HUMAN CONNEXIN43 GAP JUNCTION CHANNELS - REGULATION OF UNITARY CONDUCTANCES BY PHOSPHORYLATION

Connexin43 is the major gap protein in the heart and cardiovascular sy stem. Single channel recordings of human connexin43 gap junction chann els exogenously expressed in transfected SKHep1 cells demonstrate two discrete classes of channel events, with unitary conductances of predo minantly 60 to 70 and 90 to 100 pS when recorded with an internal solu tion containing CsCl as the major current-carrying ionic species and a t moderate transjunctional voltages (<60 mV). Human connexin43 express ed in SKHep1 cells displays multiple electrophoretic mobilities (appar ent M(r), approximate to 41 to 45 kD) when resolved in Western blots. Treatment of connexin43 from these cells with alkaline phosphatase col lapses the bands into a single 41-kD species; application of alkaline phosphatase to the cell interior through patch pipettes yields channel s that are predominantly of the larger unitary conductance. The smalle r 60- to 70-pS unitary conductance values correspond to the most commo n channel size seen in cultured rat cardiac myocytes; these channels w ere more frequently observed after treatment with the phosphatase inhi bitor okadaic acid, which was shown to increase phosphorylation of hum an connexin43 in these cells under similar conditions. Exposure to the protein kinase inhibitor staurosporine shifted the proportion of even ts toward the largest unitary conductance and resulted in decreased ph osphorylation of human connexin43 in seryl residues in these cells. Th us, 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.