A voltage-dependent Ca2+ influx pathway regulates the Ca2+-dependent Cl(-) conductance of renal IMCD-3 cells.
We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca2+-dependent Cl(-) current (I(CLCA)). Here we report that I(CLCA) activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al3+-sensitive, voltage-dependent, Ca2+ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (-60 mV), ICLCA was found to be inactive and resting currents were predominantly K+ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of ICLCA (T(0.5) approximately 500 s), while repolarization in turn resulted in a monoexponential decay in I(CLCA) (T (0.5) approximately 100 s). The activation of I(CLCA) by depolarization was reduced by lowering extracellular Ca2+ and completely inhibited by buffering cytosolic Ca2+ with EGTA, suggesting a role for Ca2+ influx in the activation of I(CLCA). However, raising bulk cytosolic Ca2+ at -60 mV did not produce sustained I(CLCA) activity. Therefore I(CLCA) is dependent on both an increase in intracellular Ca2+ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca2+ influx pathway that displays equal permeability to Ca2+ and Ba2+ ions and that is blocked by extracellular Al3+ and La3+. Furthermore, Al3+ completely and reversibly inhibited depolarization-induced activation of ICLCA, thereby directly linking Ca2+ influx to activation of I(CLCA). We speculate that during sustained membrane depolarization, calcium influx activates ICLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.
Linley
John E
JE
Epithelial Research Group, Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne NE2-4HH, UK.
Boese
Stefan H
SH
Simmons
Nicholas L
NL
Gray
Michael A
MA
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D018118:Chloride Channels / Q000502:physiology*; D002713:Chlorine / Q000378:metabolism*; D004553:Electric Conductivity; D015640:Ion Channel Gating / Q000502:physiology*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D051379:Mice
D018118:Chloride Channels; D002713:Chlorine; D002118:Calcium