Abstract
The recently cloned epithelial Ca(2+) channel (ECaC) constitutes the Ca(2+) influx pathway in 1,25-dihydroxyvitamin D(3)-responsive epithelia. We have combined patch-clamp analysis and fura-2 fluorescence microscopy to functionally characterize ECaC heterologously expressed in HEK293 cells. The intracellular Ca(2+) concentration in ECaC-expressing cells was closely correlated with the applied electrochemical Ca(2+) gradient, demonstrating the distinctive Ca(2+) permeability and constitutive activation of ECaC. Cells dialyzed with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid displayed large inward currents through ECaC in response to voltage ramps. The corresponding current-voltage relationship showed pronounced inward rectification. Currents evoked by voltage steps to potentials below -40 mV partially inactivated with a biexponential time course. This inactivation was less pronounced if Ba(2+) or Sr(2+) replaced Ca(2+) and was absent in Ca(2+)-free solutions. ECaC showed an anomalous mole fraction behavior. The permeability ratio P(Ca):P(Na) calculated from the reversal potential at 30 mM [Ca(2+)](o) was larger than 100. The divalent cation selectivity profile is Ca(2+) > Mn(2+) > Ba(2+) approximately Sr(2+). Repetitive stimulation of ECaC-expressing cells induced a decay of the current response, which was greatly reduced if Ca(2+) was replaced by Ba(2+) and was virtually abolished if [Ca(2+)](o) was lowered to 1 nM. In conclusion, ECaC is a Ca(2+) selective channel, exhibiting Ca(2+)-dependent autoregulatory mechanisms, including fast inactivation and slow down-regulation.
Highlights
The gastrointestinal tract and kidney determine the net intake and output of Ca2ϩ for the entire body and, thereby, maintain together with bone the overall Ca2ϩ balance [1]
The present study describes functional characteristics of the recently cloned epithelial Ca2ϩ channel, ECaC, that are consistent with its putative role as apical Ca2ϩ entry channel that mediates transcellular Ca2ϩ transport in 1,25(OH)2D3-responsive epithelia
Transient expression of ECaC dramatically increased the Ca2ϩ permeability of HEK293 cells, which was reflected by an elevated cytosolic Ca2ϩ level proportional to the electrochemical driving force for Ca2ϩ
Summary
The gastrointestinal tract and kidney determine the net intake and output of Ca2ϩ for the entire body and, thereby, maintain together with bone the overall Ca2ϩ balance [1]. ECaC is exclusively present in 1,25(OH)2D3-responsive epithelia, including intestine, kidney, and placenta, and is structurally related to the family of transient receptor potential channels, capsaicin receptors, and the growth factor-regulated channel (4 – 6). These Ca2ϩ-permeable cation channels contain six putative transmembrane domains, including a pore-forming region, but share only 30% homology that is mainly restricted to the pore-forming region and flanking transmembrane segments. ECaC-expressing Xenopus laevis oocytes mediate a saturable Ca2ϩ uptake determined with tracer studies and a hyperpolarization-stimulated Ca2ϩ influx measured indirectly as Ca2ϩinduced ClϪ current [3, 7]. The aim of the present study is to functionally characterize ECaC by a combined whole-cell patchclamp analysis and fura-2 fluorescence microscopy using HEK293 cells heterologously expressing ECaC
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