In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that beta-adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.

Chloride impermeability of epithelial cells can account for many of the experimental and clinical manifestations of cystic fibrosis (CF). Activation of apical-membrane Cl- channels by cyclic AMP-mediated stimuli is defective in CF airway epithelial cells, despite normal agonist-induced increases in cellular cAMP levels. This defect in Cl- channel regulation has been localized to the apical membrane by exposing the cytoplasmic surface of excised membrane patches to the catalytic subunit (C subunit) of cAMP-dependent protein kinase and ATP. In membranes from normal cells, C-subunit activated Cl- channels with properties identical to those stimulated by cAMP-dependent agonists during cell-attached recording. Activation by the C subunit was not observed in CF membranes, but the presence of Cl- channels was verified by voltage-induced activation. The failure of the C subunit to activate the Cl- channels of CF membranes indicates that the block in their cAMP-mediated activation lies distal to induction of cAMP-dependent protein kinase activity and focuses our attention on the Cl- channel and its membrane-associated regulatory proteins as the probable site of the CF defect.

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Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.

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