The carboxyl terminus of the epithelial Ca(2+) channel ECaC1 is involved in Ca(2+)-dependent inactivation.
The family of epithelial Ca(2+) channels (ECaC) is a unique group of highly Ca(2+)-selective channels consisting of two members, ECaC1 and ECaC2. We used carboxyl terminal truncations and mutants to delineate the molecular determinants of the Ca(2+)-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 Ca(2+) sensitivity significantly. Another critical sequence for Ca(2+)-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 Ca(2+)-dependent inactivation at positions 650-653. C653X showed decreased Ca(2+) sensitivity, comparable to G701X, while E649X lacked Ca(2+)-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 Ca(2+) influx. In conclusion, we have identified two domains in the carboxyl terminus of ECaC1 that control Ca(2+)-dependent inactivation.
Nilius
Bernd
B
Department of Physiology, Campus Gasthuisberg, KU Leuven, Herestraat 49, 3000 Leuven, Belgium. bernd.nilius@med.kuleuven.ac.be
Weidema
Freek
F
Prenen
Jean
J
Hoenderop
Joost G J
JG
Vennekens
Rudi
R
Hoefs
Susan
S
Droogmans
Guy
G
Bindels
René J M
RJ
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D013329:Structure-Activity Relationship; D050916:TRPV Cation Channels
D015220:Calcium Channels; C118497:ECaC protein, Oryctolagus cuniculus; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; C492240:Trpv5 protein, mouse; C446923:Trpv6 protein, mouse; D002118:Calcium