A role for the S0 transmembrane segment in voltage-dependent gating of BK channels

BK (Maxi-K) channel activity is allosterically regulated by a Ca2+ sensor, formed primarily by the channel's large cytoplasmic carboxyl tail segment, and a voltage sensor, formed by its transmembrane helices. As with other voltage-gated K channels, voltage sensing in the BK channel is accomplished through interactions of the S1-S4 transmembrane segments with the electric field. However, the BK channel is unique in that it contains an additional amino-terminal transmembrane segment, S0, which is important in the functional interaction between BK channel alpha and beta subunits. In this study, we used perturbation mutagenesis to analyze the role of S0 in channel gating. Single residues in the S0 region of the BK channel were substituted with tryptophan to give a large change in side chain volume; native tryptophans in S0 were substituted with alanine. The effects of the mutations on voltage- and Ca2+-dependent gating were quantified using patch-clamp electrophysiology. Three of the S0 mutants (F25W, L26W, and S29W) showed especially large shifts in their conductance-voltage (G-V) relations along the voltage axis compared to wild type. The G-V shifts for these mutants persisted at nominally 0 Ca2+, suggesting that these effects cannot arise simply from altered Ca2+ sensitivity. The basal open probabilities for these mutants at hyperpolarized voltages (where voltage sensor activation is minimal) were similar to wild type, suggesting that these mutations may primarily perturb voltage sensor function. Further analysis using the dual allosteric model for BK channel gating showed that the major effects of the F25W, L26W, and S29W mutations could be accounted for primarily by decreasing the equilibrium constant for voltage sensor movement. We conclude that S0 may make functional contact with other transmembrane regions of the BK channel to modulate the equilibrium between resting and active states of the channel's voltage sensor.     

An S6 mutation in BK channels reveals beta1 subunit effects on intrinsic and voltage-dependent gating

Large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels are exquisitely regulated to suit their diverse roles in a large variety of physiological processes. BK channels are composed of pore-forming alpha subunits and a family of tissue-specific accessory beta subunits. The smooth muscle-specific beta1 subunit has an essential role in regulating smooth muscle contraction and modulates BK channel steady-state open probability and gating kinetics. Effects of beta1 on channel's gating energetics are not completely understood. One of the difficulties is that it has not yet been possible to measure the effects of beta1 on channel's intrinsic closed-to-open transition (in the absence of voltage sensor activation and Ca(2+) binding) due to the very low open probability in the presence of beta1. In this study, we used a mutation of the alpha subunit (F315Y) that increases channel openings by greater than four orders of magnitude to directly compare channels' intrinsic open probabilities in the presence and absence of the beta1 subunit. Effects of beta1 on steady-state open probabilities of both wild-type alpha and the F315Y mutation were analyzed using the dual allosteric HA model. We found that mouse beta1 has two major effects on channel's gating energetics. beta1 reduces the intrinsic closed-to-open equilibrium that underlies the inhibition of BK channel opening seen in submicromolar Ca(2+). Further, P(O) measurements at limiting slope allow us to infer that beta1 shifts open channel voltage sensor activation to negative membrane potentials, which contributes to enhanced channel opening seen at micromolar Ca(2+) concentrations. Using the F315Y alpha subunit with deletion mutants of beta1, we also demonstrate that the small N- and C-terminal intracellular domains of beta1 play important roles in altering channel's intrinsic opening and voltage sensor activation. In summary, these results demonstrate that beta1 has distinct effects on BK channel intrinsic gating and voltage sensor activation that can be functionally uncoupled by mutations in the intracellular domains.     

Impact of gating modulation in CaV1.3 L-type calcium channels

CaV1.3 L-type channels control inner hair cell (IHC) sensory and sinoatrial node (SAN) function, and excitability in central neurons by means of their low-voltage activation and inactivation properties. In SAN cells CaV1.3 inward calcium current (ICa) inactivates rapidly whereas in IHCs inactivation is slow. A candidate suggested in slowing CaV1.3 channel inactivation is the presynaptically located ribbon-synapse protein RIM that is expressed in immature IHCs in presynaptic compartments also expressing CaV1.3 channels. CaV1.3 channel gating is also modulated by an intramolecular C-terminal mechanism. This mechanism was elicited during analysis of human C-terminal splice variants that differ in the length of their C-terminus and that modulates the channel's negative activation range and slows calcium-dependent inactivation.     

Hydrophobic substitution mutations in the S4 sequence alter voltage-dependent gating in Shaker K+ channels

Voltage-activated Na+, Ca2+, and K+ channels contain a common motif, the S4 sequence, characterized by a basic residue at every third position interspersed mainly with hydrophobic residues. The S4 sequence is proposed to function as the voltage sensor and to move in response to membrane depolarization, triggering conformational changes that open the channel. This hypothesis has been tested in previous studies which revealed that mutations of the S4 basic residues often shift the curve of voltage dependence of activation along the voltage axis. We find that comparable or larger shifts are caused by conservative substitutions of hydrophobic residues in the S4 sequence of the Shaker K+ channel. We suggest that the S4 structure plays an essential role in determining the relative stabilities of the closed and open states of the channel.     

Computing transient gating charge movement of voltage-dependent ion channels

The opening of voltage-gated sodium, potassium, and calcium ion channels has a steep relationship with voltage. In response to changes in the transmembrane voltage, structural movements of an ion channel that precede channel opening generate a capacitative gating current. The net gating charge displacement due to membrane depolarization is an index of the voltage sensitivity of the ion channel activation process. Understanding the molecular basis of voltage-dependent gating of ion channels requires the measurement and computation of the gating charge, Q. We derive a simple and accurate semianalytic approach to computing the voltage dependence of transient gating charge movement (Q–V relationship) of discrete Markov state models of ion channels using matrix methods. This approach allows rapid computation of Q–V curves for finite and infinite length step depolarizations and is consistent with experimentally measured transient gating charge. This computational approach was applied to Shaker potassium channel gating, including the impact of inactivating particles on potassium channel gating currents.     

Mechanism of voltage-dependent gating in skeletal muscle chloride channels.

Voltage-dependent gating was investigated in a recombinant human skeletal muscle Cl- channel, hCIC-1, heterologously expressed in human embryonic kidney (HEK-293) cells. Gating was found to be mediated by two qualitatively distinct processes. One gating step operates on a microsecond time scale and involves the rapid rearrangement of two identical intramembranous voltage sensors, each consisting of a single titratable residue. The second process occurs on a millisecond time scale and is due to a blocking-unblocking reaction mediated by a cytoplasmic gate that interacts with the ion pore of the channel. These results illustrate a rather simple structural basis for voltage sensing that has evolved in skeletal muscle Cl- channels and provides evidence for the existence of a cytoplasmic gating mechanism in an anion channel analogous to the "ball and chain" mechanism of voltage-gated cation channels.     

Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.     

Conserved gating hinge in ligand- and voltage-dependent K+ channels

Ion channels open and close their pore in a process called gating. On the basis of crystal structures of two voltage-independent K(+) channels, KcsA and MthK, a conformational change for gating has been proposed whereby the inner helix bends at a glycine hinge point (gating hinge) to open the pore and straightens to close it. Here we ask if a similar gating hinge conformational change underlies the mechanics of pore opening of two eukaryotic voltage-dependent K(+) channels, Shaker and BK channels. In the Shaker channel, substitution of the gating hinge glycine with alanine and several other amino acids prevents pore opening, but the ability to open is recovered if a secondary glycine is introduced at an adjacent position. A proline at the gating hinge favors the open state of the Shaker channel as if by preventing inner helix straightening. In BK channels, which have two adjacent glycine residues, opening is significantly hindered in a graded manner with single and double mutations to alanine. These results suggest that K(+) channels, whether ligand- or voltage-dependent, open when the inner helix bends at a conserved glycine gating hinge.     

Preventing voltage-dependent gating of anthrax toxin channels using engineered disulfides

The channel-forming component of anthrax toxin, (PA₆₃)₇, is a heptameric water-soluble protein at neutral pH, but under acidic conditions it spontaneously inserts into lipid bilayers to form a 14-stranded β-barrel ion-conducting channel. This channel plays a vital role in anthrax pathogenesis because it serves as a conduit for the membrane translocation of the two enzymatic components of anthrax toxin, lethal factor and edema factor. Anthrax channels open and close in response to changes in transmembrane voltage, a property shared by several other pore-forming toxins. We have discovered an unexpected phenomenon in cysteine-substituted channels that provides a window into this gating process: their normal voltage-dependent gating can be abolished by reaction with methanethiosulfonate (MTS) reagents or exposure to oxidizing conditions. Remarkably, this perturbation is seen with cysteines substituted at sites all along the ~100 Å length of the channel's β-barrel. In contrast, reaction with N-ethylmaleimide, a thiol-reactive compound that does not form a mixed disulfide, does not affect gating at any of the sites tested. These findings, coupled with our biochemical detection of dimers, have led us to conclude that MTS reagents are catalyzing the formation of intersubunit disulfide bonds that lock channels in a conducting state, and that voltage gating requires a conformational change that involves the entire β-barrel.     

Characterization of the gating brake in the I-II loop of CaV3 T-type calcium channels

Our interest was drawn to the I-II loop of Cav3 channels for two reasons: one, transfer of the I-II loop from a high voltage-activated channel (Cav2.2) to a low voltage-activated channel (Cav3.1) unexpectedly produced an ultra-low voltage activated channel; and two, sequence variants of the I-II loop found in childhood absence epilepsy patients altered channel gating and increased surface expression of Cav3.2 channels. To determine the roles of this loop we have studied the structure of the loop and the biophysical consequences of altering its structure. Deletions localized the gating brake to the first 62 amino acids after IS6 in all three Cav3 channels, establishing the evolutionary conservation of this region and its function. Circular dichroism was performed on a purified fragment of the I-II loop from Cav3.2 to reveal a high α-helical content. De novo computer modeling predicted the gating brake formed a helix-loop-helix structure. This model was tested by replacing the helical regions with poly-proline-glycine (PGPGPG), which introduces kinks and flexibility. These mutations had profound effects on channel gating, shifting both steady-state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure (poly-alanine, which forms α-helices) had more modest effects. Taken together, we conclude the second helix of the gating brake establishes important contacts with the gating machinery, thereby stabilizing a closed state of T-channels, and that this interaction is disrupted by depolarization, allowing the S6 segments to spread open and Ca (2+) ions to flow through.     

Impact of gating modulation in CaV1.3 L-type calcium channels

Ca_V1.3 L-type channels control inner hair cell (IHC) sensory and sinoatrial node (SAN) function, and excitability in central neurons by means of their low-voltage activation and inactivation properties. In SAN cells Ca_V1.3 inward calcium current (I_Ca) inactivates rapidly whereas in IHCs inactivation is slow. A candidate suggested in slowing Ca_V1.3 channel inactivation is the presynaptically located ribbon-synapse protein RIM that is expressed in immature IHCs in presynaptic compartments also expressing Ca_V1.3 channels. Ca_V1.3 channel gating is also modulated by an intramolecular C-terminal mechanism. This mechanism was elicited during analysis of human C-terminal splice variants that differ in the length of their C-terminus and that modulates the channel’s negative activation range and slows calcium-dependent inactivation.     

Swapping the I-II intracellular linker between L-type CaV1.2 and R-type CaV2.3 high-voltage gated calcium channels exchanges activation attributes

Calcium entry through voltage-gated calcium channels (VGCC) initiates diverse cellular functions. VGCC pore-forming subunit (Ca_Vα1) contains four homology repeats, each encompassing a voltage sensor and a pore domain. Three main classes of Ca_Vα1 subunits have been described, Ca_V1, Ca_V2 and Ca_V3 that differ in their voltage-dependence of activation and in the extent in which this process is modulated by the auxiliary β-subunit (Ca_Vβ). Association of Ca_Vβ induces a coil-to-helix conformation of the I-II intracellular linker joining the first and second repeat of CaVα1 that is thought to be crucial for modulation of channel function. When expressed in Xenopus laevis oocytes in the absence of Ca_Vβ the voltage to reach 50% activation (V_0.5) for Ca_V1.2 and Ca_V2.3 differs by more than 60 mV and the channel current-carrying capacity by more than thirty-fold. Here we report that the difference in V_0.5 is reduced to about 30 mV and the current-carrying capacity becomes virtually identical when the I-II linkers of Ca_V1.2 and Ca_V2.3 are swapped. Co-expression with Ca_Vβ increases the current-carrying capacity of chimeric channels by the same extent, while the difference in V_0.5 with respect to their corresponding parental channels vanishes. Our findings indicate that Ca_Vβ modulatory potency is determined by both, the nature of the I-II linker and the pore-forming subunit background. Moreover, they demonstrate that the I-II linker encodes self-reliant molecular determinants for channel activation and suggest that besides to the secondary structure adopted by this segment upon Ca_Vβ association, its chemical nature is as well relevant.     

Role of putative voltage-sensor countercharge D4 in regulating gating properties of CaV1.2 and CaV1.3 calcium channels

Voltage-dependent calcium channels (Ca_V) activate over a wide range of membrane potentials, and the voltage-dependence of activation of specific channel isoforms is exquisitely tuned to their diverse functions in excitable cells. Alternative splicing further adds to the stunning diversity of gating properties. For example, developmentally regulated insertion of an alternatively spliced exon 29 in the fourth voltage-sensing domain (VSD IV) of Ca_V1.1 right-shifts voltage-dependence of activation by 30 mV and decreases the current amplitude several-fold. Previously we demonstrated that this regulation of gating properties depends on interactions between positive gating charges (R1, R2) and a negative countercharge (D4) in VSD IV of Ca_V1.1. Here we investigated whether this molecular mechanism plays a similar role in the VSD IV of Ca_V1.3 and in VSDs II and IV of Ca_V1.2 by introducing charge-neutralizing mutations (D4N or E4Q) in the corresponding positions of Ca_V1.3 and in two splice variants of Ca_V1.2. In both channels the D4N (VSD IV) mutation resulted in a ?5 mV right-shift of the voltage-dependence of activation and in a reduction of current density to about half of that in controls. However in Ca_V1.2 the effects were independent of alternative splicing, indicating that the two modulatory processes operate by distinct mechanisms. Together with our previous findings these results suggest that molecular interactions engaging D4 in VSD IV contribute to voltage-sensing in all examined Ca_V1 channels, however its striking role in regulating the gating properties by alternative splicing appears to be a unique property of the skeletal muscle Ca_V1.1 channel.     

Differential roles of S6 domain hinges in the gating of KCNQ potassium channels

Voltage-gated K+ channel activation is proposed to result from simultaneous bending of all S6 segments away from the central axis, enlarging the aperture of the pore sufficiently to permit diffusion of K+ into the water-filled central cavity. The hinge position for the bending motion of each S6 segment is proposed to be a Gly residue and/or a Pro-Val-Pro motif in Kv1-Kv4 channels. The KCNQ1 (Kv7.1) channel has Ala-336 in the Gly-hinge position and Pro-Ala-Gly. Here we show that mutation of Ala-336 to Gly in KCNQ1 increased current amplitude and shifted the voltage dependence of activation to more negative potentials, consistent with facilitation of hinge activity that favors the open state. In contrast, mutation of Ala-336 to Cys or Thr shifted the voltage dependence of activation to more positive potentials and reduced current amplitude. Mutation of the putative Gly hinge to Ala in KCNQ2 (Kv7.2) abolished channel function. Mutation-dependent changes in current amplitude, but not kinetics, were found in heteromeric KCNQ1/KCNE1 channels. Mutation of the Pro or Gly of the Pro-Ala-Gly motif to Ala abolished KCNQ1 function and introduction of Gly in front of the Ala-mutations partially recovered channel function, suggesting that flexibility at the PAG is important for channel activation.     

Antagonism of 4-substituted 1,4-dihydropyridine-3,5-dicarboxylates toward voltage-dependent L-type Ca2+ channels CaV1.3 and CaV1.2

L-type Ca²⁺ channels in mammalian brain neurons have either a Ca_V1.2 or Ca_V1.3 pore-forming subunit. Recently, it was shown that Ca_V1.3 Ca²⁺ channels underlie autonomous pacemaking in adult dopaminergic neurons in the substantia nigra pars compacta, and this reliance renders them sensitive to toxins used to create animal models of Parkinson’s disease. Antagonism of these channels with the dihydropyridine antihypertensive drug isradipine diminishes the reliance on Ca²⁺ and the sensitivity of these neurons to toxins, pointing to a potential neuroprotective strategy. However, for neuroprotection without an antihypertensive side effect, selective Ca_V1.3 channel antagonists are required. In an attempt to identify potent and selective antagonists of Ca_V1.3 channels, 124 dihydropyridines (4-substituted-1,4-dihydropyridine-3,5-dicarboxylic diesters) were synthesized. The antagonism of heterologously expressed Ca_V1.2 and Ca_V1.3 channels was then tested using electrophysiological approaches and the FLIPR Calcium 4 assay. Despite the large diversity in substitution on the dihydropyridine scaffold, the most Ca_V1.3 selectivity was only about twofold. These results support a highly similar dihydropyridine binding site at both Ca_V1.2 and Ca_V1.3 channels and suggests that other classes of compounds need to be identified for Ca_V1.3 selectivity.     

Activation gating of hERG potassium channels: S6 glycines are not required as gating hinges

The opening of ion channels is proposed to arise from bending of the pore inner helices that enables them to pivot away from the central axis creating a cytosolic opening for ion diffusion. The flexibility of the inner helices is suggested to occur either at a conserved glycine located adjacent to the selectivity filter (glycine gating hinge) and/or at a second site occupied by glycine or proline containing motifs. Sequence alignment with other K+ channels shows that hERG possesses glycine residues (Gly648 and Gly657) at each of these putative hinge sites. In apparent contrast to the hinge hypotheses, substitution of both glycine residues for alanine causes little effect on either the voltage-dependence or kinetics of channel activation, and open state block by intracellular blockers. Substitution of the glycines with larger hydrophobic residues causes a greater propensity for the channel to open. We propose that in contrast to Shaker the pore of hERG is intrinsically more stable in the open than the closed conformation and that substitution at Gly648 or Gly657 further shifts the gating equilibrium to favor the open state. Molecular dynamics simulations indicate the S6 helices of hERG are inherently flexible, even in the absence of the glycine residues. Thus hERG activation gating exhibits important differences to other Kv channels. Our findings indicate that the hERG inner helix glycine residues are required for the tight packing of the channel helices and that the flexibility afforded by glycine or proline residues is not universally required for activation gating.     

Artificial membrane excitability revisited and implications for the gating of voltage-dependent ion channels

Excitability phenomena in planar lipid bilayers doped with alamethicin and protamines have been first described by Mueller and Rudin (Nature 217, 713-719, 1968). These properties are reinvestigated here with virtually solvent-free bilayers made of synthetic phospholipids doped with alamethicin charged component (Glu18) and protamine or other synthetic basic polypeptides. After retrieving the narrow set of experimental requisites allowing negative resistance and action potentials to develop, the potencies of different basic polypeptides were compared. Poly-arginines were found to be by far the most efficient. We also describe a transient increase of current amplitude upon addition of calcium that may reflect a lateral phase separation and conversely a gradual decrease of negative resistance due to tetrodotoxin, a potent sodium channel blocker. Functional modulations are correlated with conformational changes assayed in circular dichroism: alamethicin ellipticity in small unilamellar vesicles is markedly reduced upon protamine addition, only if the ionic strength is in the same low range that is compatible with regenerative conductance properties. These results are discussed in the framework of current models of ion channels gating.     

An evolutionarily conserved network of amino acids mediates gating in voltage-dependent potassium channels

A novel sequence-analysis technique for detecting correlated amino acid positions in intermediate-size protein families (50-100 sequences) was developed, and applied to study voltage-dependent gating of potassium channels. Most contemporary methods for detecting amino acid correlations within proteins use very large sets of data, typically comprising hundreds or thousands of evolutionarily related sequences, to overcome the relatively low signal-to-noise ratio in the analysis of co-variations between pairs of amino acid positions. Such methods are impractical for voltage-gated potassium (Kv) channels and for many other protein families that have not yet been sequenced to that extent. Here, we used a phylogenetic reconstruction of paralogous Kv channels to follow the evolutionary history of every pair of amino acid positions within this family, thus increasing detection accuracy of correlated amino acids relative to contemporary methods. In addition, we used a bootstrapping procedure to eliminate correlations that were statistically insignificant. These and other measures allowed us to increase the method's sensitivity, and opened the way to reliable identification of correlated positions even in intermediate-size protein families. Principal-component analysis applied to the set of correlated amino acid positions in Kv channels detected a network of inter-correlated residues, a large fraction of which were identified as gating-sensitive upon mutation. Mapping the network of correlated residues onto the 3D structure of the Kv channel from Aeropyrum pernix disclosed correlations between residues in the voltage-sensor paddle and the pore region, including regions that are involved in the gating transition. We discuss these findings with respect to the evolutionary constraints acting on the channel's various domains. The software is available on our website