The long-term objectives of this laboratory remain the delineatio of the structure/function relationships involved in the gating (opening and closing) of voltage-gated ion channels. Three major gating mechanisms have bee described in Shaker potassium channels: i. Activation/deactivation- the normal voltage sensitive opening and closing mechanism. ii. Fast (N-type) inactivatio which closes channels during short maintained depolarizations (tens of milliseconds duration). iii. Slow (C-type) inactivation which closes channels during longer periods of depolarization (seconds). Only the second of these mechanisms has been effectively characterized in previous work, as involving a tethered ball domain which blocks the channel by binding to an internal bindin site near the internal mouth of the permeation path. However, recent work from our group has established that slow, C-type, inactivation does not involve either closing or blocking of the permeation pathway in Shaker channels. This unusual inactivation mechanism results from a change of channel selectivity such that channels no longer conduct K+ ions. On the other hand, macroscopic Na+ ions can be recorded in K-free solutions even after full development of C-type inactivation. This proposal aims: A, to further characterize both the selectivity change and the properties of activation and deactivation gating in C-type inactivated channels and B, to us site-directed mutagenesis of pore domain residues to evaluate the molecular mechanisms involved in the change from normal to C-type selectivity. This work will be carried out using Xenopus oocyte-expressed Shaker channels. Ionic and gating currents will be evaluated using standard patch clamp methods This work is strongly health-related, in that C-type inactivation is the primary mechanism controlling the inactivation and recovery rates of the cardiac potassium channels responsible for both IKS and IKF currents. Defects in this mechanism are responsible for the LQT1 and LQT2 syndromes associated with potentially lethal cardiac arrhythmias.
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