Cloning of a new mouse two-P domain channel subunit and a human homologue with a unique pore structure.

Mouse KCNK6 is a new subunit belonging to the TWIK channel family. This 335-amino acid polypeptide has four transmembrane segments, two pore-forming domains, and a Ca2+-binding EF-hand motif. Expression of KCNK6 transcripts is principally observed in eyes, lung, stomach and embryo. In the eyes, immunohistochemistry reveals protein expression only in some of the retina neurons. Although KCNK6 is able to dimerize as other functional two-P domain K+ channels when it is expressed in COS-7 cells, it remains in the endoplasmic reticulum and is unable to generate ionic channel activity. Deletions, mutations, and chimera constructions suggest that KCNK6 is not an intracellular channel but rather a subunit that needs to associate with a partner, which remains to be discovered, in order to reach the plasma membrane. A closely related human KCNK7-A subunit has been cloned. KCNK7 displays an intriguing GLE sequence in its filter region instead of the G(Y/F/L)G sequence, which is considered to be the K+ channel signature. This subunit is alternatively spliced and gives rise to the shorter forms KCNK7-B and -C. None of the KCNK7 structures can generate channel activity by itself. The KCNK7 gene is situated on chromosome 11, in the q13 region, where several candidate diseases have been identified.

Molecular properties of neuronal G-protein-activated inwardly rectifying K+ channels.

Four cDNA-encoding G-activated inwardly rectifying K+ channels have been cloned recently (Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. (1993) Nature 364, 802-806; Lesage, F., Duprat, F., Fink, M., Guillemare, E., Coppola, T., Lazdunski, M., and Hugnot, J. P. (1994) FEBS Lett. 353, 37-42; Krapivinsky, G., Gordon, E. A., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). We report the cloning of a mouse GIRK2 splice variant, noted mGIRK2A. Both channel proteins are functionally expressed in Xenopus oocytes upon injection of their cRNA, alone or in combination with the GIRK1 cRNA. Three GIRK channels, mGIRK1-3, are shown to be present in the brain. Colocalization in the same neurons of mGIRK1 and mGIRK2 supports the hypothesis that native channels are made by an heteromeric subunit assembly. GIRK3 channels have not been expressed successfully, even in the presence of the other types of subunits. However, GIRK3 chimeras with the amino- and carboxyl-terminal of GIRK2 are functionally expressed in the presence of GIRK1. The expressed mGIRK2 and mGIRK1, -2 currents are blocked by Ba2+ and Cs+ ions. They are not regulated by protein kinase A and protein kinase C. Channel activity runs down in inside-out excised patches, and ATP is required to prevent this rundown. Since the nonhydrolyzable ATP analog AMP-PCP is also active and since addition of kinases A and C as well as alkaline phosphatase does not modify the ATP effect, it is concluded that ATP hydrolysis is not required. An ATP binding process appears to be essential for maintaining a functional state of the neuronal inward rectifier K+ channel. A Na+ binding site on the cytoplasmic face of the membrane acts in synergy with the ATP binding site to stabilize channel activity.

Receptor binding characterization in kidney membrane of [3H]U-37883, a novel ATP-sensitive K+ channel blocker with diuretic/natriuretic properties.

U-37883 (4-morpholinecarboximidine-N-1-adamantyl-N-cyclohexyl), a known blocker of ATP-sensitive K+ (KATP) channels, produces natriuresis/diuresis in vivo by a direct effect on the kidney. In the present study, the binding characteristics of the U-37883 receptor were investigated using pig kidney cortex microsomes. [3H]U-37883 (0.5-5 nM, 50 Ci/mmol) exhibited specific binding, which was reversible, increased linearly with protein concentration (50-500 micrograms/ml), and was destroyed after treatment with proteases. Scatchard plots derived from the competition experiments suggested the presence of a single class of low affinity binding sites, with a Kd of 225 nM and a Bmax of 7.8 pmol/mg of protein. A similar Kd value was derived from complementary studies dealing with association and dissociation kinetics. The binding of [3H]U-37883 was tissue specific, because very little specific binding could be detected in microsomes from rat insulinoma cells (RINm5F) and brain. In contrast, these membranes displayed high affinity specific binding of [3H]glyburide, another KATP channel blocker. Finally, analogs of U-37883 that were found to be active KATP channel blockers in isolated rabbit mesenteric artery and active in vivo as diuretics/natriuretics were also found to be active in displacing specific binding of [3H]U-37883, whereas the inactive analogs (no vascular KATP channel-blocking activity and no in vivo diuresis/natriuresis) were inactive in this binding assay. We suggest that the U-37883 binding site represents a functional receptor that mediates the KATP channel antagonism and natriuresis observed with this class of compounds.