The selectivity filter of a potassium channel, murine kir2.1, investigated using scanning cysteine mutagenesis.
We have produced a structural model of the pore-forming H5 (or P) region of the strong inward rectifier K+ channel, Kir2.1, based initially on an existing molecular model of the pore region of the voltage-gated K+ channel, Kv1.3. Cysteine-scanning mutagenesis and subsequent blockage by Ag+ was used to test our model by determining the residues in H5 whose side chains line the ion conduction pathway. Mutations made in eight positions within the highly conserved H5 region resulted in apparently non-functional channels. Constructing covalently linked dimers, which carry a cysteine substitution in only one of the linked subunits, rescued six of these mutants; a covalently linked tetramer, carrying a cysteine substitution on only one of the linked subunits, rescued a further mutant. Our results using the dimers and tetramers suggest that residues Thr141, Thr142, Ile143, Tyr145, Phe147 and Cys149 are accessible to externally applied Ag+ (100-200 nM) and therefore that their side chains line the channel pore. We conclude that the topology of the Kir pore is similar, but not identical, to that of Kv channels. Additionally, the molecular model suggests that selectivity may be conferred both by aromatic residues (Tyr145 and Phe147) via cation-pi interactions and by backbone carbonyl groups (Thr142 and Gly144).
Ion Channel Group, Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, Leicester LE1 9HN, UK.
C
C
Dart
M L
ML
Leyland
P J
PJ
Spencer
P R
PR
Stanfield
M J
MJ
Sutcliffe
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D003196:Computer Graphics; D006224:Cricetinae; D003545:Cysteine; D046911:Macromolecular Substances; D008564:Membrane Potentials; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D015221:Potassium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D046911:Macromolecular Substances; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D003545:Cysteine