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.     

Nonindependent K+ Movement through the Pore in IRK1 Potassium Channels

We measured unidirectional K⁺ in- and efflux through an inward rectifier K channel (IRK1) expressed in Xenopus oocytes. The ratio of these unidirectional fluxes differed significantly from expectations based on independent ion movement. In an extracellular solution with a K⁺ concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n′, of ∼2.2 and was constant over a voltage range from −50 to −25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n′ value of 2.2 is significantly smaller than the value of 3.5 obtained for Shaker K channels under identical conditions. To determine if other permeation properties that reflect multi-ion behavior differed between these two channel types, we measured the conductance (at 0 mV) of single IRK1 channels as a function of symmetrical K⁺ concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K⁺ activity of 40 mM, substantially less than the reported value of 300 mM for Shaker K channels. We investigated the ability of simple permeation models based on absolute reaction rate theory to simulate IRK1 current–voltage, conductance, and flux-ratio data. Certain classes of four-barrier, three-site permeation models are inconsistent with the data, but models with high lateral barriers and a deep central well were able to account for the flux-ratio and single channel data. We conclude that while the pore in IRK1 and Shaker channels share important similarities, including K⁺ selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K⁺ concentration, and may differ in the number of K⁺ ions that can simultaneously occupy the pore: IRK1 channels may contain three ions, but the pore in Shaker channels can accommodate four or more ions.     

Selectivity Changes during Activation of Mutant Shaker Potassium Channels

Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH₂-terminal–truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179–182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in ∼40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of ∼1.6 e₀. The fully open T442S chimeric channel has the conductance sequence Rb⁺ > NH₄ ⁺ > K⁺. The opposite conductance sequence, K⁺ > NH₄ ⁺ > Rb⁺, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb⁺.     

Ion Conduction through C-Type Inactivated Shaker Channels

C-type inactivation of Shaker potassium channels involves entry into a state (or states) in which the inactivated channels appear nonconducting in physiological solutions. However, when Shaker channels, from which fast N-type inactivation has been removed by NH₂-terminal deletions, are expressed in Xenopus oocytes and evaluated in inside-out patches, complete removal of K⁺ ions from the internal solution exposes conduction of Na⁺ and Li⁺ in C-type inactivated conformational states. The present paper uses this observation to investigate the properties of ion conduction through C-type inactivated channel states, and demonstrates that both activation and deactivation can occur in C-type states, although with slower than normal kinetics. Channels in the C-type states appear “inactivated” (i.e., nonconducting) in physiological solutions due to the summation of two separate effects: first, internal K⁺ ions prevent Na⁺ ions from permeating through the channel; second, C-type inactivation greatly reduces the permeability of K⁺ relative to the permeability of Na⁺, thus altering the ion selectivity of the channel.     

Macroscopic Na+ Currents in the “Nonconducting” Shaker Potassium Channel Mutant W434F

C-type inactivation in Shaker potassium channels inhibits K⁺ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na⁺ and Li⁺ currents in K⁺-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na⁺ and Li⁺ currents (in K⁺-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na⁺ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.     

Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K⁺ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K⁺ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.     

Structure of the inactivating gate from the Shaker voltage gated K+ channel analyzed by NMR spectroscopy

Rapid inactivation of voltage-gated K⁺ (K_V) channels is mediated by an N-terminal domain (inactivating ball domain) which blocks the open channel from the cytoplasmic side. Inactivating ball domains of various K_V channels are also biologically active when synthesized separately and added as a peptide to the solution. Synthetic inactivating ball domains from different K_V channels with hardly any sequence homology mediate quite similar effects even on unrelated K_V channel subtypes whose inactivation domain has been deleted. The solution structure of the inactivating ball peptide from Shaker (Sh-P22) was analyzed with NMR spectroscopy. The NMR data indicate a non-random structure in an aqueous environment. However, while other inactivating ball peptides showed well-defined three-dimensional structures under these conditions, Sh-P22 does not have a unique, compactly folded structure in solution.     

Role of Outer-pore Residue Y380 in U-type Inactivation of KV2.1 Channels

The interpretation of slow inactivation in potassium channels has been strongly influenced by work on C-type inactivation in Shaker channels. Slow inactivation in Shaker and some other potassium channels can be dramatically modulated by the state of the pore, including mutations at outer pore residue T449, which altered inactivation kinetics up to 100-fold. K_V2.1, another voltage-dependent potassium channel, exhibits a biophysically distinct inactivation mechanism with a U-shaped voltage-dependence and preferential closed-state inactivation, termed U-type inactivation. However, it remains to be demonstrated whether U-type and C-type inactivation have different molecular mechanisms. This study examines mutations at Y380 (homologous to Shaker T449) to investigate whether C-type and U-type inactivation have distinct molecular mechanisms, and whether C-type inactivation can occur at all in K_V2.1. Y380 mutants do not introduce C-type inactivation into K_V2.1 and have little effect on U-type inactivation of K_V2.1. Interestingly, two of the mutants tested exhibit twofold faster recovery from inactivation compared to wild-type channels. The observation that mutations have little effect suggests K_V2.1 lacks C-type inactivation as it exists in Shaker and that C-type and U-type inactivation have different molecular mechanisms. Kinetic modeling predicts that all mutants inactivate preferentially, but not exclusively, from partially activated closed states. Therefore, K_V2.1 exhibits a single U-type inactivation process including some inactivation from open as well as closed states.     

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.     

More gating charges are needed to open a Shaker K+ channel than are needed to open an rBIIA Na+ channel

This study presents what is, to our knowledge, a novel technique by means of which the ratio of the single gating charges of voltage-gated rat brain IIA (rBIIA) sodium and Shaker potassium ion channels was estimated. In the experiment, multiple tandems of enhanced green fluorescent protein were constructed and inserted into the C-terminals of Na⁺ and K⁺ ion channels. cRNA of Na⁺ and K⁺ ion channels was injected and expressed in Xenopus laevis oocytes. The two electrode voltage-clamp technique allowed us to determine the total gating charge of sodium and potassium ion channels, while a relative measure of the amount of expressed channels could be established on the basis of the quantification of the fluorescence intensity of membrane-bound channels marked by enhanced green fluorescent proteins. As a result, gating charge and fluorescence intensity were found to be positively correlated. A relative comparison of the single gating charges of voltage-gated sodium and potassium ion channels could thus be established: the ratio of the single gating charges of the Shaker potassium channel and the rBIIA sodium channel was found to be 2.5 ± 0.4. Assuming the single channel gating charge of the Shaker K⁺ channel to be ∼13 elementary charges (well supported by other studies), this leads to approximately six elementary charges for the rBIIA sodium channel, which includes a fraction of gating charge that is missed during inactivation.     

Bupivacaine blocks N-type inactivating Kv channels in the open state: no allosteric effect on inactivation kinetics

Local anesthetics bind to ion channels in a state-dependent manner. For noninactivating voltage-gated K channels the binding mainly occurs in the open state, while for voltage-gated inactivating Na channels it is assumed to occur mainly in inactivated states, leading to an allosterically caused increase in the inactivation probability, reflected in a negative shift of the steady-state inactivation curve, prolonged recovery from inactivation, and a frequency-dependent block. How local anesthetics bind to N-type inactivating K channels is less explored. In this study, we have compared bupivacaine effects on inactivating (Shaker and K_v3.4) and noninactivating (Shaker-IR and K_v3.2) channels, expressed in Xenopus oocytes. Bupivacaine was found to block these channels time-dependently without shifting the steady-state inactivation curve markedly, without a prolonged recovery from inactivation, and without a frequency-dependent block. An analysis, including computational testing of kinetic models, suggests binding to the channel mainly in the open state, with affinities close to those estimated for corresponding noninactivating channels (300 and 280 μM for Shaker and Shaker-IR, and 60 and 90 μM for K_v3.4 and K_v3.2). The similar magnitudes of K_d, as well as of blocking and unblocking rate constants for inactivating and noninactivating Shaker channels, most likely exclude allosteric interactions between the inactivation mechanism and the binding site. The relevance of these results for understanding the action of local anesthetics on Na channels is discussed.     

Properties of Shaker-type Potassium Channels in Higher Plants

Potassium (K⁺), the most abundant cation in biological organisms, plays a crucial role in the survival and development of plant cells, modulation of basic mechanisms such as enzyme activity, electrical membrane potentials, plant turgor and cellular homeostasis. Due to the absence of a Na⁺/K⁺ exchanger, which widely exists in animal cells, K⁺ channels and some type of K⁺ transporters function as K⁺ uptake systems in plants. Plant voltage-dependent K⁺ channels, which display striking topological and functional similarities with the voltage-dependent six-transmembrane segment animal Shaker-type K⁺ channels, have been found to play an important role in the plasma membrane of a variety of tissues and organs in higher plants. Outward-rectifying, inward-rectifying and weakly-rectifying K⁺ channels have been identified and play a crucial role in K⁺ homeostasis in plant cells. To adapt to the environmental conditions, plants must take advantage of the large variety of Shaker-type K⁺ channels naturally present in the plant kingdom. This review summarizes the extensive data on the structure, function, membrane topogenesis, heteromerization, expression, localization, physiological roles and modulation of Shaker-type K⁺ channels from various plant species. The accumulated results also help in understanding the similarities and differences in the properties of Shaker-type K⁺ channels in plants in comparison to those of Shaker channels in animals and bacteria.     

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.     

Functional role of the N-terminus of Na,K-ATPase α-subunit as an inactivation gate of palytoxin-induced pump channel

The N-terminus of the Na⁺,K⁺-ATPase α-subunit shows some homology to that of Shaker-B K⁺ channels; the latter has been shown to mediate the N-type channel inactivation in a ball-and-chain mechanism. When the Torpedo Na⁺,K⁺-ATPase is expressed in Xenopus oocytes and the pump is transformed into an ion channel with palytoxin (PTX), the channel exhibits a time-dependent inactivation gating at positive potentials. The inactivation gating is eliminated when the N-terminus is truncated by deleting the first 35 amino acids after the initial methionine. The inactivation gating is restored when a synthetic N-terminal peptide is applied to the truncated pumps at the intracellular surface. Truncated pumps generate no electrogenic current and exhibit an altered stoichiometry for active transport. Thus, the N-terminus of the α-subunit appears to act like an inactivation gate and performs a critical step in the Na⁺,K⁺-ATPase pumping function.     

KCNQ1 Channels Do Not Undergo Concerted but Sequential Gating Transitions in Both the Absence and the Presence of KCNE1 Protein

The co-assembly of KCNQ1 with KCNE1 produces I_KS, a K⁺ current, crucial for the repolarization of the cardiac action potential. Mutations in these channel subunits lead to life-threatening cardiac arrhythmias. However, very little is known about the gating mechanisms underlying KCNQ1 channel activation. Shaker channels have provided a powerful tool to establish the basic gating mechanisms of voltage-dependent K⁺ channels, implying prior independent movement of all four voltage sensor domains (VSDs) followed by channel opening via a last concerted cooperative transition. To determine the nature of KCNQ1 channel gating, we performed a thermodynamic mutant cycle analysis by constructing a concatenated tetrameric KCNQ1 channel and by introducing separately a gain and a loss of function mutation, R231W and R243W, respectively, into the S4 helix of the VSD of one, two, three, and four subunits. The R231W mutation destabilizes channel closure and produces constitutively open channels, whereas the R243W mutation disrupts channel opening solely in the presence of KCNE1 by right-shifting the voltage dependence of activation. The linearity of the relationship between the shift in the voltage dependence of activation and the number of mutated subunits points to an independence of VSD movements, with each subunit incrementally contributing to channel gating. Contrary to Shaker channels, our work indicates that KCNQ1 channels do not experience a late cooperative concerted opening transition. Our data suggest that KCNQ1 channels in both the absence and the presence of KCNE1 undergo sequential gating transitions leading to channel opening even before all VSDs have moved.     

The Link between Ion Permeation and Inactivation Gating of Kv4 Potassium Channels

Kv4 potassium channels undergo rapid inactivation but do not seem to exhibit the classical N-type and C-type mechanisms present in other Kv channels. We have previously hypothesized that Kv4 channels preferentially inactivate from the preopen closed state, which involves regions of the channel that contribute to the internal vestibule of the pore. To further test this hypothesis, we have examined the effects of permeant ions on gating of three Kv4 channels (Kv4.1, Kv4.2, and Kv4.3) expressed in Xenopus oocytes. Rb⁺ is an excellent tool for this purpose because its prolonged residency time in the pore delays K⁺ channel closing. The data showed that, only when Rb⁺ carried the current, both channel closing and the development of macroscopic inactivation are slowed (1.5- to 4-fold, relative to the K⁺ current). Furthermore, macroscopic Rb⁺ currents were larger than K⁺ currents (1.2- to 3-fold) as the result of a more stable open state, which increases the maximum open probability. These results demonstrate that pore occupancy can influence inactivation gating in a manner that depends on how channel closing impacts inactivation from the preopen closed state. By examining possible changes in ionic selectivity and the influence of elevating the external K⁺ concentration, additional experiments did not support the presence of C-type inactivation in Kv4 channels.     

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.     

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.     

Regulation of Deactivation by an Amino Terminal Domain in Human Ether-à-go-go?–related Gene Potassium Channels

Abnormalities in repolarization of the cardiac ventricular action potential can lead to life-threatening arrhythmias associated with long QT syndrome. The repolarization process depends upon the gating properties of potassium channels encoded by the human ether-à-go-go–related gene (HERG), especially those governing the rate of recovery from inactivation and the rate of deactivation. Previous studies have demonstrated that deletion of the NH₂ terminus increases the deactivation rate, but the mechanism by which the NH₂ terminus regulates deactivation in wild-type channels has not been elucidated. We tested the hypothesis that the HERG NH₂ terminus slows deactivation by a mechanism similar to N-type inactivation in Shaker channels, where it binds to the internal mouth of the pore and prevents channel closure. We found that the regulation of deactivation by the HERG NH₂ terminus bears similarity to Shaker N-type inactivation in three respects: (a) deletion of the NH₂ terminus slows C-type inactivation; (b) the action of the NH₂ terminus is sensitive to elevated concentrations of external K⁺, as if its binding along the permeation pathway is disrupted by K⁺ influx; and (c) N-ethylmaleimide, covalently linked to an aphenotypic cysteine introduced within the S4–S5 linker, mimics the N deletion phenotype, as if the binding of the NH₂ terminus to its receptor site were hindered. In contrast to N-type inactivation in Shaker, however, there was no indication that the NH₂ terminus blocks the HERG pore. In addition, we discovered that separate domains within the NH₂ terminus mediate the slowing of deactivation and the promotion of C-type inactivation. These results suggest that the NH₂ terminus stabilizes the open state and, by a separate mechanism, promotes C-type inactivation.