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|Title:||Structure-function studies of inwardly rectifying potassium channels|
|Authors:||Passmore, Gayle Martine|
|Presented at:||University of Leicester|
|Abstract:||The patch clamp technique was used to investigate the effect of mutations in the P-region on permeation characteristics and ionic selectivity in members of the Kir2.0 subfamily. Kir2.2 exhibits electrophysiological characteristics similar to those of Kir2.1 though there are differences in both unitary conductance and the kinetics of Ba2+ blockage. The P-region of these channels is virtually identical with the exception that leucine (L) at position 148 in Kir2.2 is replaced by phenylalanine (F) in Kir2.1. The effects of mutating L148 to phenylalanine in Kir2.2 and F147 to leucine in Kir2.1 on unitary conductance and channel sensitivity to Ba2+ were investigated. Neither mutation altered unitary conductance from that seen in the wild-type channel. However, mutation L148F in Kir2.2 reduced the association rate constant for Ba2+ blockage without affecting affinity. In contrast, mutation F147L in Kir2.1 increased channel affinity for Ba2+ without affecting the association rate constant. Thus, resides outside the P-region are responsible for the difference in unitary conductance and some of the differences in Ba2+ block in Kir2.2 and Kir2.1.;The effects on ionic selectivity of substituting single amino acid residues at position 143 in the P-region of Kir2.1 were also investigated. Substitution of isoleucine by hydrophobic residues such as valine (I143V) and leucine (I143L) raised the relative Rb+ permeability, whilst substitution of the more hydrophilic residue threonine (I143T) enhanced K+ selectivity. Two further mutants, I143C and I143S, failed to yield currents, but could be rescued by bathing cells in extracellular solution containing 10mM dithiothreitol (DTT). The permeability ratios were then similar to wild-type. The rescue of mutant channels by DTT suggests that the pore of Kir channels may undergo conformational changes. In vitro translation studies suggest that channel function is rescued through the disruption of an intra-subunit disulphide bond.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Cell Physiology and Pharmacology|
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