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Modulation of Kv1.5 slow inactivation by external cations Kwan, Daniel Cheuk Hang

Abstract

Slow inactivation is an intrinsic biophysical property of voltage-gated potassium (Kv) channels that results in a non-conducting state under physiological conditions. It limits the amount of current through Kv channels and affects cellular excitability. However, the molecular basis of slow inactivation is not well understood. In this thesis investigation, the modulation of slow inactivation in the human Kvl .5 channel by extracellular Zn²⁺, protons (H⁺), Ni²⁺, and other divalent-cations was studied using standard voltage clamp techniques. Zn²⁺, H⁺, and Ni²⁺ accelerated slow inactivation and caused a current inhibition in Kvl.5 expressed in HEK-293 and mouse ltk cells. The current inhibition was hypothesized to result from the binding of Zn²⁺, H⁺, and Ni²⁺ to the turret histidine residue (H463) which in turn promoted a slow inactivation process involving the outer pore mouth arginine residue (R487). The current inhibition induced by Zn²⁺, H⁺, and Ni²⁺ was attenuated either by increasing extracellular [K⁺] or by mutating H463 to glutamine (H463Q) or R487 to valine (R487V). Unitary current analysis revealed H⁺ and Ni²⁺ did not change the single channel current at +100 mV or the single channel conductance between 0 and +100 mV, but the number of blank (null) sweeps recorded with depolarizing pulses lasting up to 1 s was increased. The proportion of null sweeps correlated well with the extent of inhibition of macroscopic Kvl .5 current by external H⁺. A model incorporating two modes of gating was employed to describe the transitions between the active sweeps (mode A) and the null sweeps (mode U), and external H⁺ was proposed to inhibit Kv 1.5 current by promoting mode U gating. Consistent with this model was the finding that external K⁺ antagonized mode U gating induced by external H⁺. Channels were observed to switch from mode U back to mode A during prolonged depolarizations (> 6 s), and the delay in opening (first latency) was correlated with the dwell time in a depolarization-induced slow inactivated state. Together, the results suggest that Zn²⁺, H⁺, and Ni²⁺ inhibit Kvl .5 current by promoting a slow (P/C- type) inactivation process proceeding from closed states.

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