AJP - Cell Physiology, Vol 264, Issue 4 C977-C985, Copyright © 1993 by American Physiological Society

ARTICLES

Ca(2+)-dependent Cl- channels in undifferentiated human colonic cells (HT-29). II. Regulation and rundown

A. P. Morris and R. A. Frizzell
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294-0005.

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.

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