of Work: We demonstrated previously that neuropeptide stimulation of BK channel activity in rat pituitary tumor cells (GH4C1) requires protein phosphatase (PP) activity (R.E. White et al., 1991; 1993), but the substrate remains unknown. When inside-out membrane patches from GH4C1 cells are excised into an ATP-free solution, BK channel activity gradually increases through a process that is reversed by Mg-ATP, but not by Mg, ADP or GTP, and is blocked by okadaic acid (OKA), a PP inhibitor. This indicates that the substrate is the pore forming protein or a closely associated regulatory protein. To address this, Dr. Romeo has isolated a cDNA encoding a BK channel alpha subunit by PCR from total GH4C1 mRNA, which Dr. Bauer has studied with the patch clamp technique. Cotransfection of HEK293 cells with cDNAs encoding BK alpha and the green fluorescent protein increased the sustained voltage- dependent outward current in fluorescent cells. This current was reduced by 8-chlorophenylthio-cAMP and by phorbol 12,13 dibutyrate, which stimulate protein kinase activity. In cell-attached patches on fluorescent HEK cells, channel activity was dominated by 150 pS channels with the same voltage-dependence as native BK channels. When these patches were excised into ATP-free solutions, activity increased, and in some cases was reversed by Mg-ATP. We are investigating the structural basis of these effects. Dr. Wang has investigated the mechanism of potassium channel (GIRK) stimulation by acetylcholine (ACh) in atrial myocytes where G-proteins are believed to bind directly to GIRK. When cell-attached patches were formed under conditions that do not deplete intracellular calcium, bath application of ACh stimulated the activity of 35 pS, inwardly rectifying K-selective channels. This effect was not reduced by atropine but was blocked by OKA, indicating the involvement of a diffusible messenger downstream of G-protein activation. When cells were current-clamped at 35 degrees C through nystatin-perforated patches, where both activated G-proteins and protein phosphatases could access the channels, OKA inhibited shortening of action potential duration by ACh. OKA was not simply disrupting G-protein activation by muscarinic receptors because OKA also inhibited GIRK activation by GTPgammaS in cell-free patches. Furthermore, application of purified PP2A was sufficient to stimulate GIRK activity in cell-free patches without adding guanine nucleotides. These results do not rule out direct effects of G- protein binding on GIRK activity in vivo, but they make such binding unnecessary to explain ACh action on intact atrial myocytes.
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| Erxleben, Christian; Gomez-Alegria, Claudio; Darden, Thomas et al. (2003) Modulation of cardiac Ca(V)1.2 channels by dihydropyridine and phosphatase inhibitor requires Ser-1142 in the domain III pore loop. Proc Natl Acad Sci U S A 100:2929-34 |
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