An Intersubunit Interaction between S4-S5 Linker and S6 Is Responsible for the Slow Off-gating Component in Shaker K+ Channels

Abstract

Voltage-gated ion channels are controlled by the membrane potential, which is sensed by peripheral, positively charged voltage sensors. The movement of the charged residues in the voltage sensor may be detected as gating currents. In Shaker K ؉ channels, the gating currents are asymmetric; although the on-gating currents are fast, the off-gating currents contain a slow component. This slow component is caused by a stabilization of the activated state of the voltage sensor and has been suggested to be linked to ion permeation or C-type inactivation. The molecular determinants responsible for the stabilization, however, remain unknown. Here, we identified an interaction between Arg-394, Glu-395, and Leu-398 on the C termini of the S4-S5 linker and Tyr-485 on the S6 of the neighboring subunit, which is responsible for the development of the slow off-gating component. Mutation of residues involved in this intersubunit interaction modulated the strength of the associated interaction. Impairment of the interaction still led to pore opening but did not exhibit slow gating kinetics. Development of this interaction occurs under physiological ion conduction and is correlated with pore opening. We, thus, suggest that the above residues stabilize the channel in the open state.The voltage dependence of ion channels is the basis for all electrical signaling in the central nervous system. In tetrameric voltage-gated K ϩ channels, each subunit is composed of six transmembrane ␣-helices (S1-S6), with S1-S4 forming the voltage sensing domain and S5-S6 of all four subunits forming the pore. The voltage-sensing domains are covalently connected to the S5 of the pore region by the S4-S5 linker. The intracellular gate is made up of the S6 C-terminal ends that cross each other, forming a bundle that occludes the pore when the channel is closed. Pore opening in voltage-gated K ϩ channels is controlled by the movement of the voltage sensor in which charged residues of the S4 respond to changes in membrane potential. During this conformational change, the charges are moved through the electric field, generating the transient gating currents (for review, see Ref. 1). Gating currents were first predicted by Hodgkin and Huxley and were first detected in sodium channels by Armstrong et al. (2, 3). The movement is transferred to the pore domain (electromechanical coupling) and subsequently leads to pore opening. Voltage sensor movement precedes pore opening so that the transitions the channel undergoes during electromechanical coupling are reflected in the gating currents.Activation (on) and deactivation (off) gating currents for the non-conducting Shaker-IR channel, W434F (4 -6) have been previously described (6 -8). Briefly, on-gating currents rise and decay quickly after small depolarizations but rise more slowly and exhibit more prolonged and complex decay kinetics after intermediate depolarizations and, finally, develop and decay rapidly after depolarizations large enough to activate all channels. In contrast, offgating currents, which de...

Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker

“…In support of this idea, voltage sensor relaxation in Shaker is also sensitive to both the length and composition of the S3-S4 linker (22). Other studies have shown that mutations in the S4-S5 linker and S6 of Shaker channels, which uncouple the voltage sensor from the pore gate, also impede mode-shift behavior, suggesting that mode-shift might originate from the mechanical load placed on the voltage sensor domain by the pore (23,24). Others have also suggested that voltage-independent gating steps might underlie mode-shift behavior, rather than voltage sensor-mediated relaxation (25).…”

A Limited 4 Å Radial Displacement of the S4-S5 Linker Is Sufficient for Internal Gate Closing in Kv Channels

“…3C). The interaction is mediated by annealing of the S4-S5 linker to the C-terminal end of S6 (S6T (9,10,11,13,14,51)). The S4-S5 linker "presses" against the S6 at the level of its PVP motif, and accordingly, the pore closes with a "hydrophobic seal" by pushing the isoleucine 398 residues into the pathway, excluding most water molecules from the central cavity in the closed state (Fig.…”

Tetrameric assembly of KvLm K + channels with defined numbers of voltage sensors

“…Although it is well established that each sensor moves independently during the early transitions in activation (8), the nature of the interactions between subunits in Kv channels underlying the transition from activated-not-open to open (the opening transition) remains unsettled (6,(9)(10)(11)(12). Structurally, the late kinetic transitions are considered to arise from conformational changes in the S4-S5 linker (7,13), whereas the final opening transition (6, 7) entails a change in conformation of S6, which forms the bundle-crossing of the pore (activation gate).…”