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 K_d 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 (δ) were 0.94 ± 0.06, 1.0 ± 0.05 and 1.37 ± 0.06 respectively. The δ 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 K_d in sfROMK2 suggests that the streptavidin tag, and perhaps the C-terminus, also affect the pore.