the NHE1 cytosolic tail mediates activation of the Na/H exchanger by

mammalian Na/H exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a single proton (H) in exchange for one extracellular Na. The human protein contains a500-amino acid membrane domain and a regulatory, 315-amino acid cytosolic domain. NHE1 is activated by a number of hormones including endothelin (ET) and by Ca2. The regulatory tail possesses an inhibitory calmodulin (CaM)-binding domain, and inhibi-tion of NHE1 is relieved by binding of a Ca2-CaM complex. We examined the dynamics of ET-1 and Ca2 regulation of binding to NHE1 in vivo. CFP was linked to the NHE1 protein cytoplasmic COOH terminus. This was stably transfected into AP-1 cells that are devoid of their own NHE1 protein. The protein was expressed and targeted prop-erly and retained NHE1 activity comparable to the wild-type protein. We examined the in vivo coupling of NHE1 to CaM by Förster resonance energy transfer using CaM linked to the fluorescent protein Venus. Ca