Influences of the N- and C-termini of the distal nephron inward rectifier, ROMK.
The inward rectifying potassium channels of the ROMK family are present in the distal nephron of the kidney. These channels have two membrane spanning portions, between which lies a hydrophobic domain thought to confer the majority of the conductive properties of the channel. The N- and C-termini are both intracellular. In this paper we have examined the contribution of the N- and C-termini to the pore by examining the interaction of Cs+ with the channels. ROMK1 has an additional 19 amino acids on its N-terminus in comparison to ROMK2. The C-terminus of ROMK2 was extended by addition of a streptavidin tag (sfROMK2). Currents were measured following expression in Xenopus oocytes using two-electrode voltage clamp. ROMK1, ROMK2 and sfROMK2 exhibited concentration- and voltage-dependent block of inward currents by extracellular Cs+. The Hill coefficients were not significantly different from one. The mean Kd values at 0 mV were 100.6 +/- 10.6, 63.1 +/- 3.9 and 40.6 +/- 9.4, respectively (p < 0.05). The electric distances (delta) were 0.94 +/- 0.06, 1.0 +/- 0.05 and 1.37 +/- 0.06 respectively. The delta of sfROMK2 was greater than either ROMK1 or ROMK2 (p < 0.001). ROMK1, ROMK2 and sfROMK2 are sensitive to extracellular Cs+. Block was both concentration- and voltage-dependent. sfROMK2 is most Cs+-sensitive. ROMK1 contains an additional N-terminal 19 amino acids. Thus the pore properties of these two isoforms are subtly different, and influenced by the N-terminus. The lower Kd in sfROMK2 suggests that the streptavidin tag, and perhaps the C-terminus, also affect the pore.
Bhandari
S
S
School of Biomedical Sciences, Department of Physiology, Worsley Medical Building, University of Leeds, UK. Sunil.Bhandari@medix-uk.com
Hunter
M
M
D000818:Animals; D002586:Cesium / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D007202:Indicators and Reagents; D007686:Kidney Tubules, Distal / Q000502:physiology*; D008954:Models, Biological; D009865:Oocytes; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000737:chemistry* / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D016324:Sequence Tagged Sites; D019809:Streptavidin; D013329:Structure-Activity Relationship; D014981:Xenopus
D007202:Indicators and Reagents; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D002586:Cesium; D019809:Streptavidin