THE SELECTIVITY FILTER OF A POTASSIUM CHANNEL, MURINE KIR2.1, INVESTIGATED USING SCANNING CYSTEINE MUTAGENESIS

1. We have produced a structural model of the pore-forming H5 (or P) r egion of the strong inward rectifier K+ channel, Kir2.1, based initial ly on an existing molecular model of the pore region of the voltage-ga ted K+ channel, Kv1.3. 2. Cysteine-scanning mutagenesis and subsequent blockage by Ag+ was used to test our model by determining the residue s in H5 whose side chains line the ion conduction pathway. 3. Mutation s made in eight positions within the highly conserved H5 region result ed in apparent ly non-functional channels. Constructing covalently lin ked dimers, which carry a cysteine substitution in only one of the lin ked subunits, rescued six of these mutants; a covalently linked tetram er, carrying a cysteine substitution on only one of the linked subunit s, rescued a further mutant. 4. Our results using the dimers and tetra mers suggest that residues Thr141, Thr142, Ile143, Tyr145, Phe147 and Cys149 are accessible to externally applied Ag+ (100-200 nM) and there fore that their side chains line the channel pore. 5. We conclude that the topology of the Kir pore is similar, but not identical, to that o f Kv channels. Additionally, the molecular model suggests that selecti vity may be conferred both by aromatic residues (Tyr145 and Phe147) vi a cation-pi interactions and by backbone carbonyl groups (Thr142 and G ly144).