A NONSELECTIVE CATION CURRENT ACTIVATED VIA, THE MULTIFUNCTIONAL CA2+-CALMODULIN-DEPENDENT PROTEIN-KINASE IN HUMAN EPITHELIAL-CELLS

1. Activation of macroscopic membrane currents by intracellular calcium ([Ca2+](i)) signalling pathways was examined in human T-84 epithelial cells, a model secretory cell line. Elevation of [Ca2+](i) by either the calcium ionophore A23187 (1 mu M) or the cholinergic agonist carbachol, led to the transient activation of both a chloride and cation current in single voltage clamped cells. The channels underlying the cation conductance were found to be equally permeable to external Na+, K+ and Cs+, but impermeable to the large organic cations tetraethylammonium and N-methyl-D-glucamine (NMDG). These observations indicate that the cation channels are non-selective with respect to monovalent cations. 3. Persistent activation of both the chloride and non-selective cation currents by [Ca2+](i) was observed following inhibition of cellular phosphatase activity by the phosphatase inhibitor microcystin LR or the ATP analogue ATP gamma S. This finding strongly suggests the presence of a phosphorylation event in the calcium-dependent activation pathway for both currents. 4. Intracellular dialysis with peptide inhibitors of the multifunctional C2+-calmodulin-dependent protein kinase (CaM kinase) blocked the activation of both the chloride and cation conductances by elevated [Ca2+](i). Dialysis with an inactive control peptide had no effect on the activation of either current. CaM kinase thus appears to be critically involved in the calcium-dependent activation of both the chloride and cation currents in these cells. 5. Associated with the whole-cell cation conductance were macroscopic tail currents observed at the chloride reversal potential. The distinct kinetic properties of these tail currents were used as a biophysical 'signature' of the whole-cell conductance. 6. In excised, inside-out membrane patches, [Ca2+](i) activated single cation channel activity. These channels had a mean conductance of 20 pS, were impermeable to NMDG, and their mean open probability increased at positive membrane potentials. The properties of these single channel events thus closely resemble those reported previously for calcium-activated cation channels in epithelia. 7. Using a novel 'tail current' voltage clamp protocol in excised membrane patches, we observed that ensemble averages of single cation channel events reproduced the behaviour and kinetic properties of the macroscopic tail currents of the calcium-activated cation conductance. This finding provides evidence that the observed single channel events probably underlie the macroscopic cation current recorded from intact cells. 8. The results from this study demonstrate that CaM kinase mediates the calcium-dependent activation of both a chloride and a non-selective cation current in human T-84 epithelial cells. Using single channel recordings, we believe we have identified the corresponding whole-cell current for the 20-40 pS calcium-activated cation channel activity reported previously in epithelia and other cell preparations. Physiologically: a calcium-activated inward cation current would allow sodium influx in association with calcium-dependent electrolyte and protein secretion. Thus CaM kinase-dependent activation of cation channels may serve as a co-ordinated influx pathway to balance the efflux and influx of osmotically active solutes as part of an overall cell volume regulatory mechanism.