Being flexible : the voltage-controllable activation gate of Kv channels

Kv channels form voltage-dependent potassium selective pores in the outer cell membrane and are composed out of four alpha-subunits, each having six membrane-spanning alpha-helices (S1-S6). The alpha-subunits tetramerize such that the S5-S6 pore domains co-assemble into a centrally located K+ pore which is surrounded by four operational voltage-sensing domains (VSD) that are each formed by the S1-S4 segments. Consequently, each subunit is capable of responding to changes in membrane potential and dictates whether the pore should be conductive or not. K+ permeation through the pore can be sealed off by two separate gates in series: (a) at the inner S6 bundle crossing (BC gate) and (b) at the level of the selectivity filter (SF gate) located at the extracellular entrance of the pore. Within the last years a general consensus emerged that a direct communication between the S4S5-linker and the bottom part of S6 (S6(c)) constitutes the coupling with the VSD thus making the BC gate the main voltage-controllable activation gate. While the BC gate listens to the VSD, the SF changes its conformation depending on the status of the BC gate. Through the eyes of an entering K+ ion, the operation of the BC gate apparatus can be compared with the iris-like motion of the diaphragm from a camera whereby its diameter widens. Two main gating motions have been proposed to create this BC gate widening: (1) tilting of the helix whereby the S6 converts from a straight a-helix to a tilted one or (2) swiveling of the S60 whereby the S6 remains bent. Such motions require a flexible hinge that decouples the pre-and post-hinge segment. Roughly at the middle of the S6 there exists a highly conserved glycine residue and a tandem proline motif that seem to fulfill the role of a gating hinge which allows for tilting/swiveling/rotations of the post-hinge S6 segment. In this review we delineate our current view on the operation of the BC gate for controlling K+ permeation in Kv channels

Coupling of Voltage Sensing to Channel Opening Reflects Intrasubunit Interactions in Kv Channels

Voltage-gated K+ channels play a central role in the modulation of excitability. In these channels, the voltage-dependent movement of the voltage sensor (primarily S4) is coupled to the (S6) gate that opens the permeation pathway. Because of the tetrameric structure, such coupling could occur within each subunit or between adjacent subunits. To discriminate between these possibilities, we analyzed various combinations of a S4 mutation (R401N) and a S6 mutation (P511G) in hKv1.5, incorporated into tandem constructs to constrain subunit stoichiometry. R401N shifted the voltage dependence of activation to negative potentials while P511G did the opposite. When both mutations were introduced in the same α-subunit of the tandem, the positive shift of P511G was compensated by the negative shift of R401N. With each mutation in a separate subunit of a tandem, this compensation did not occur. This suggests that for Kv channels, the coupling between voltage sensing and gating reflects primarily an intrasubunit interaction

Negative charges in the loop between the A and B box of the T1 domain are involved in Kv2.1 and Kv2.1/Kv6.3 channel tetramerization

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Modulation of closed−state inactivation in Kv2.1/Kv6.4 heterotetramers as mechanism for 4−AP induced potentiation

The voltage-gated K+ (Kv) channel subunits Kv2.1 and Kv2.2 are expressed in almost every tissue. The diversity of Kv2 current is increased by interacting with the electrically silent Kv (KvS) subunits Kv5-Kv6 and Kv8-Kv9, into functional heterotetrameric Kv2/KvS channels. These Kv2/KvS channels possess unique biophysical properties and display a more tissue-specific expression pattern, making them more desirable pharmacological and therapeutic targets. However, little is known about the pharmacological properties of these heterotetrameric complexes. We demonstrate that Kv5.1, Kv8.1 and Kv9.3 currents were inhibited differently by the channel blocker 4 -aminopyridine (4-AP) compared to Kv2.1 homotetramers. In contrast, Kv6.4 currents were potentiated by 4-AP while displayingmoderately increased affinities for the channel pore blockers quinidine and flecainide. We found that the 4-AP induced potentiation of Kv6.4 currents was caused bymodulation of the Kv6.4-mediated closed-state inactivation: suppression by 4-AP of the Kv2.1/Kv6.4 closed-state inactivation recovered a population of Kv2.1/Kv6.4 channels that was inactivated at resting conditions, i.e. at a holding potential of -80 mV. This modulation also resulted in a slower initiation and faster recovery from closed-state inactivation. Using chimeric substitutions between Kv6.4 and Kv9.3 subunits, we demonstrated that the lower half of the S6 domain (S6c) plays a crucial role in the 4-AP induced potentiation. These results demonstrate that KvS subunits modify the pharmacological response of Kv2 subunits when assembled in heterotetramers and illustrate the potential of KvS subunits to provide unique pharmacological properties to the heterotetramers, as is the case for 4-AP on Kv2.1/Kv6.4 channels

The Electrically Silent Kv6.4 Subunit Confers Hyperpolarized Gating Charge Movement in Kv2.1/Kv6.4 Heterotetrameric Channels

The voltage-gated K+ (Kv) channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ∼40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K+ equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits

Alkanols inhibit voltage-gated K+ channels via a distinct gating modifying mechanism that prevents gate opening

Alkanols are small aliphatic compounds that inhibit voltage-gated K+ (K-v) channels through a yet unresolved gating mechanism. K-v channels detect changes in the membrane potential with their voltage-sensing domains (VSDs) that reorient and generate a transient gating current. Both 1-Butanol (1-BuOH) and 1-Hexanol (1-HeOH) inhibited the ionic currents of the Shaker K-v channel in a concentration dependent manner with an IC50 value of approximately 50 mM and 3 mM, respectively. Using the non-conducting Shaker-W434F mutant, we found that both alkanols immobilized approximately 10% of the gating charge and accelerated the deactivating gating currents simultaneously with ionic current inhibition. Thus, alkanols prevent the final VSD movement(s) that is associated with channel gate opening. Applying 1-BuOH and 1-HeOH to the Shaker-P475A mutant, in which the final gating transition is isolated from earlier VSD movements, strengthened that neither alkanol affected the early VSD movements. Drug competition experiments showed that alkanols do not share the binding site of 4-aminopyridine, a drug that exerts a similar effect at the gating current level. Thus, alkanols inhibit Shaker-type K-v channels via a unique gating modifying mechanism that stabilizes the channel in its non-conducting activated state

Kv3.1 uses a timely resurgent K+ current to secure action potential repolarization

High-frequency action potential (AP) transmission is essential for rapid information processing in the central nervous system. Voltage-dependent K(v)3 channels play an important role in this process thanks to their high activation threshold and fast closure kinetics, which reduce the neuron's refractory period. However, premature Kv3 channel closure leads to incomplete membrane repolarization, preventing sustainable AP propagation. Here, we demonstrate that K(v)3.1b channels solve this problem by producing resurgent K+ currents during repolarization, thus ensuring enough repolarizing power to terminate each AP. Unlike previously described resurgent Na+ and K+ currents, K(v)3.1b's resurgent current does not originate from recovery of channel block or inactivation but results from a unique combination of steep voltage-dependent gating kinetics and ultra-fast voltage-sensor relaxation. These distinct properties are readily transferrable onto an orthologue K-v channel by transplanting the voltage-sensor's S3-S4 loop, providing molecular insights into the mechanism by which K(v)3 channels contribute to high-frequency AP transmission

Hydrophobic drug/toxin binding sites in voltage-dependent K+ and Na+ channels

In the Na(v)channel family the lipophilic drugs/toxins binding sites and the presence of fenestrations in the channel pore wall are well defined and categorized. No such classification exists in the much larger K(v)channel family, although certain lipophilic compounds seem to deviate from binding to well-known hydrophilic binding sites. By mapping different compound binding sites onto 3D structures of Kv channels, there appear to be three distinct lipid-exposed binding sites preserved in K(v)channels: the front and back side of the pore domain, and S2-S3/S3-S4 clefts. One or a combination of these sites is most likely the orthologous equivalent of neurotoxin site 5 in Na(v)channels. This review describes the different lipophilic binding sites and location of pore wall fenestrations within the K(v)channel family and compares it to the knowledge of Na(v)channels