Ca(2+)-dependent Cl- channels in undifferentiated human colonic cells (HT-29). II. Regulation and rundown.
The regulation of 15-pS Cl- channels by Ca(2+)-mobilizing agonists was investigated by simultaneous cell-attached patch and intracellular Ca2+ concentration ([Ca2+]i) measurements. Cells were loaded with a synthetic peptide made from the calmodulin binding domain of Ca2+/calmodulin-dependent protein kinase II. This caused inhibition of Cl- channel activity without any corresponding effect on either agonist-induced [Ca2+]i mobilization or K+ channel activation. Calmodulin therefore confers Ca2+ sensitivity to the 15-pS channel. When patches were excised from the cell, Cl- channel activity ran down. Channel rundown was not reversed by ATP or calmodulin. When recording from cell-attached patches of detergent-treated cells, similar phenomenology was observed. Therefore, other factors that are lost upon plasma membrane permeabilization are required for the functioning of Ca(2+)-dependent Cl- channels. After rundown of these channels, a large-conductance, multistate, Ca(2+)-insensitive Cl- channel was seen. The smallest subconductance state of this channel was of similar magnitude to that of the Ca(2+)-dependent Cl- channel. Furthermore, its voltage and halide sensitivities were similar to those reported for the 15-pS Cl- channel and Ca(2+)-dependent whole cell Cl- currents. Because this channel is not observed in the intact cell, this may be a remnant conductance of the Ca(2+)-sensitive 15-pS Cl- channel.
Morris
A P
AP
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294-0005.
Frizzell
R A
RA
D000230:Adenocarcinoma; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000502:physiology; D002454:Cell Differentiation; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D018118:Chloride Channels; D003110:Colonic Neoplasms; D004072:Digitonin / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006706:Homeostasis; D006801:Humans; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008536:Meglumine / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology*; D009496:Neurotensin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D018118:Chloride Channels; D007473:Ion Channels; D008565:Membrane Proteins; D009496:Neurotensin; D004533:Egtazic Acid; D015759:Ionomycin; D008536:Meglumine; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D004072:Digitonin; D002118:Calcium