Modulation of the epithelial calcium channel, ECaC, by intracellular Ca2+

We have studied the modulation by intracellular Ca2+ of the epithelial Ca2 channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and i nside-out patch clamp current recordings were combined with Furall-Ca2+ mea surements: 1. Currents through ECaC were dramatically inhibited if Ca2+ was the charge carrier. This inhibition was dependent on the extracellular Ca2+ concentra tion and occurred also in cells buffered intracellularly with 10 mM BAPTA. 2. Application of 30 mM [Ca2+](e) induced in non-Ca2+ buffered HEK 293 cell s at -80 mV an increase in intracellular Ca2+ ([Ca2+](i)) with a maximum ra te of rise of 241 +/- 15 nM/s (n = 18 cells) and a peak value of 891 +/- 10 6 nM. The peak of the concomitant current with a density of 12.3 +/-2.6pA/p F was closely correlated with the peak of the first-time derivative of the Ca2+ transient, as expected if the Ca2+ transient is due to influx of Ca2+. Consequently, no Ca2+ signal was observed in cells transfected with the Ca 2+ impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized. 3. Increasing [Ca2+](i) by dialyzing the cell with pipette solutions contai ning various Ca2+ concentrations, all buffered with 10 mM BAPTA, inhibited currents through ECaC carried by either Nat or Ca2+ ions. Half maximal inhi bition of Ca2+ currents in the absence of monovalent cations occurred at 67 nM (n between 6 and 8), whereas Naf currents in the absence of Ca2+ and Mg 2+ were inhibited with an IC50 of 89 nM (n between 6 and 10). Currents thro ugh ECaC in the presence of 1 mM Ca2+ and Naf, which are mainly carried by Ca2+, are inhibited by [Ca2+](i) with an IC50 of 82 nM (n between 6 and 8). Monovalent cation currents through the Ca2+ impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca2+](i) (IC50 =123 nM, n between 7 and 18). 4. The sensitivity of ECaC currents in inside-out patches for [Ca2+](i) was slightly shifted to higher concentrations as compared with whole cell meas urements. Half-maximal inhibition occurred at 169 nM if Na+ was the charge carrier (n between 4 and 11) and 228 nM at 1 mM [Ca2+](e) (n between 4 and 8). 5. Recovery from inhibition upon washout of extracellular Ca2+ (whole-cell configuration) or removal of Ca2+ from the inner side of the channel (insid e-out patches) was slow in both conditions. Half-maximal recovery was reach ed after 96 +/- 34 s (n = 15) in whole-cell mode and after 135 +/- 23s (n = 17) in inside-out patches. 6. We conclude that influx of Ca2+ through ECaC and [Ca2+](i) induce feedba ck inhibition of ECaC currents, which is controlled by the concentration of Ca2+ in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca2+ from an internal Ca2+ binding site at ECaC. (C) 2001 Harcourt Publishers Ltd.