EFFECTS OF INSULIN AND PHOSPHATASE ON A CA2+-DEPENDENT CL- CHANNEL IN A DISTAL NEPHRON CELL-LINE (A6)

A Cl− channel with a small single-channel conductance (3 pS) was observed in cell-attached patches formed on the apical membrane of cells from the distal nephron cell line (A6) cultured on permeable supports. The current-voltage (I-V) relationship from cell-attached patches or inside-out patches with 1 µM cytosolic Ca2+ strongly rectified with no inward current at potentials more negative than Ecl. However, the rectification decreased (i.e., inward current increased) when the cytosolic Ca2+ concentration ([Ca2+]) was increased above 1 µM. If [Ca2+]i is increased to 800 µM, the I-V relationship became linear. Besides the change in the I-V relationship, an increase in [Ca2+]i also increases the open probability of the channel. Regardless of the recording condition, the channel has one open and one closed state. Both closing and opening rates were dependent on [Ca2+]i an increase of [Ca2+]i decreased the closing rate and increased the opening rate. The Ca2+ dependence of transition rates at positive membrane potentials (cell interior with respect to external surface) were much larger than the dependence at negative intracellular potentials. The I-V relationship of chloride channels in inside-out patches from cells pretreated with insulin was linear even with 1 µM [Ca2+]i while channel currents from cells under similar conditions but without insulin still strongly rectified. Alkaline phosphatase applied to the intracellular surface of inside-out patches altered the outward rectification of single channels in a manner qualitatively similar to that of insulin pretreatment. These observations suggest that phosphorylation/dephosphorylation of the channel modulates the sensitivity of the Cl− channel to cytosolic Ca2+ and that insulin produces its effect by promoting dephosphorylation of the channel. © 1990, Rockefeller University Press., All rights reserved.