CA-2+-DEPENDENT CL- CHANNELS IN UNDIFFERENTIATED HUMAN COLONIC CELLS (HT-29) .2. REGULATION AND RUNDOWN

The regulation of 15-pS Cl- channels by Ca2+-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 Ca2+-dependent Cl- channels. After rundown of these channels, a large-conductance, multistate, Ca2+-insensitive Cl- channel was seen. The smallest subconductance state of this channel was of similar magnitude to that of the Ca2+-dependent Cl- channel. Furthermore, its voltage and halide sensitivities were similar to those reported for the 15-pS Cl- channel and Ca2+-dependent whole cell Cl- currents. Because this channel is not observed in the intact cell, this may be a remnant conductance of the Ca2+-sensitive 15-pS Cl- channel.