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 calciu m ([Ca2+](i)) signalling pathways was examined in human T-84 epithelia l cells, a model secretory cell line. Elevation of [Ca2+](i) by either the calcium ionophore A23187 (1 mu M) or the cholinergic agonist carb achol, led to the transient activation of both a chloride and cation c urrent in single voltage clamped cells. The channels underlying the ca tion conductance were found to be equally permeable to external Na+, K + and Cs+, but impermeable to the large organic cations tetraethylammo nium and N-methyl-D-glucamine (NMDG). These observations indicate that the cation channels are non-selective with respect to monovalent cati ons. 3. Persistent activation of both the chloride and non-selective c ation currents by [Ca2+](i) was observed following inhibition of cellu lar phosphatase activity by the phosphatase inhibitor microcystin LR o r the ATP analogue ATP gamma S. This finding strongly suggests the pre sence of a phosphorylation event in the calcium-dependent activation p athway for both currents. 4. Intracellular dialysis with peptide inhib itors of the multifunctional C2+-calmodulin-dependent protein kinase ( CaM kinase) blocked the activation of both the chloride and cation con ductances by elevated [Ca2+](i). Dialysis with an inactive control pep tide had no effect on the activation of either current. CaM kinase thu s appears to be critically involved in the calcium-dependent activatio n of both the chloride and cation currents in these cells. 5. Associat ed with the whole-cell cation conductance were macroscopic tail curren ts observed at the chloride reversal potential. The distinct kinetic p roperties of these tail currents were used as a biophysical 'signature ' of the whole-cell conductance. 6. In excised, inside-out membrane pa tches, [Ca2+](i) activated single cation channel activity. These chann els had a mean conductance of 20 pS, were impermeable to NMDG, and the ir mean open probability increased at positive membrane potentials. Th e properties of these single channel events thus closely resemble thos e reported previously for calcium-activated cation channels in epithel ia.7. Using a novel 'tail current' voltage clamp protocol in excised m embrane patches, we observed that ensemble averages of single cation c hannel events reproduced the behaviour and kinetic properties of the m acroscopic 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 med iates the calcium-dependent activation of both a chloride and a non-se lective cation current in human T-84 epithelial cells. Using single ch annel recordings, we believe we have identified the corresponding whol e-cell current for the 20-40 pS calcium-activated cation channel activ ity reported previously in epithelia and other cell preparations. Phys iologically: a calcium-activated inward cation current would allow sod ium influx in association with calcium-dependent electrolyte and prote in secretion. Thus CaM kinase-dependent activation of cation channels may serve as a co-ordinated influx pathway to balance the efflux and i nflux of osmotically active solutes as part of an overall cell volume regulatory mechanism.