Residues beyond the selectivity filter of the K+ channel Kir2.1 regulate permeation and block by external Rb+ and Cs+

1. Kir2.1 channels are blocked by Rb+ and Cs+ in a voltage-dependent manner , characteristic: of many inward rectifier K+ channels. Mutation of Ser165 in the transmembrane domain M2 to Leu (S165L) abolished Rb+ blockage and lo wered Cs+ blocking affinity. At negative voltages Rb+ carried large inward currents. 2. A model of the Kir2.1 channel, built by homology with the structure of t he Streptomyces lividans K+ channel KcsA, suggested the existence of an int ersubunit hydrogen bond between Ser165 and Thr141 in the channel pore-formi ng P-region that helps stabilise the structure of this region. However, mut ations of Thr141 and Xer165 did not produce effects consistent with a hydro gen bond between these residues being essential for blockage. 3. An alternative alignment between the M2 regions of Kir2.1 and KcsA sugge sted that Xer165 is itself a pore-lining residue, more directly affecting b lockage. We were able to replace Ser165 with a variety of polar and non-pol ar residues, consistent with this residue being pore lining. Some of these changes affected channel blockage. 4. We tested the hypothesis that Asp172 - a residue implicated in channel g ating by polyamines - formed an additional selectivity filter by using the triple mutant T141A/X165L/D172N. Large Rb+ and Cs+ currents were measured i n this mutant. 5. We propose that both Thr141 and Xer165 are likely to provide binding sit es for monovalent blocking cations in wild-type channels. These residues li e beyond the carbonyl oxygen tunnel thought to form the channel selectivity filter, which the blocking cations must therefore traverse.