ca(V)1.2 channels regulate multiple vascular functions and are implicated in the pathogenesis of human disease, including hypertension. However, the molecular identity of Ca(V)1.2 channels expressed in myocytes of myogenic arteries that regulate vascular pressure and blood flow is unknown. Here, we cloned Ca(V)1.2 subunits from resistance size cerebral arteries and demonstrate that myocytes contain a novel, cysteine rich N terminus that is derived from exon 1 (termed "exon 1c"), which is located within CACNA1C, the Ca(V)1.2 gene. Quantitative PCR revealed that exon 1c was predominant in arterial myocytes, but rare in cardiac myocytes, where exon 1a prevailed. When co-expressed with alpha(2)delta subunits, Ca(V)1.2 channels containing the novel exon 1c-derived N terminus exhibited: 1) smaller whole cell current density, 2) more negative voltages of half activation (V(1/2,act)) and half-inactivation (V(1/2,inact)), and 3) reduced plasma membrane insertion, when compared with channels containing exon 1b. beta(1b) and beta(2a) subunits caused negative shifts in the V(1/2,act) and V(1/2,inact) of exon 1b-containing Ca(V)1.2alpha(1)/alpha(2)delta currents that were larger than those in exon 1c-containing Ca(V)1.2alpha(1)/alpha(2)delta currents. In contrast, beta(3) similarly shifted V(1/2,act) and V(1/2,inact) of currents generated by exon 1b- and exon 1c-containing channels. beta subunits isoform-dependent differences in current inactivation rates were also detected between N-terminal variants. Data indicate that through novel alternative splicing at exon 1, the Ca(V)1.2 N terminus modifies regulation by auxiliary subunits. The novel exon 1c should generate distinct voltage-dependent Ca(2+) entry in arterial myocytes, resulting in tissue-specific Ca(2+) signaling.     

Mutations in the EF-hand motif impair the inactivation of barium currents of the cardiac alpha1C channel.

Calcium-dependent inactivation has been described as a negative feedback mechanism for regulating voltage-dependent calcium influx in cardiac cells. Most recent evidence points to the C-terminus of the alpha1C subunit, with its EF-hand binding motif, as being critical in this process. The EF-hand binding motif is mostly conserved between the C-termini of six of the seven alpha1 subunit Ca2+ channel genes. The role of E1537 in the C-terminus of the alpha1C calcium channel inactivation was investigated here after expression in Xenopus laevis oocytes. Whole-cell currents were measured in the presence of 10 mM Ba2+ or 10 mM Ca2+ after intracellular injection of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Against all expectations, our results showed a significant reduction in the rate of voltage-dependent inactivation as measured in Ba2+ solutions for all E1537 mutants, whereas calcium-dependent inactivation appeared unscathed. Replacing the negatively charged glutamate residue by neutral glutamine, glycine, serine, or alanine significantly reduced the rate of Ba2+-dependent inactivation by 1.5-fold (glutamine) to 3.5-fold (alanine). The overall rate of macroscopic inactivation measured in Ca2+ solutions was also reduced, although a careful examination of the distribution of the fast and slow time constants suggests that only the slow time constant was significantly reduced in the mutant channels. The fast time constant, the hallmark of Ca2+-dependent inactivation, remained remarkably constant among wild-type and mutant channels. Moreover, inactivation of E1537A channels, in both Ca2+ and Ba2+ solutions, appeared to decrease with membrane depolarization, whereas inactivation of wild-type channels became faster with positive voltages. All together, our results showed that E1537 mutations impaired voltage-dependent inactivation and suggest that the proximal part of the C-terminus may play a role in voltage-dependent inactivation in L-type alpha1C channels.     

G protein modulation of N-type calcium channels is facilitated by physical interactions between syntaxin 1A and Gbetagamma

The direct modulation of N-type calcium channels by G protein betagamma subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of N-type channels and Gbetagamma have recently been identified (see, i.e., Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446); however, little is known about cellular mechanisms that modulate this interaction. Here we report that a protein of the presynaptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by Gbetagamma. When syntaxin 1A was coexpressed with (N-type) alpha(1B) + alpha(2)-delta + beta(1b) channels in tsA-201 cells, the channels underwent a 18 mV negative shift in half-inactivation potential, as well as a pronounced tonic G protein inhibition as assessed by its reversal by strong membrane depolarizations. This tonic inhibition was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the enhanced G protein modulation. However, when G protein betagamma subunits were concomitantly coexpressed, the toxin became ineffective in removing G protein inhibition, suggesting that syntaxin 1A optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that Gbetagamma physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with Gbetagamma and the synprint motif of the N-type channel II-III linker. Taken together, our experiments suggest a mechanism by which syntaxin 1A mediates a colocalization of G protein betagamma subunits and N-type calcium channels, thus resulting in more effective G protein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the structural specialization of the presynaptic terminal.     

Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.     

Fast and slow inactivation kinetics of the Ca2+ channels ECaC1 and ECaC2 (TRPV5 and TRPV6). Role of the intracellular loop located between transmembrane segments 2 and 3

The Ca(2+) channels ECaC1 and ECaC2 (TRPV5 and TRPV6) share several functional properties including permeation profile and Ca(2+)-dependent inactivation. However, the kinetics of ECaC2 currents notably differ from ECaC1 currents. The initial inactivation is much faster in ECaC2 than in ECaC1, and the kinetic differences between Ca(2+) and Ba(2+) currents are more pronounced for ECaC2 than ECaC1. Here, we identify the structural determinants for these functional differences. Chimeric proteins were expressed heterologously in HEK 293 cells and studied by patch clamp analysis. Both channels retained their phenotype after exchanging the complete N termini, the C termini, or even both N and C termini, i.e. ECaC1 with the ECaC2 N or C terminus still showed the ECaC1 phenotype and vice versa. The substitution of the intracellular loop between the transmembrane domains 2 and 3 of ECaC2 with that of ECaC1 induced a delay of inactivation. Three amino acid residues (Leu-409, Val-411 and Thr-412) present in this loop determine the fast inactivation behavior. When this intracellular loop between the transmembrane domains 2 and 3 of ECaC1 was exchanged with the TM2-TM3 loop of ECaC2, the ECaC1 kinetics were analogous to ECaC2. In conclusion, the TM2-TM3 loop is a critical determinant of the inactivation in ECaC1 and ECaC2.     

Molecular determinants of inactivation within the I-II linker of alpha1E (CaV2.3) calcium channels

Voltage-dependent inactivation of CaV2.3 channels was investigated using point mutations in the beta-subunit-binding site (AID) of the I-II linker. The quintuple mutant alpha1E N381K + R384L + A385D + D388T + K389Q (NRADK-KLDTQ) inactivated like the wild-type alpha1E. In contrast, mutations of alpha1E at position R378 (position 5 of AID) into negatively charged residues Glu (E) or Asp (D) significantly slowed inactivation kinetics and shifted the voltage dependence of inactivation to more positive voltages. When co-injected with beta3, R378E inactivated with tau(inact) = 538 +/- 54 ms (n = 14) as compared with 74 +/- 4 ms (n = 21) for alpha1E (p < 0.001) with a mid-potential of inactivation E(0.5) = -44 +/- 2 mV (n = 10) for R378E as compared with E(0.5) = -64 +/- 3 mV (n = 9) for alpha1E. A series of mutations at position R378 suggest that positively charged residues could promote voltage-dependent inactivation. R378K behaved like the wild-type alpha1E whereas R378Q displayed intermediate inactivation kinetics. The reverse mutation E462R in the L-type alpha1C (CaV1.2) produced channels with inactivation properties comparable to alpha1E R378E. Hence, position 5 of the AID motif in the I-II linker could play a significant role in the inactivation of Ca(V)1.2 and CaV2.3 channels.     

C-terminal fragments of the alpha 1C (CaV1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated alpha 1C subunits

L-type Ca(2+) channels in native tissues have been found to contain a pore-forming alpha(1) subunit that is often truncated at the C terminus. However, the C terminus contains many important domains that regulate channel function. To test the hypothesis that C-terminal fragments may associate with and regulate C-terminal-truncated alpha(1C) (Ca(V)1.2) subunits, we performed electrophysiological and biochemical experiments. In tsA201 cells expressing either wild type or C-terminal-truncated alpha(1C) subunits in combination with a beta(2a) subunit, truncation of the alpha(1C) subunit by as little as 147 amino acids led to a 10-15-fold increase in currents compared with those obtained from control, full-length alpha(1C) subunits. Dialysis of cells expressing the truncated alpha(1C) subunits with C-terminal fragments applied through the patch pipette reconstituted the inhibition of the channels seen with full-length alpha(1C) subunits. In addition, C-terminal deletion mutants containing a tethered C terminus also exhibited the C-terminal-induced inhibition. Immunoprecipitation assays demonstrated the association of the C-terminal fragments with truncated alpha(1C) subunits. In addition, glutathione S-transferase pull-down assays demonstrated that the C-terminal inhibitory fragment could associate with at least two domains within the C terminus. The results support the hypothesis the C- terminal fragments of the alpha(1C) subunit can associate with C-terminal-truncated alpha(1C) subunits and inhibit the currents through L-type Ca(2+) channels.     

Ultra-slow inactivation in mu1 Na+ channels is produced by a structural rearrangement of the outer vestibule

While studying the adult rat skeletal muscle Na+ channel outer vestibule, we found that certain mutations of the lysine residue in the domain III P region at amino acid position 1237 of the alpha subunit, which is essential for the Na+ selectivity of the channel, produced substantial changes in the inactivation process. When skeletal muscle alpha subunits (micro1) with K1237 mutated to either serine (K1237S) or glutamic acid (K1237E) were expressed in Xenopus oocytes and depolarized for several minutes, the channels entered a state of inactivation from which recovery was very slow, i.e., the time constants of entry into and exit from this state were in the order of approximately 100 s. We refer to this process as "ultra-slow inactivation". By contrast, wild-type channels and channels with the charge-preserving mutation K1237R largely recovered within approximately 60 s, with only 20-30% of the current showing ultra-slow recovery. Coexpression of the rat brain beta1 subunit along with the K1237E alpha subunit tended to accelerate the faster components of recovery from inactivation, as has been reported previously of native channels, but had no effect on the mutation-induced ultra-slow inactivation. This implied that ultra-slow inactivation was a distinct process different from normal inactivation. Binding to the pore of a partially blocking peptide reduced the number of channels entering the ultra-slow inactivation state, possibly by interference with a structural rearrangement of the outer vestibule. Thus, ultra-slow inactivation, favored by charge-altering mutations at site 1237 in micro1 Na+ channels, may be analogous to C-type inactivation in Shaker K+ channels.     

Steady-state and closed-state inactivation properties of inactivating BK channels

Calcium-dependent potassium (BK-type) Ca2+ and voltage-dependent K+ channels in chromaffin cells exhibit an inactivation that probably arises from coassembly of Slo1 alpha subunits with auxiliary beta subunits. One goal of this work was to determine whether the Ca2+ dependence of inactivation arises from any mechanism other than coupling of inactivation to the Ca2+ dependence of activation. Steady-state inactivation and the onset of inactivation were studied in inside-out patches and whole-cell recordings from rat adrenal chromaffin cells with parallel experiments on inactivating BK channels resulting from cloned alpha + beta2 subunits. In both cases, steady-state inactivation was shifted to more negative potentials by increases in submembrane [Ca2+] from 1 to 60 microM. At 10 and 60 microM Ca2+, the maximal channel availability at negative potentials was similar despite a shift in the voltage of half availability, suggesting there is no strictly Ca2+-dependent inactivation. In contrast, in the absence of Ca2+, depolarization to potentials positive to +20 mV induces channel inactivation. Thus, voltage-dependent, but not solely Ca2+-dependent, kinetic steps are required for inactivation to occur. Finally, under some conditions, BK channels are shown to inactivate as readily from closed states as from open states, indicative that a key conformational change required for inactivation precedes channel opening.     

Functional coupling between the Kv1.1 channel and aldoketoreductase Kvbeta1

The Shaker family voltage-dependent potassium channels (Kv1) assemble with cytosolic beta-subunits (Kvbeta) to form a stable complex. All Kvbeta subunits have a conserved core domain, which in one of them (Kvbeta2) is an aldoketoreductase that utilizes NADPH as a cofactor. In addition to this core, Kvbeta1 has an N terminus that closes the channel by the N-type inactivation mechanism. Point mutations in the putative catalytic site of Kvbeta1 alter the on-rate of inactivation. Whether the core of Kvbeta1 functions as an enzyme and whether its enzymatic activity affects N-type inactivation had not been explored. Here, we show that Kvbeta1 is a functional aldoketoreductase and that oxidation of the Kvbeta1-bound cofactor, either enzymatically by a substrate or non-enzymatically by hydrogen peroxide or NADP(+), induces a large increase in open channel current. The modulation is not affected by deletion of the distal C terminus of the channel, which has been suggested in structural studies to interact with Kvbeta. The rate of increase in current, which reflects NADPH oxidation, is approximately 2-fold faster at 0-mV membrane potential than at -100 mV. Thus, cofactor oxidation by Kvbeta1 is regulated by membrane potential, presumably via voltage-dependent structural changes in Kv1.1 channels.     

S1–S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1–S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1–S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNa_V1.4. These show that side chains of putative countercharges in hNa_V1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I–III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.     

The HOOK-domain between the SH3 and the GK domains of Cavbeta subunits contains key determinants controlling calcium channel inactivation

Ca(v)beta subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3)-domain and a guanylate kinase-like (GK)-domain. The SH3-domain is split, with its final (fifth) beta-strand separated from the rest of the domain by an intervening sequence termed the HOOK-domain, whose sequence varies between Ca(v)beta subunits. Here we have been guided by the recent structural studies of Ca(v)beta subunits in the design of specific truncated constructs, with the goal of investigating the role of the HOOK-domain of Ca(v)beta subunits in the modulation of inactivation of N-type calcium channels. We have coexpressed the beta subunit constructs with Ca(v)2.2 and alpha(2)delta-2, using the N-terminally palmitoylated beta(2a) subunit, because it supports very little voltage-dependent inactivation, and made comparisons with beta(1b) domains. Deletion of the variable region of the beta(2a) HOOK-domain resulted in currents with a rapidly inactivating component, and additional mutation of the beta(2a) palmitoylation motif further enhanced inactivation. The isolated GK-domain of beta(2a) alone enhanced current amplitude, but the currents were rapidly and completely inactivating. When the beta(2a)-GK-domain construct was extended proximally, by including the HOOK-domain and the epsilon-strand of the SH3-domain, inactivation was about four-fold slower than in the absence of the HOOK domain. When the SH3-domain of beta(2a) truncated prior to the HOOK-domain was coexpressed with the (HOOK+epsilonSH3+GK)-domain of beta(2a), all the properties of beta(2a) were restored, in terms of loss of inactivation. Furthermore, removal of the HOOK sequence from the (HOOK+epsilonSH3+GK)-beta(2a) construct increased inactivation. Together, these results provide evidence that the HOOK domain is an important determinant of inactivation.     

The voltage dependence of hEag currents is not determined solely by membrane-spanning domains

The ether-à-go-go potassium channels hEag1 and hEag2 are highly homologous. Even though both possess identical voltage-sensing domain S4, the channels act differently in response to voltage. Therefore we asked whether transmembrane domains other than the voltage sensor could contribute to the voltage-dependent behaviour of these potassium channels. For this chimaeras were created, in which each single transmembrane domain of hEag1 was replaced by the corresponding segment of hEag2. The voltage-dependent properties of the chimaeras were analysed after expression in Xenopus laevis oocytes using the two-electrode voltage-clamp method. By this we found, that only the mutations in transmembrane domains S5 and S6 are able to change the voltage sensitivity of hEag1 by shifting the half-activation potential (V ₅₀) to values intermediate between the two wild types. Moreover, the presence of Mg²⁺ has strong effects on the voltage sensitivity of hEag2 shifting V ₅₀ by more than 50 mV to more positive values. Interestingly, despite the identical binding site Mg²⁺ showed only little effects on hEag1 or the chimaeras. Altogether, our data suggest that not only transmembrane spanning regions, but also non-membrane spanning regions are responsible for differences in the behaviour of the hEag1 and hEag2 potassium channels. EBSA Satellite meeting: Ion channels, Leeds, July 2007.