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

G. A. Thompson, M. L. Leyland, I. Ashmole, M. J. Sutcliffe * and P. R. Stanfield

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 lowered 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 the Streptomyces lividans K⁺ channel KcsA, suggested the existence of an intersubunit hydrogen bond between Ser165 and Thr141 in the channel pore-forming P-region that helps stabilise the structure of this region. However, mutations of Thr141 and Ser165 did not produce effects consistent with a hydrogen bond between these residues being essential for blockage.

3. An alternative alignment between the M2 regions of Kir2.1 and KcsA suggested that Ser165 is itself a pore-lining residue, more directly affecting blockage. We were able to replace Ser165 with a variety of polar and non-polar 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 gating by polyamines - formed an additional selectivity filter by using the triple mutant T141A/S165L/D172N. Large Rb⁺ and Cs⁺ currents were measured in this mutant.

5. We propose that both Thr141 and Ser165 are likely to provide binding sites for monovalent blocking cations in wild-type channels. These residues lie beyond the carbonyl oxygen tunnel thought to form the channel selectivity filter, which the blocking cations must therefore traverse.

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