title: Multiple residues in the p-region and m2 of murine kir 2.1 regulate blockage by external ba.
abstract: We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on Ba(2+) blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary (CHO) cells and used the whole cell patch-clamp technique to record K(+) currents in the absence and presence of externally applied Ba(2+). Wild type Kir2.1 was blocked by externally applied Ba(2+) in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of Ba(2+) blockage. The estimated K(d)(0) was 108 microM for Kir2.1 wild type, 124 microM for a concatameric WT-Y145V dimer, 109 microM for a WT-Y145L dimer, and 267 microM for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for Ba(2+), also showing two components with very different time courses. Mutants D172K and D172R (lining the central, aqueous cavity of the channel) showed both a decreased affinity to Ba(2+) and a decrease in the on transition rate constant (k(on)). These results imply that residues stabilising the cytoplasmic end of the selectivity filter (T141, S165) and in the central cavity (D172) are major determinants of high affinity Ba(2+) blockage in Kir 2.1.
authors.affiliation: Department of Physiology and Biophysics, Seoul National University, College of Medicine, Seoul 110-799, Korea.
authors.forename: Young Mee
authors.initials: YM
authors.lastname: Lee
authors.forename: Gareth A
authors.initials: GA
authors.lastname: Thompson
authors.forename: Ian
authors.initials: I
authors.lastname: Ashmole
authors.forename: Mark
authors.initials: M
authors.lastname: Leyland
authors.forename: Insuk
authors.initials: I
authors.lastname: So
authors.forename: Peter R
authors.initials: PR
authors.lastname: Stanfield