Permeation and gating properties of the novel epithelial Ca(2+) channel.
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.
Vennekens
R
R
Department of Physiology, Campus Gasthuisberg, Katholieke Universiteit Leuven, Leuven B-3000, Belgium.
Hoenderop
J G
JG
Prenen
J
J
Stuiver
M
M
Willems
P H
PH
Droogmans
G
G
Nilius
B
B
Bindels
R J
RJ
D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D016257:Fura-2; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D050916:TRPV Cation Channels
D015220:Calcium Channels; D002413:Cations, Divalent; D002614:Chelating Agents; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; D002118:Calcium; D016257:Fura-2