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 ma jority of the conductive properties of the channel. The N- and C-termini ar e both intracellular. In this paper we have examined the contribution of th e Nand C-termini to the pore by examining the interaction of Cs+ with the c hannels. ROMK1 has an additional 19 amino acids on its N-terminus in compar ison to ROMK2. The C-terminus of ROMK2 was extended by addition of a strept avidin tag (sfROMK2). Currents were measured following expression in Xenopu s oocytes using two-electrode voltage clamp. ROMK1, ROMK2 and sfROMK2 exhib ited concentration- and voltage-dependent block of inward currents by extra cellular Cs'. The Hill coefficients were not significantly different from o ne. 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 sfRCMK2 w as greater than either ROMK1 or ROMK2 (p < 0.001). ROMK1, ROMK2 and sfRCMK2 are sensitive to extracellular Cs+. Block was both concentration- and volt age-dependent. sfRCMK2 is most Cs-sensitive. ROMK1 contains an additional N -terminal 19 amino acids. Thus the pore properties of these two isoforms ar e subtly different, and influenced by the N-terminus. The lower K-d in sfRO MK2 suggests that the streptavidin tag, and perhaps the C-terminus, also af fect the pore. Copyright (C) 2001 S. Karger AG, Basel.