A non-selective cation current activated via the multifunctional Ca(2+)-calmodulin-dependent protein kinase in human epithelial cells

• Published in: The Journal of physiology 1995-10, Vol.488 (Pt 1), p.37-55
• Main Authors: Braun, A P, Schulman, H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anions - metabolism; Calcium-Calmodulin-Dependent Protein Kinases - antagonists & inhibitors; Calcium-Calmodulin-Dependent Protein Kinases - physiology; Cations - metabolism; Cell Line - cytology; Cell Line - enzymology; Cell Membrane Permeability - physiology; Chloride Channels - physiology; Epithelial Cells; Epithelium - enzymology; Humans; Molecular Sequence Data; Patch-Clamp Techniques; Peptides - physiology; Phosphoric Monoester Hydrolases - antagonists & inhibitors
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.1995.sp020944

1. Activation of macroscopic membrane currents by intracellular calcium ([Ca2+]i) signalling pathways was examined in human T84 epithelial cells, a model secretory cell line. 2. Elevation of [Ca2+]i by either the calcium ionophore A23187 (1 microM) 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 Ca(2+)-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 sin