title: Influences of the N- and C-termini of the distal nephron inward rectifier, ROMK.
abstract: 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.
authors.affiliation: School of Biomedical Sciences, Department of Physiology, Worsley Medical Building, University of Leeds, UK. Sunil.Bhandari@medix-uk.com
authors.forename: S
authors.initials: S
authors.lastname: Bhandari
authors.forename: M
authors.initials: M
authors.lastname: Hunter
mesh_terms: 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
chemical_list: D007202:Indicators and Reagents; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D002586:Cesium; D019809:Streptavidin