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A charged residue in the turret or outer pore mouth of hKv1.5 alters macroscopic current kinetics Lee, Logan Eric Elmer

Abstract

Using hKvl.5 expressed in HEK-293 cells the roles of R487 and H463 in the processes of permeation and inactivation were studied. Mutation of the arginine at position 487 to a valine (R487V) alters the I-Vrelationship through Kvl.5 such that the normalized current at positive voltages, in symmetrical 140 mM K+ , is less than at negative voltages. The magnitude of current reduction at positive voltages was unaffected by extracellular acidification suggesting that the protonation state of H463 does not effect the rate of K + exit or entry at the outer pore mouth. Using an Eyring-rate model we have ascertained that the reduction in the normalized outward currents through R487V occurs through a change at the outer pore mouth that alters the permeation process. In addition to the role of R487V in the process of permeation, it has also been shown that a histidine residue at position 463 in Kvl.5 confers sensitivity to external protons and affects the rate, of inactivation. To further investigate the role these residues, R487 and H463, on Kvl.5 macroscopic current kinetics we engineered a double mutant that swapped the residues at positions 463 and 487. In Kvl.5 H463R/R487H there was a decrease in the time constant for inactivation and an increase in the time constant for recovery from inactivation. Elevation of K + 0 (140 mM) increased the time constant for inactivation and deactivation through a 'foot-in-the-door' mechanism by which elevated K + 0 occupies an outer and inner binding site, respectively, at the selectivity filter and prevents the pore from collapsing. These results support the hypothesis that processes of activation/deactivation and inactivation are not separate entities, but rather, alterations of one parameter can have dramatic effects on the other in Kvl.5.

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