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.
Highlights
EXPERIMENTAL PROCEDURESMolecular Biology and Channel Expression—All experiments were done using the pBSTA vector into which the Shaker IR (“inactivation-removed,” containing an N-terminal deletion (⌬6 – 46) that removes its fast inactivation properties [4, 35]) was cloned
The RELY Interaction Develops under Physiological Ion Permeation—We have demonstrated that in the non-conducting mutant W434F an interaction develops between the resi
In the present study we identified an interaction in Shaker Kϩ channels (RELY interaction) involving residues located at the junction of the C-terminal S4-S5 linker and the S5 (Arg-394, Glu-395, Leu-398) of one subunit and the C-terminal S6 of the adjacent subunit (Y485A)
Summary
Molecular Biology and Channel Expression—All experiments were done using the pBSTA vector into which the Shaker IR (“inactivation-removed,” containing an N-terminal deletion (⌬6 – 46) that removes its fast inactivation properties [4, 35]) was cloned. V1⁄2 and VЈ 1⁄2 for the first and second transition, respectively This follows after short derivation from Equation 4 below with voltage-dependent rate constants. The initial conditions for the on-gating transition (start of the pulse) are given by the steady state of the rate constants at resting potential (off-rate constants), and for the off-gating transition the predicted values after the depolarizing pulse according to the on-rate constants were used. With this model the traces could be well fitted except for possible delays in the immediate onset of on-gating, which require additional steps in the charge movement. The V1⁄2 of the entire system will always be more negative than that of the charge carrying step(s), and the QV will always be shifted to more negative potentials
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