Biophysical effects of pore mutations of ROMK1.
Potassium channels are ubiquitous, being present in all living organisms. These proteins share common structural elements, which confer common functional features. In general, all K+ channels have a high selectivity for K+, and are blocked by cations of similar dimensions, such as Cs+ and Ba2+. Mutations in the pore region tend to lead to either the total loss of function or K+ selectivity. We have made mutations to one of the most highly conserved residues of the pore, glycine-143, of the inward rectifier ROMK1 (Kir1.1), and examined the resulting channel properties in the Xenopus oocyte expression system with a two-electrode voltage clamp. Mutations G143A and G143R resulted in failure to express functional channels. Co-injection of wild-type ROMK1 cRNA with these mutants led to rescue of channel function, which was different from wild-type ROMK1. In both mutants, the sensitivity to Ba2+ and Cs+ was increased, the rate of onset of block by Ba2+ was enhanced, and the selectivity to potassium was reduced. Whereas the crystallographic evidence shows that cations bind to the carbonyl backbone of the pore-lining residues, the present results indicate that the side chains of these amino acids, which face away from the pore lining, also affect permeation.
Bhandari
S
S
School of Biomedical Sciences, Department of Physiology, Worsley Medical Building, University of Leeds, Leeds LS2 9NQ, UK. sunil.bhandari@medix-uk.com
Hunter
M
M
D000818:Animals; D001464:Barium / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000235:genetics; D002586:Cesium / Q000378:metabolism; D005260:Female; D005998:Glycine / Q000235:genetics*; D016014:Linear Models; D009154:Mutation / Q000235:genetics*; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D015221:Potassium Channels / Q000235:genetics* / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying; D018075:RNA, Complementary / Q000235:genetics; D014981:Xenopus
D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D018075:RNA, Complementary; D002586:Cesium; D001464:Barium; D005998:Glycine