Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba<SUP>2＋</SUP>

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba<SUP>2＋</SUP>

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&#1356;M for Kir2.1 wild type, 124&#1356;M for a concatameric WT-Y145V dimer, 109&#1356;M for a WT-Y145L dimer, and 267&#1356;M 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.