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Pore properties and ionic block of the rabbit epithelial calcium channel expressed in HEK 293 cells

Rudi Vennekens, Jean Prenen, Joost G. J. Hoenderop*, René J. M. Bindels*, Guy Droogmans and Bernd Nilius

1. 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.

2. 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).

3. 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.

4. Extracellular Mg²⁺ blocked both divalent and monovalent currents with an IC₅₀ of 62 ± 9 µM (n = 4) in Ca²⁺-free conditions and 328 ± 50 µM (n = 4-9) in 100 µM Ca²⁺ solutions.

5. Mono- and divalent currents through ECaCs were blocked by gadolinium, lanthanum and cadmium, with a blocking order of Cd²⁺ >> Gd³⁺ > La³⁺.

6. 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.

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