Protein Rearrangements Underlying Slow Inactivation of the Shaker K+ Channel

Voltage-dependent ion channels transduce changes in the membrane electric field into protein rearrangements that gate their transmembrane ion permeation pathways. While certain molecular elements of the voltage sensor and gates have been identified, little is known about either the nature of their conformational rearrangements or about how the voltage sensor is coupled to the gates. We used voltage clamp fluorometry to examine the voltage sensor (S4) and pore region (P-region) protein motions that underlie the slow inactivation of the Shaker K⁺ channel. Fluorescent probes in both the P-region and S4 changed emission intensity in parallel with the onset and recovery of slow inactivation, indicative of local protein rearrangements in this gating process. Two sequential rearrangements were observed, with channels first entering the P-type, and then the C-type inactivated state. These forms of inactivation appear to be mediated by a single gate, with P-type inactivation closing the gate and C-type inactivation stabilizing the gate''s closed conformation. Such a stabilization was due, at least in part, to a slow rearrangement around S4 that stabilizes S4 in its activated transmembrane position. The fluorescence reports of S4 and P-region fluorophore are consistent with an increased interaction of the voltage sensor and inactivation gate upon gate closure, offering insight into how the voltage-sensing apparatus is coupled to a channel gate.     

Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

Prolonged depolarization induces a slow inactivation process in some K⁺ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K⁺ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K⁺ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.     

A direct demonstration of closed-state inactivation of K+ channels at low pH

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K⁺ or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.     

Voltage-insensitive Gating after Charge-neutralizing Mutations in the S4 Segment of Shaker Channels

Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and close from −160 to +80 mV with a constant opening probability (P _o). Although P _o is low (∼0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are partially blocked by external Ca²⁺ ions at very negative potentials. Furthermore, mean open time is approximately doubled, in both mutant channels and control Shaker channels, when Rb⁺ is substituted for K⁺ as the permeant ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus, mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive P _o seen in these mutant channels suggests that important determinants of normal channel opening derive from electrostatic coupling between S4 charges and the pore domain.     

Slow Inactivation in Shaker K Channels Is Delayed by Intracellular Tetraethylammonium

After removal of the fast N-type inactivation gate, voltage-sensitive Shaker (Shaker IR) K channels are still able to inactivate, albeit slowly, upon sustained depolarization. The classical mechanism proposed for the slow inactivation observed in cell-free membrane patches—the so called C inactivation—is a constriction of the external mouth of the channel pore that prevents K⁺ ion conduction. This constriction is antagonized by the external application of the pore blocker tetraethylammonium (TEA). In contrast to C inactivation, here we show that, when recorded in whole Xenopus oocytes, slow inactivation kinetics in Shaker IR K channels is poorly dependent on external TEA but severely delayed by internal TEA. Based on the antagonism with internally or externally added TEA, we used a two-pulse protocol to show that half of the channels inactivate by way of a gate sensitive to internal TEA. Such gate had a recovery time course in the tens of milliseconds range when the interpulse voltage was −90 mV, whereas C-inactivated channels took several seconds to recover. Internal TEA also reduced gating charge conversion associated to slow inactivation, suggesting that the closing of the internal TEA-sensitive inactivation gate could be associated with a significant amount of charge exchange of this type. We interpreted our data assuming that binding of internal TEA antagonized with U-type inactivation (Klemic, K.G., G.E. Kirsch, and S.W. Jones. 2001. Biophys. J. 81:814–826). Our results are consistent with a direct steric interference of internal TEA with an internally located slow inactivation gate as a “foot in the door” mechanism, implying a significant functional overlap between the gate of the internal TEA-sensitive slow inactivation and the primary activation gate. But, because U-type inactivation is reduced by channel opening, trapping the channel in the open conformation by TEA would also yield to an allosteric delay of slow inactivation. These results provide a framework to explain why constitutively C-inactivated channels exhibit gating charge conversion, and why mutations at the internal exit of the pore, such as those associated to episodic ataxia type I in hKv1.1, cause severe changes in inactivation kinetics.     

The zinc binding site of the Shaker channel KDC1 from Daucus carota

KDC1 is a voltage-dependent Shaker-like potassium channel subunit cloned from Daucus carota which produces conductive channels in Xenopus oocytes only when coexpressed with other plant Shaker potassium subunits, such as KAT1 from Arabidopsis thaliana. External Zn²⁺ determines a potentiation of the current mediated by the dimeric construct KDC1-KAT1, which has been ascribed to zinc binding at a site comprising three histidines located at the S3-S4 (H161, H162) and S5-S6 (H224) linkers of KDC1. Here we demonstrate that also glutamate 164, located in close proximity of the KDC1 S4 segment, is an essential component of the zinc-binding site. On the contrary, glutamate 159, located in symmetrical position with respect to E164 in the sequence E₁₅₉XHHXE₁₆₄ but more distant from the voltage sensor, does not play any role in zinc binding. The effects of Zn²⁺ can be expressed as a “shift” of the gating parameters along the voltage axis. Kinetic modeling shows that Zn²⁺ slows the closing kinetics of KDC1-KAT1 without affecting the opening kinetics. Possibly, zinc affects the movement of the voltage sensor in and out of the membrane phase through electrostatic modification of a site close to the voltage sensor.     

Alanine Scanning of the S6 Segment Reveals a Unique and cAMP-sensitive Association between the Pore and Voltage-dependent Opening in HCN Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels resemble Shaker K⁺ channels in structure and function. In both, changes in membrane voltage produce directionally similar movement of positively charged residues in the voltage sensor to alter the pore structure at the intracellular side and gate ion flow. However, HCNs open when hyperpolarized, whereas Shaker opens when depolarized. Thus, electromechanical coupling between the voltage sensor and gate is opposite. A key determinant of this coupling is the intrinsic stability of the pore. In Shaker, an alanine/valine scan of residues across the pore, by single point mutation, showed that most mutations made the channel easier to open and steepened the response of the channel to changes in voltage. Because most mutations likely destabilize protein packing, the Shaker pore is most stable when closed, and the voltage sensor works to open it. In HCN channels, the pore energetics and vector of work by the voltage sensor are unknown. Accordingly, we performed a 22-residue alanine/valine scan of the distal pore of the HCN2 isoform and show that the effects of mutations on channel opening and on the steepness of the response of the channel to voltage are mixed and smaller than those in Shaker. These data imply that the stabilities of the open and closed pore are similar, the voltage sensor must apply force to close the pore, and the interactions between the pore and voltage sensor are weak. Moreover, cAMP binding to the channel heightens the effects of the mutations, indicating stronger interactions between the pore and voltage sensor, and tips the energetic balance toward a more stable open state.Hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channels are similar in structure and function to Shaker K⁺ channels (1–3). As in Shaker, HCN channels are comprised of four subunits, which each consist of six predicted membrane-spanning segments (S1–S6). The S1–S4 segments form the voltage-sensing domain, and the S5 and S6 segments, the pore-forming domain. The S4 segment in both channels contains positive charges that move similarly in response to changes in membrane voltage (4–6), to then alter the pore structure at the intracellular side of the S6 segment; this region functions as a voltage-controlled gate to cation flow (7–10). Despite these similarities, HCN channels are opened by hyperpolarization of the membrane potential, whereas Shaker channels open in response to depolarization. Thus, the electromechanical coupling between the voltage sensor and the gate is reversed in these two channels.A key determinant of this coupling is the intrinsic stability of the closed and open conformations of the pore. In Shaker channels, it has been proposed that the pore is intrinsically most stable when closed and that the voltage sensor works to open the pore during depolarization (11, 12). Results from an alanine/valine scan of residues across the entire Shaker pore, by single point mutation, showed that most mutations made the channel easier to open and steepened the response of the channel to changes in voltage. It was argued that, because most mutations likely destabilize protein packing, the closed conformation must be the stable state; this is consistent with the observed crystal structures of Shaker-related channels KcsA and MthK, in the closed and open states, respectively, wherein more optimally and tightly packed helices were seen in the closed conformation (13–15).Because of presumed shared architecture of the gate between HCN and Shaker channels, HCN channels might also be most stable when closed, and thus the voltage sensor would work to open the pore upon hyperpolarization. To test this hypothesis, we performed an alanine/valine scan of the C-terminal 22 amino acids of the S6 segment in HCN2, used as a prototype, and examined pore energetics as described previously in Shaker (11). Choice of this region for mutation was based on: 1) in Shaker, the corresponding region harbors one of two clusters of gating-sensitive residues and 2) it contains the voltage-controlled gate. Surprisingly, the effects of the mutations on channel opening and on the steepness of the channel''s response to voltage are mixed and smaller than those in Shaker. These findings imply that, in HCN2, the stabilities of the open and closed pore are similar, the interactions between the pore and voltage sensor, both structural and functional, are weaker than in Shaker, and that the voltage sensor must apply force to the pore to close it. Thus, Shaker is closed and HCN2 is open in the absence of input from the voltage sensor. Moreover, cAMP binding to the HCN2 channel heightens the effects of the mutations, indicating stronger interactions between the pore and voltage sensor, and tips the energetic balance toward a more stable open state.     

Functional interactions of voltage sensor charges with an S2 hydrophobic plug in hERG channels

Human ether-à-go-go–related gene (hERG, Kv11.1) potassium channels have unusually slow activation and deactivation kinetics. It has been suggested that, in fast-activating Shaker channels, a highly conserved Phe residue (F290) in the S2 segment forms a putative gating charge transfer center that interacts with S4 gating charges, i.e., R362 (R1) and K374 (K5), and catalyzes their movement across the focused electric field. F290 is conserved in hERG (F463), but the relevant residues in the hERG S4 are reversed, i.e., K525 (K1) and R537 (R5), and there is an extra positive charge adjacent to R537 (i.e., K538). We have examined whether hERG channels possess a transfer center similar to that described in Shaker and if these S4 charge differences contribute to slow gating in hERG channels. Of five hERG F463 hydrophobic substitutions tested, F463W and F463Y shifted the conductance–voltage (G-V) relationship to more depolarized potentials and dramatically slowed channel activation. With the S4 residue reversals (i.e., K525, R537) taken into account, the closed state stabilization by F463W is consistent with a role for F463 that is similar to that described for F290 in Shaker. As predicted from results with Shaker, the hERG K525R mutation destabilized the closed state. However, hERG R537K did not stabilize the open state as predicted. Instead, we found the neighboring K538 residue to be critical for open state stabilization, as K538R dramatically slowed and right-shifted the voltage dependence of activation. Finally, double mutant cycle analysis on the G-V curves of F463W/K525R and F463W/K538R double mutations suggests that F463 forms functional interactions with K525 and K538 in the S4 segment. Collectively, these data suggest a role for F463 in mediating closed–open equilibria, similar to that proposed for F290 in Shaker channels.     

Glutamine Substitution at Alanine1649 in the S4–S5 Cytoplasmic Loop of Domain 4 Removes the Voltage Sensitivity of Fast Inactivation in the Human Heart Sodium Channel

Normal activation–inactivation coupling in sodium channels insures that inactivation is slow at small but rapid at large depolarizations. M1651Q/M1652Q substitutions in the cytoplasmic loop connecting the fourth and fifth transmembrane segments of Domain 4 (S4–S5/D4) of the human heart sodium channel subtype 1 (hH1) affect the kinetics and voltage dependence of inactivation (Tang, L., R.G. Kallen, and R. Horn. 1996. J. Gen. Physiol. 108:89–104.). We now show that glutamine substitutions NH₂-terminal to the methionines (L¹⁶⁴⁶, L¹⁶⁴⁷, F¹⁶⁴⁸, A¹⁶⁴⁹, L¹⁶⁵⁰) also influence the kinetics and voltage dependence of inactivation compared with the wild-type channel. In contrast, mutations at the COOH-terminal end of the S4–S5/D4 segment (L¹⁶⁵⁴, P¹⁶⁵⁵, A¹⁶⁵⁶) are without significant effect. Strikingly, the A1649Q mutation renders the current decay time constants virtually voltage independent and decreases the voltage dependences of steady state inactivation and the time constants for the recovery from inactivation. Single-channel measurements show that at negative voltages latency times to first opening are shorter and less voltage dependent in A1649Q than in wild-type channels; peak open probabilities are significantly smaller and the mean open times are shorter. This indicates that the rate constants for inactivation and, probably, activation are increased at negative voltages by the A1649Q mutation reminiscent of Y1494Q/ Y1495Q mutations in the cytoplasmic loop between the third and fourth domains (O''Leary, M.E., L.Q. Chen, R.G. Kallen, and R. Horn. 1995. J. Gen. Physiol. 106:641–658.). Other substitutions, A1649S and A1649V, decrease but fail to eliminate the voltage dependence of time constants for inactivation, suggesting that the decreased hydrophobicity of glutamine at either residues A¹⁶⁴⁹ or Y¹⁴⁹⁴Y¹⁴⁹⁵ may disrupt a linkage between S4–S5/D4 and the interdomain 3–4 loop interfering with normal activation–inactivation coupling.     

Sodium Channel Inactivation Is Altered by Substitution of Voltage Sensor Positive Charges

The role of the voltage sensor positive charges in fast and slow inactivation of the rat brain IIA sodium channel was investigated by mutating the second and fourth conserved positive charges in the S4 segments of all four homologous domains. Both charge-neutralizing mutations (by glutamine substitution) and charge-conserving mutations were constructed in a cDNA encoding the sodium channel α subunit. To determine if fast inactivation altered the effects of the mutations on slow inactivation, the mutations were also constructed in a channel that had fast inactivation removed by the incorporation of the IFMQ3 mutation in the III–IV linker (West, J.W., D.E. Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A. Catterall. 1992. Proc. Natl. Acad. Sci. USA. 89:10910– 10914). Most of the mutations shifted the v_1/2 of fast inactivation in the negative direction, with the largest effects resulting from mutations in domains I and II. These shifts were in the opposite direction compared with those observed for activation. The effects of the mutations on slow inactivation depended on whether fast inactivation was intact or not. When fast inactivation was eliminated, most of the mutations resulted in positive shifts in the v_1/2 of slow inactivation. The largest effects again resulted from mutations in domains I and II. When fast inactivation was intact, the mutations in domains II and III resulted in negative shifts in the v_1/2 of slow inactivation. Neutralization of the fourth charge in domain I or II resulted in the appearance of a second component in the voltage dependence of slow inactivation that was only observable when fast inactivation was intact. These results suggest the S4 regions of all four domains of the sodium channel are involved in the voltage dependence of inactivation, but to varying extents. Fast inactivation is not strictly coupled to activation, but it derives some independent voltage sensitivity from the charges in the S4 domains. Finally, there is an interaction between the fast and slow inactivation processes.     

Structural Implications of Fluorescence Quenching in the Shaker K+ Channel

When attached to specific sites near the S4 segment of the nonconducting (W434F) Shaker potassium channel, the fluorescent probe tetramethylrhodamine maleimide undergoes voltage-dependent changes in intensity that correlate with the movement of the voltage sensor (Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Science. 271:213–216; Cha, A., and F. Bezanilla. 1997. Neuron. 19:1127–1140). The characteristics of this voltage-dependent fluorescence quenching are different in a conducting version of the channel with a different pore substitution (T449Y). Blocking the pore of the T449Y construct with either tetraethylammonium or agitoxin removes a fluorescence component that correlates with the voltage dependence but not the kinetics of ionic activation. This pore-mediated modulation of the fluorescence quenching near the S4 segment suggests that the fluorophore is affected by the state of the external pore. In addition, this modulation may reflect conformational changes associated with channel opening that are prevented by tetraethylammonium or agitoxin. Studies of pH titration, collisional quenchers, and anisotropy indicate that fluorophores attached to residues near the S4 segment are constrained by a nearby region of protein. The mechanism of fluorescence quenching near the S4 segment does not involve either reorientation of the fluorophore or a voltage-dependent excitation shift and is different from the quenching mechanism observed at a site near the S2 segment. Taken together, these results suggest that the extracellular portion of the S4 segment resides in an aqueous protein vestibule and is influenced by the state of the external pore.     

The Binding of κ-Conotoxin PVIIA and Fast C-Type Inactivation of Shaker K+ Channels are Mutually Exclusive

κ-Conotoxin PVIIA (κ-PVIIA), a 27-amino acid peptide identified from the venom of Conus purpurascens, inhibits the Shaker K⁺ channel by blocking its outer pore. The toxin appears as a gating modifier because its binding affinity decreases with relatively fast kinetics upon channel opening, but there is no indication that it interferes with the gating transitions of the wild-type channels (WT), including the structural changes of the outer pore that underlie its slow C-type inactivation. In this report we demonstrate that in two outer pore mutants of Shaker-IR (M448K and T449S), that have high toxin sensitivity and fast C-type inactivation, the latter process is instead antagonized by and incompatible with κ-PVIIA binding. Inactivation is slowed by the necessary preliminary unbinding of κ-PVIIA, whereas toxin rebinding must await recovery from inactivation causing a double-exponential relaxation of the second response to double-pulse stimulations. Compared with the lack of similar effects in WT, these results demonstrate the ability of peptide toxins like κ-PVIIA to reveal possibly subtle differences in structural changes of the outer pore of K⁺ channels; however, they also warn against a naive use of fast inactivating mutants as models for C-type inactivation. Unfolded from the antagonistic effect of inactivation, toxin binding to mutant noninactivated channels shows state- and voltage-dependencies similar to WT: slow and high affinity for closed channels; relatively fast dissociation from open channels at rate increasing with voltage. This supports the idea that these properties depend mainly on interactions with pore-permeation processes that are not affected by the mutations. In mutant channels the state-dependence also greatly enhances the protection of toxin binding against steady-state inactivation at low depolarizations while still allowing large responses to depolarizing pulses that relieve toxin block. Although not obviously applicable to any known combination of natural channel and outer-pore blocker, our biophysical characterization of such highly efficient mechanism of protection from steady-state outer-pore inactivation may be of general interest.     

Direct Interaction between the Voltage Sensors Produces Cooperative Sustained Deactivation in Voltage-gated H+ Channel Dimers

The voltage-gated H⁺ channel (Hv) is a voltage sensor domain-like protein consisting of four transmembrane segments (S1–S4). The native Hv structure is a homodimer, with the two channel subunits functioning cooperatively. Here we show that the two voltage sensor S4 helices within the dimer directly cooperate via a π-stacking interaction between Trp residues at the middle of each segment. Scanning mutagenesis showed that Trp situated around the original position provides the slow gating kinetics characteristic of the dimer''s cooperativity. Analyses of the Trp mutation on the dimeric and monomeric channel backgrounds and analyses with tandem channel constructs suggested that the two Trp residues within the dimer are functionally coupled during Hv deactivation but are less so during activation. Molecular dynamics simulation also showed direct π-stacking of the two Trp residues. These results provide new insight into the cooperative function of voltage-gated channels, where adjacent voltage sensor helices make direct physical contact and work as a single unit according to the gating process.     

Widening the scope of constriction

JGP modeling study suggests that selectivity filter constriction is a plausible mechanism for C-type inactivation of the Shaker voltage-gated potassium channel.

In response to prolonged activation, many K⁺ channels spontaneously reduce the membrane conductance by undergoing C-type inactivation, a kinetic process crucial for the pacing of cardiac action potentials and the modulation of neuronal firing patterns. In the pH-activated bacterial channel KcsA, C-type inactivation appears to involve constriction of the channel’s selectivity filer that prohibits ion conduction, but whether voltage-gated channels like Drosophila Shaker use a similar mechanism is controversial (1). In this issue of JGP, a computational study by Li et al. suggests that filter constriction is indeed a plausible mechanism for the C-type inactivation of Shaker (2).(Left to right) Jing Li, Benoît Roux, and colleagues use computational modeling to show that selectivity filter constriction, allosterically promoted by opening of the intracellular activation gate, is a plausible mechanism for the C-type inactivation of voltage-gated K⁺ channels such as Drosophila Shaker. The selectivity filter is conductive (left) when the intracellular gate is partially open, but adopts a constricted conformation (right) when the gate is open wide.Various structural approaches have shown that C-type inactivation of KcsA channels is associated with the symmetrical constriction of all four channel subunits at the level of the central glycine residue in the selectivity filter. Benoît Roux and colleagues at The University of Chicago used MD simulations to show that the KcsA pore can transition from the conductive to the constricted conformation on an appropriate timescale, and that this transition is allosterically promoted by the wide opening of the pore’s intracellular gate (3). Modeling by Roux and colleagues suggests that C-type inactivation of cardiac hERG channels could also involve selectivity filter constriction, though in this case it appears to be an asymmetric process in which only two of the channel’s subunits move closer together (4).“In view of the high similarity between the pore domains of Shaker and KcsA (almost 40% sequence identity), we wanted to examine if it’s possible for the Shaker selectivity filter to constrict and, if so, how similar it is to KcsA,” Roux explains. Led by first author Jing Li—now an assistant professor at the University of Mississippi—Roux and colleagues developed several homology models of the Shaker pore domain with the intracellular gate open to various degrees (2).MD simulations and free energy calculations revealed that the Shaker selectivity filter can dynamically transition from a conductive to a constricted conformation, and that this transition is allosterically coupled to the intracellular gate; the constricted conformation is stable when the gate is wide open. “Our computations strongly suggest that constriction is a plausible mechanism for the C-type inactivation of Shaker,” Roux says. “There’s no reason based on the currently available information to reject the existence of a constricted state in Shaker channels.”As with KcsA, Shaker channels appear to constrict symmetrically at the level of the selectivity filter’s central glycine. But Li et al.’s simulations revealed some small variations between the two channels, including differences in the number of water molecules bound to each channel subunit and the arrangement of the hydrogen-bond network they form to stabilize the constricted state.Li et al. also modeled the pore domain of the Shaker W434F mutant, which is widely assumed to be trapped in a C-type inactivated state. The simulation suggests that the mutant channel’s filter adopts a stable constricted conformation even when the intracellular gate is only partially open, although the constriction is asymmetric and occurs at the level of a different filter residue (2).Constriction may therefore be a universal mechanism of C-type inactivation, even if the exact conformation varies from channel to channel. But, says Roux, confirming this will require more experimental work using the right conditions and mutations to capture the structure of inactivated channels.     

Role of Arginine Residues on the S4 Segment of the <Emphasis Type="Italic">Bacillus halodurans</Emphasis> Na<Superscript>+</Superscript> Channel in Voltage-sensing

The one-domain voltage-gated sodium channel of Bacillus halodurans (NaChBac) is composed of six transmembrane segments (S1–S6) comprising a pore-forming region flanked by segments S5 and S6 and a voltage-sensing element composed of segment S4. To investigate the role of the S4 segment in NaChBac channel activation, we used the cysteine mutagenesis approach where the positive charges of single and multiple arginine (R) residues of the S4 segment were replaced by the neutrally charged amino acid cysteine (C). To determine whether it was the arginine residue itself or its positive charge that was involved in channel activation, arginine to lysine (R to K) mutations were constructed. Wild-type (WT) and mutant NaChBac channels were expressed in tsA201 cells and Na⁺ currents were recorded using the whole-cell configuration of the patch-clamp technique. The current/voltage (I-V) and conductance/voltage (G-V) relationships steady-state inactivation (h _∞) and recovery from inactivation were evaluated to determine the effects of the S4 mutations on the biophysical properties of the NaChBac channel. R to C on the S4 segment resulted in a slowing of both activation and inactivation kinetics. Charge neutralization of arginine residues mostly resulted in a shift toward more positive potentials of G-V and h _∞ curves. The G-V curve shifts were associated with a decrease in slope, which may reflect a decrease in the gating charge involved in channel activation. Single neutralization of R114, R117, or R120 by C resulted in a very slow recovery from inactivation. Double neutralization of R111 and R129 confirmed the role of R111 in activation and suggested that R129 is most probably not part of the voltage sensor. Most of the R to K mutants retained WT-like current kinetics but exhibited an intermediate G-V curve, a steady-state inactivation shifted to more hyperpolarized potentials, and intermediate time constants of recovery from inactivation. This indicates that R, at several positions, plays an important role in channel activation. The data are consistent with the notion that the S4 is most probably the voltage sensor of the NaChBac channel and that both positive charges and the nature of the arginine residues are essential for channel activation.This revised version was published online in June 2005 with a corrected cover date.     

Orientation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> KAT1 Channel in the Plasma Membrane

The Arabidopsis thaliana KAT1, an inward-rectifying potassium channel, shares molecular features with the Shaker family of outward rectifier K⁺ channels. The KAT1 amino-acid sequence reveals the presence of a positively charged S4 and a segment containing the TXGYGD signature sequence in the pore (P) region. To test whether the inward-rectifying properties of KAT1 are due to reverse orientation in the membrane, such that the voltage sensor is oriented in the opposite direction of the electric field compared with the Shaker K⁺ channel, we have inserted a flag epitope in the NH₂ terminus or the S3–S4 loop. The KAT1 and tagged constructs expressed functional channels in whole cells, Xenopus oocytes and COS-7. The electrophysiological properties of both tagged constructs were similar to those of the wild type. Immunofluorescence with an antibody against the flag epitope and an anti-C terminal KAT1 determined the membrane localization of these epitopes and the orientation of the KAT1 channel in the membrane. Our data confirm that KAT1 in eukaryotic cells has an orientation similar to the Shaker K⁺ channel.     

Intermediate Conductances during Deactivation of Heteromultimeric Shaker Potassium Channels

A previous study of the T442S mutant Shaker channel revealed activation-coupled subconductance levels that apparently represent kinetic intermediates in channel activation (Zheng, J., and F.J. Sigworth. 1997. J. Gen. Physiol. 110:101–117). We have now extended the study to heteromultimeric channels consisting of various numbers of mutant subunits as well as channels without mutant subunits, all in the background of a chimeric Shaker channel having increased conductance. It has been found that activation-coupled sublevels exist in all these channel types, and are traversed in at least 80% of all deactivation time courses. In symmetric K⁺ solutions, the currents in the two sublevels have a linear voltage dependence, being 23–44% and 54–70% of the fully open conductance. Sublevels in different channel types share similar voltage dependence of the mean lifetime and similar ion selectivity properties. However, the mean lifetime of each current level depends approximately geometrically on the number of mutant subunits in the channel, becoming shorter in channels having fewer mutant subunits. Each mutant subunit appears to stabilize all of the conducting states by ∼0.5 kcal/mol. Consistent with previous results in the mutant channel, sublevels in channels with two or no mutant subunits also showed ion selectivities that differ from that of the fully open level, having relatively higher K⁺ than Rb⁺ conductances. A model is presented in which Shaker channels have two coupled activation gates, one associated with the selectivity filter and a second associated with the S6 helix bundle.     

A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb

Voltage-gated sodium channels undergo slow inactivation during repetitive depolarizations, which controls the frequency and duration of bursts of action potentials and prevents excitotoxic cell death. Although homotetrameric bacterial sodium channels lack the intracellular linker-connecting homologous domains III and IV that causes fast inactivation of eukaryotic sodium channels, they retain the molecular mechanism for slow inactivation. Here, we examine the functional properties and slow inactivation of the bacterial sodium channel NavAb expressed in insect cells under conditions used for structural studies. NavAb activates at very negative membrane potentials (V_1/2 of approximately −98 mV), and it has both an early phase of slow inactivation that arises during single depolarizations and reverses rapidly, and a late use-dependent phase of slow inactivation that reverses very slowly. Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation. The gating charge R3 interacts with Asn49 in the crystal structure of NavAb, and mutation of this residue to Cys causes a similar positive shift in the voltage dependence of activation and block of the late phase of slow inactivation as mutation N49K. Prolonged depolarizations that induce slow inactivation also cause hysteresis of gating charge movement, which results in a requirement for very negative membrane potentials to return gating charges to their resting state. Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation. Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.     

Fast Inactivation in Shaker K+ Channels : Properties of Ionic and Gating Currents

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as “charge immobilization” (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567–590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at −90 mV return potential changed from a single fast component to at least two components, the slower requiring ∼200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at −120 and −90 mV. In contrast, at higher potentials (−70 and −50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of “parallel” inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH₂-terminus region.     

Activation of Shaker Potassium Channels : III. An Activation Gating Model for Wild-Type and V2 Mutant Channels

A functional kinetic model is developed to describe the activation gating process of the Shaker potassium channel. The modeling in this paper is constrained by measurements described in the preceding two papers, including macroscopic ionic and gating currents and single channel ionic currents. These data were obtained from the normally activating wild-type channel as well as a mutant channel V2, in which the leucine at position 382 has been mutated to a valine. Different classes of models that incorporate Shaker''s symmetrical tetrameric structure are systematically examined. Many simple gating models are clearly inadequate, but a model that can account for all of the qualitative features of the data has the channel open after its four subunits undergo three transitions in sequence, and two final transitions that reflect the concerted action of the four subunits. In this model, which we call Scheme 3+2′, the channel can also close to several states that are not part of the activation path. Channel opening involves a large total charge movement (10.8 e₀), which is distributed among a large number of small steps each with rather small charge movements (between 0.6 and 1.05 e₀). The final two transitions are different from earlier steps by having slow backward rates. These steps confer a cooperative mechanism of channel opening at Shaker''s activation voltages. In the context of Scheme 3+2′, significant effects of the V2 mutation are limited to the backward rates of the final two transitions, implying that L382 plays an important role in the conformational stability of the final two states.