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...
The voltage sensors of voltage-gated ion channels undergo a conformational change upon depolarization of the membrane that leads to pore opening. This conformational change can be measured as gating currents and is thought to be transferred to the pore domain via an annealing of the covalent link between voltage sensor and pore (S4-S5 linker) and the C terminus of the pore domain (S6). Upon prolonged depolarizations, the voltage dependence of the charge movement shifts to more hyperpolarized potentials. This mode shift had been linked to C-type inactivation but has recently been suggested to be caused by a relaxation of the voltage sensor itself. In this study, we identified two ShakerIR mutations in the S4-S5 linker (I384N) and S6 (F484G) that, when mutated, completely uncouple voltage sensor movement from pore opening. Using these mutants, we show that the pore transfers energy onto the voltage sensor and that uncoupling the pore from the voltage sensor leads the voltage sensors to be activated at more negative potentials. This uncoupling also eliminates the mode shift occurring during prolonged depolarizations, indicating that the pore influences entry into the mode shift. Using voltage-clamp fluorometry, we identified that the slow conformational change of the S4 previously correlated with the mode shift disappears when uncoupling the pore. The effects can be explained by a mechanical load that is imposed upon the voltage sensors by the pore domain and allosterically modulates its conformation. Mode shift is caused by the stabilization of the open state but leads to a conformational change in the voltage sensor.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.