Explore the vast range of titles available.

Voltage gated potassium (Kv) channels regulate processes from cellular excitability to immune response and are major pharmaceutical targets. Despite recent structural advances, the closed state structure of the strictly coupled Kv1 family remains elusive. Here, we capture the structure of the Shaker potassium channel with a closed pore by uncoupling its voltage sensor domains from the pore domain. Structural determination of the uncoupled I384R mutant by single particle Cryo-EM reveals a fully closed pore coexisting with activated, non-relaxed voltage sensors. Comparison with the open pore structure suggests a roll-and-turn movement along the length of the pore-forming S6 helices, contrasting with canonical gating models based on limited movements of S6. These rotational-translational motions place two hydrophobic residues, one in the inner cavity and the other at the bundle crossing region, directly at the permeation pathway, limiting the pore radius to less than 1 Å. The selectivity filter is captured in a noncanonical state, partially expanded at G446, unlike previously described dilated or pinched filter conformations. Together, these findings suggest a reinterpretation of the mechanism of activation gating for strictly coupled Kv1 channels, highlighting the strictly sensor-pore coupling that underlies different functional states. Voltage-gated potassium channels control excitability, but their non-conductive structure remained elusive. Here, authors reveal the Shaker channel’s closed-pore structure, showing distinct “roll-and-turn” helix movements that redefine activation gating in the Kv1 family.

Voltage-dependent potassium channels (Kv) play a crucial role in membrane repolarization during action potentials. They undergo voltage-dependent structural conformational transitions according to their distribution across their energy landscape. Understanding these transitions helps us comprehend their molecular function. Here, we used sudden and sustained temperature changes (Tstep) combined with different voltage protocols and mutations to dissect the energy landscape of the Shaker K + channel. We used two mutations, ILT (V369I, I372L, and S376T) and I384N, that affect the coupling between the voltage sensor (VSD) and the pore domain (PD), to obtain the temperature dependence of VSD last transition and the intrinsic temperature dependence of the pore, respectively. Our findings support a loose or tight conformation of the electromechanical coupling. In the loose conformation, the movement of the VSD is necessary but not sufficient to efficiently propagate the electromechanical energy to open the pore. In contrast, this movement is effectively translated into pore opening in the tight conformation. Our results describe the energy landscape of the Shaker channel and how its temperature dependence can be modulated by affecting its electromechanical coupling. Voltage‐gated potassium channels allow nerve communication by coupling voltage sensing to pore opening. Here, the authors applied rapid temperature steps and mutations to map the channel’s energy landscape, revealing how distinct energy barriers contribute to channel activation.

The S4–S5 linker physically links voltage sensor and pore domain in voltage-gated ion channels and is essential for electromechanical coupling between both domains. Little dynamic information is available on the movement of the cytosolic S4–S5 linker due to lack of a direct electrical or optical readout. To understand the movements of the gating machinery during activation and inactivation, we incorporated fluorescent unnatural amino acids at four positions along the linker of the Shaker KV channel. Using two-color voltage-clamp fluorometry, we compared S4–S5 linker movements with charge displacement, S4 movement, and pore opening. We found that the proximal S4–S5 linker moves with the S4 helix throughout the gating process, whereas the distal portion undergoes a separate motion related to late gating transitions. Both pore and S4–S5 linker undergo rearrangements during C-type inactivation. In presence of accelerated C-type inactivation, the energetic coupling between movement of the distal S4–S5 linker and pore opening disappears.

Membrane-embedded voltage-sensor domains in voltage-dependent potassium channels (Kv channels) contain an impressive number of charged residues. How can such highly charged protein domains be efficiently inserted into biological membranes? In the plant Kv channel KAT1, the S2, S3, and S4 transmembrane helices insert cooperatively, because the S3, S4, and S3-S4 segments do not have any membrane insertion ability by themselves. Here we show that, in the Drosophila Shaker Kv channel, which has a more hydrophobic S3 helix than KAT1, S3 can both insert into the membrane by itself and mediate the insertion of the S3-S4 segment in the absence of S2. An engineered KAT1 S3-S4 segment in which the hydrophobicity of S3 was increased or where S3 was replaced by Shaker S3 behaves as Shaker S3-S4. Electrostatic interactions among charged residues in S2, S3, and S4, including the salt bridges between E283 or E293 in S2 and R368 in S4, are required for fully efficient membrane insertion of the Shaker voltage-sensor domain. These results suggest that cooperative insertion of the voltage-sensor transmembrane helices is a property common to Kv channels and that the degree of cooperativity depends on a balance between electrostatic and hydrophobic forces.

Voltage sensors regulate the conformations of voltage-dependent ion channels and enzymes. Their nearly switchlike response as a function of membrane voltage comes from the movement of positively charged amino acids, arginine or lysine, across the membrane field. We used mutations with natural and unnatural amino acids, electrophysiological recordings, and x-ray crystallography to identify a charge transfer center in voltage sensors that facilitates this movement. This center consists of a rigid cyclic \"cap\" and two negatively charged amino acids to interact with a positive charge. Specific mutations induce a preference for lysine relative to arginine. By placing lysine at specific locations, the voltage sensor can be stabilized in different conformations, which enables a dissection of voltage sensor movements and their relation to ion channel opening.

Potassium channels: the active-to-inactive switch Switching between conductive and non-conductive states is central to the function of ion channels. In potassium channels, inactivation gating occurs by two distinct molecular mechanisms: N-type inactivation (a rapid autoinhibitory process in which an N-terminal particle blocks conduction by binding to the open pore) and C-type inactivation (originating from conformational transitions at the selectivity filter). In the first of two papers, Eduardo Perozo and co-workers solve the X-ray crystal structure of the K + channel KcsA in an 'open-inactivated' conformation together with a series of crystal structures of channels that are 'trapped' in a set of partially open conformations. In the second paper, the authors identify the underlying mechanism by which movements in the inner gate of this channel trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. K + channels can convert between conductive and non-conductive forms through mechanisms that range from flicker transitions (which occur in microseconds) to C-type inactivation (which occurs on millisecond to second timescales). Here, the mechanisms are revealed through which movements of the inner gate of the K + channel KcsA trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. The coupled interplay between activation and inactivation gating is a functional hallmark of K + channels 1 , 2 . This coupling has been experimentally demonstrated through ion interaction effects 3 , 4 and cysteine accessibility 1 , and is associated with a well defined boundary of energetically coupled residues 2 . The structure of the K + channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation–inactivation gating 5 . Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter 6 , 7 , allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K + channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K + channels.

Most ion channels are multimeric (typically comprising 3–5 subunits). The subunits are encoded by homologous members of a gene family, generating an enormous set of possible heteromeric combinations. In this study, we provide evidence that the preferred target of conopeptide κM-RIIIJ is a heteromeric K v 1 channel consisting of three K v 1.2 subunits and one K v 1.1 or K v 1.6 subunit. We define the molecular interaction of κM-RIIIJ with these asymmetric K v 1 channels and show that in dorsal root ganglia (DRG) neurons, different κM-RIIIJ concentrations can distinguish discrete subpopulations of neurons. Our results highlight the potential of natural products and venom components, such as conopeptides, to generally elucidate native physiological roles of specific heteromeric ion channel isoforms at the cellular, circuit, and systems level. The vast complexity of native heteromeric K + channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K + channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this “zone of ignorance” in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets “asymmetric” K v channels composed of three K v 1.2 subunits and one K v 1.1 or K v 1.6 subunit with 100-fold higher apparent affinity compared with homomeric K v 1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional K v 1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative K v 1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by ( i ) covalent linkage of members of the mammalian Shaker-related K v 1 family to K v 1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K + channel-mediated currents in heterologous expression systems; ( ii ) molecular dynamics simulations of asymmetric K v 1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and ( iii ) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric K v channel complexes that operate in native tissues.

A Shaker Kv-channel V478W mutant shows enhanced C-type inactivation with disruption of the outermost K + site in the selectivity filter (IS1). The crystal structure of Kv1.2-2.1 bearing the equivalent mutation reveals an empty IS1. C-type inactivation underlies important roles played by voltage-gated K + (Kv) channels. Functional studies have provided strong evidence that a common underlying cause of this type of inactivation is an alteration near the extracellular end of the channel's ion-selectivity filter. Unlike N-type inactivation, which is known to reflect occlusion of the channel's intracellular end, the structural mechanism of C-type inactivation remains controversial and may have many detailed variations. Here we report that in voltage-gated Shaker K + channels lacking N-type inactivation, a mutation enhancing inactivation disrupts the outermost K + site in the selectivity filter. Furthermore, in a crystal structure of the Kv1.2-2.1 chimeric channel bearing the same mutation, the outermost K + site, which is formed by eight carbonyl-oxygen atoms, appears to be slightly too small to readily accommodate a K + ion and in fact exhibits little ion density; this structural finding is consistent with the functional hallmark of C-type inactivation.

The voltage-dependent potassium channels (Kv channels) show several different types of inactivation. N-type inactivation is a fast inactivating mechanism, which is essentially an open pore blockade by the amino-terminal structure of the channel itself or the auxiliary subunit. There are several functionally discriminatable slow inactivation (C-type, P-type, U-type), the mechanism of which is supposed to include rearrangement of the pore region. In some Kv1 channels, the actual inactivation is brought about by coupling of N-type and C-type inactivation (N-C coupling). In the present study, we focused on the N-C coupling of the Aplysia Kv1 channel (AKv1). AKv1 shows a robust N-type inactivation, but its recovery is almost thoroughly from C-type inactivated state owing to the efficient N-C coupling. In the I8Q mutant of AKv1, we found that the inactivation as well as its recovery showed two kinetic components apparently correspond to N-type and C-type inactivation. Also, the cumulative inactivation which depends on N-type mechanism in AKv1 was hindered in I8Q, suggesting that N-type inactivation of I8Q is less stable. We also found that Zn2+ specifically accelerates C-type inactivation of AKv1 and that H382 in the pore turret is involved in the Zn2+ binding. Because the region around Ile8 (I8) in AKv1 has been suggested to be involved in the pre-block binding of the amino-terminal structure, our results strengthen a hypothesis that the stability of the pre-block state is important for stable N-type inactivation as well as the N-C coupling in the Kv1 channel inactivation.

Voltage-gated ion channels open and close in response to changes in membrane potential, thereby enabling electrical signaling in excitable cells. The voltage sensitivity is conferred through four voltage-sensor domains (VSDs) where positively charged residues in the fourth transmembrane segment (S4) sense the potential. While an open state is known from the Kv1.2/2.1 X-ray structure, the conformational changes underlying voltage sensing have not been resolved. We present 20 additional interactions in one open and four different closed conformations based on metal-ion bridges between all four segments of the VSD in the voltage-gated Shaker K channel. A subset of the experimental constraints was used to generate Rosetta models of the conformations that were subjected to molecular simulation and tested against the remaining constraints. This achieves a detailed model of intermediate conformations during VSD gating. The results provide molecular insight into the transition, suggesting that S4 slides at least 12 Å along its axis to open the channel with a 310 helix region present that moves in sequence in S4 in order to occupy the same position in space opposite F290 from open through the three first closed states.