The carboxyl terminus of the epithelial Ca2+ channel ECaC1 is involved in Ca2+-dependent inactivation

The family of epithelial Ca2+ channels (ECaC) is a unique group of highly Ca2+-selective channels consisting of two members, ECaC1 and ECaC2. We used carboxyl terminal truncations and mutants to delineate the molecular determinants of the Ca2+-dependent inhibition of ECaC. To this end, rabbit ECaC1 was expressed heterologously with green fluorescent protein (GFP) in human embryonic kidney 293 (HEK293) cells using a bicistronic vector. Deletion of the last 30 amino acids of the carboxyl terminus of ECaC1 (G701X) decreased the Ca2+ sensitivity significantly. Another critical sequence for Ca2+-dependent inactivation of ECaC1 was found upstream in the carboxyl terminus. Analysis of truncations at amino acid 635, 639, 646, 649 and 653 disclosed a critical sequence involved in Ca2+-dependent inactivation at positions 650-653. C653X showed decreased Ca2+ sensitivity, comparable to G701X, while E649X lacked Ca2+-dependent inactivation. Interestingly, the number of green fluorescent cells, which is an index of the number of transfected cells, was significantly smaller for cells transfected with truncations shorter than E649 than for cells transfected with wild-type ECaC. However, the expression level of GFP was restored in the presence of the ECaC blocker ruthenium red, suggesting that these truncations resulted in deleterious Ca2+ influx. In conclusion, we have identified two domains in the carboxyl terminus of ECaC1 that control Ca2+-dependent inactivation.