Human Connexin43 Gap Junction Channels Regulation of Unitary Conductances by Phosphoiylation

Connexin43 is the major gap protein in the heart and cardiovascular system. Single channel recordings of human connexin43 gap junction channels exogenously expressed in transfected SKHepl cells demonstrate two discrete classes of channel events, with unitary conductances of predominantly 60 to 70 and 90 to 100 pS when recorded with an internal solution containing CsCl as the major current-carrying ionic species and at moderate transjunctional voltages (<60 mV). Human connexin43 expressed in SKHepl cells displays multiple electrophoretic mobilities (apparent Mr, ~~'41 to 45 kD) when resolved in Western blots. Treatment of connexin43 from these cells with alkaline phosphatase collapses the bands into a single 41-kD species; application of alkaline phosphatase to the cell interior through patch pipettes yields channels that are predominantly of the larger unitary conductance. The smaller 60to 70-pS unitary conductance values correspond to the most common channel size seen in cultured rat cardiac ap junction proteins are encoded by the conG T nexin multigene family.1'2 Based on both RNA hybridization analyses and immunodetection studies, connexin43 appears to be the most abundant and widespread of the connexins thus far characterized. Connexin43 is expressed throughout the cardiovascular system, most prominently in the heart and in vascular smooth muscle and endothelial cells (for reviews see References 3 through 5). Recent studies using both nucleic acid and immunological probes demonstrate that individual tissues or even cell pairs from these tissues can express multiple connexin isoforms.3,6-8 Hence, the use of dissociated cells to characterize the physiological properties of channels composed of individual connexins is problematic. To evaluate properties of distinct connexin isoforms, including those that are ordinarily inaccessible to voltage-clamp analysis (such as human cardiac myocytes), exogenous expression systems have been developed. A major advance in understanding gap junction channel function has been the expression of complementary RNAs in Xenopus oocytes.9-12 This system has clearly demonstrated that the expression of a single connexin type is sufficient to establish functional intercellular Received December 14, 1993; accepted January 28, 1994. From the Departments of Neuroscience (A.P.M., J.C.S., D.C.S.), Medicine (G.I.F., D.C.S.), and Molecular Genetics (G.I.F.), Albert Einstein College of Medicine, Bronx, NY, and the Departamento de Ciencias Fisiol6gicas, Pontificia Universidad Cat6lica de Chile, Santiago, Chile (J.C.S.). Correspondence to David C. Spray, PhD, Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461. myocytes; these channels were more frequently observed after treatment with the phosphatase inhibitor okadaic acid, which was shown to increase phosphorylation of human connexin43 in these cells under similar conditions. Exposure to the protein kinase inhibitor staurosporine shifted the proportion of events toward the largest unitary conductance and resulted in decreased phosphorylation of human connexin43 in seryl residues in these cells. Thus, the unitary conductance of human connexin43 gap junction channels covaries with the phosphorylation state of the protein. This change in unitary conductance appears to be a unique effect of phosphorylation on gap junction channels, since it has not been observed for other ion channels that have thus far been evaluated. (Circ Res.