AJP - Cell Physiology, Vol 261, Issue 2 C387-C392, Copyright © 1991 by American Physiological Society

ARTICLES

Regulation of Ca(2+)-activated K+ channels by protein kinase A and phosphatase inhibitors

A. Carl, J. L. Kenyon, D. Uemura, N. Fusetani and K. M. Sanders
Department of Physiology, University of Nevada, School of Medicine, Reno 89557.

Many proteins including ion channels are regulated by phosphorylation. We tested the effect of 10 U/ml catalytic subunit protein kinase A on 260-pS Ca(2+)-activated K+ channels in excised inside-out membrane patches from freshly dispersed smooth muscle cells of the canine proximal colon. At +50 mV with 10(-7) M Ca2+ and -50 mV with 10(-6) M Ca2+, open probability of the channels was increased to 270 +/- 48% of control (n = 12). This increase was due to a shift in voltage-dependent activation by 13.9 +/- 3.2 mV (n = 3) to more negative potentials. Protein kinase A in the absence of ATP had no effect on channel activity (n = 3). Regulation by phosphorylation must be accompanied by dephosphorylation. We tested the effect of two potent inhibitors of protein phosphatases, calyculin A and okadaic acid. Application of 10(-9) to 10(-6) M of each inhibitor in the presence of protein kinase A further increased open probability by up to 250%. Calyculin A appeared to be less effective in increasing open probability than okadaic acid, suggesting that the phosphatase involved is neither type 1, 2A, nor 2B. Calyculin A in the absence of protein kinase A was ineffective. These data suggest that endogenous phosphatases are found in excised membrane patches and that a balance between phosphorylation and dephosphorylation may provide an important control of colonic motility.

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