MOLECULAR-PROPERTIES OF NEURONAL G-PROTEIN-ACTIVATED INWARDLY RECTIFYING K+ CHANNELS

Four cDNA-encoding G-activated inwardly rectifying K+ channels have be en 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) FEES Lett. 353, 37-42; Krapivinsky, G., Gor don, E. A., Wickm an, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Na ture 374, 135-141). We report the cloning of a mouse GIRK2 splice vari ant, noted mGIRK2A. Both channel proteins are functionally expressed i n Xenopus oocytes upon injection of their cRNA, alone or in combinatio n 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 he teromeric subunit assembly. GIRK3 channels have not been expressed suc cessfully, even in the presence of the other types of subunits. Howeve r, GIRK3 chimeras with the amino and carboxyl-terminal of GIRK2 are fu nctionally expressed in the presence of GIRK1. The expressed mGIRK2 an d mGIRK1, -2 currents are blocked by Ba2+ and Cs+ ions. They are not r egulated by protein kinase A and protein kinase C. Channel activity ru ns down in inside-out excised patches, and ATP is required to prevent this rundown. Since the nonhydrolyzable ATP analog AMP-PCP is also act ive and since addition of kinases A and C as well as alkaline phosphat ase does not modify the ATP effect, it is concluded that ATP hydrolysi s is not required. An ATP binding process appears to be essential for maintaining a functional state of the neuronal inward rectifier K+ cha nnel. A Na+ binding site on the cytoplasmic face of the membrane acts in synergy with the ATP binding site to stabilize channel activity.