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 microM 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+]i) was increased above 1 microM. If [Ca2+]i is increased to 800 microM, 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 microM [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.
Marunaka
Y
Y
Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322.
Eaton
D C
DC
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism*; D009399:Nephrons / Q000187:drug effects / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000494:pharmacology*
D018118:Chloride Channels; D002712:Chlorides; D007328:Insulin; D008565:Membrane Proteins; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium