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      Influences of the N- and C-Termini of the Distal Nephron Inward Rectifier, ROMK

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          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<sup>+</sup> 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<sup>+</sup>. The Hill coefficients were not significantly different from one. The mean K<sub>d</sub> 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<sup>+</sup>. Block was both concentration- and voltage-dependent. sfROMK2 is most Cs<sup>+</sup>-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<sub>d</sub> in sfROMK2 suggests that the streptavidin tag, and perhaps the C-terminus, also affect the pore.

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          Most cited references 9

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          Cloning and expression of an inwardly rectifying ATP-regulated potassium channel.

          A complementary DNA encoding an ATP-regulated potassium channel has been isolated by expression cloning from rat kidney. The predicted 45K protein, which features two potential membrane-spanning helices and a proposed ATP-binding domain, represents a major departure from the basic structural design characteristic of voltage-gated and second messenger-gated ion channels. But the presence of an H5 region, which is likely to form the ion conduction pathway, indicates that the protein may share a common origin with voltage-gated potassium channel proteins.
            • Record: found
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            • Article: not found

            Putative receptor for the cytoplasmic inactivation gate in the Shaker K+ channel.

            Inactivation of ion channels is important in the control of membrane excitability. For example, delayed-rectifier K+ channels, which regulate action potential repolarization, are inactivated only slowly, whereas A-type K+ channels, which affect action potential duration and firing frequency, have both fast and slow inactivation. Fast inactivation of Na+ and K+ channels may result from the blocking of the permeation pathway by a positively charged cytoplasmic gate such as the one encoded by the first 20 amino acids of the Shaker B (ShB) K+ channel. We report here that mutation of five highly conserved residues between the proposed membrane-spanning segments S4 and S5 (also termed H4) of ShB affects the stability of the inactivated state and alters channel conductance. One such mutation stabilizes the inactivated state of ShB as well as the inactivated state induced in the delayed-rectifier type K+ channel drk1 by the cytoplasmic application of the ShB N-terminal peptide. The S4-S5 loop, therefore, probably forms part of a receptor for the inactivation gate and lies near the channel's permeation pathway.
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              • Abstract: found
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              Alteration of ionic selectivity of a K+ channel by mutation of the H5 region.

               D A Yool,  T. Schwarz (1991)
              The high ionic selectivity of K+ channels is a unifying feature of this diverse class of membrane proteins. Though K+ channels differ widely in regulation and kinetics, physiological studies have suggested a common structure: a single file pore containing multiple ion-binding sites and having broader vestibules at both ends. We have used site-directed mutagenesis and single-channel recordings to identify a molecular region that influences ionic selectivity in a cloned A-type K+ channel from Drosophila. Single amino-acid substitutions in H5, the fifth hydrophobic region, enhanced the passage of NH4+ and Rb+, ions with diameters larger than K+, without compromising the ability of the channel to exclude the smaller cation, Na+. The mutations that substantially altered selectivity had little effect on the gating properties of the channel. We conclude that the H5 region is likely to line the pore of the K+ channel.

                Author and article information

                Kidney Blood Press Res
                Kidney and Blood Pressure Research
                S. Karger AG
                29 August 2001
                : 24
                : 3
                : 142-148
                School of Biomedical Sciences, Department of Physiology, Worsley Medical Building, University of Leeds, UK
                54221 Kidney Blood Press Res 2001;24:142–148
                © 2001 S. Karger AG, Basel

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                Figures: 5, References: 29, Pages: 7
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