Abstract
We have used the whole-cell patch-clamp technique to analyse the permeation properties and ionic block of the epithelial Ca2+ channel ECaC heterologously expressed in human embryonic kidney (HEK) 293 cells. Cells dialysed with 10 mM BAPTA and exposed to Ca2+-containing, monovalent cation-free solutions displayed large inwardly rectifying currents. Their reversal potential depended on the extracellular Ca2+ concentration, [Ca2+]o. The slope of the relationship between reversal potential and [Ca2+]o on a logarithmic scale was 21 +/- 4 mV, compared with 29 mV as predicted by the Nernst equation (n = 3-5 cells). Currents in mixtures of Ca2+ and Na+ or Ca2+ and Ba2+ showed anomalous mole fraction behaviour. We have described the current-concentration plot for Ca2+ and Na+ by a kinetic permeation model, i.e. the "step" model. Extracellular Mg2+ blocked both divalent and monovalent currents with an IC50 of 62 +/- 9 microM(n = 4) in Ca2+-free conditions and 328 +/- 50 microM (n = 4-9) in 100 microM Ca2+ solutions. Mono- and divalent currents through ECaCs were blocked by gadolinium, lanthanum and cadmium, with a blocking order of Cd2+ >> Gd3+ > La3+. We conclude that the permeation of monovalent and divalent cations through ECaCs shows similarities with L-type voltage-gated Ca2+ channels, the main differences being a higher Ca2+ affinity and a significantly higher current density in micromolar Ca2+ concentrations in the case of ECaCs.
