Ca2+-Induced Conformational Changes of Na+-Ca2+ Exchanger Dimers: Role of Ca2+ Binding Domains

Abstract

The Na+-Ca2+ exchanger is activated by the binding of cytoplasmic Ca2+ to two Ca2+ binding domains (CBD1 and CBD2). How binding of Ca2+ is translated into exchanger activation is unknown. We investigated Ca2+-dependent movements as changes in FRET between exchanger dimers tagged with CFP or YFP at positions 266 within the large cytoplasmic loop of NCX1.1. The biophysical properties of the fluorescent exchangers are identical to those of the untagged NCX. Fluorescent exchangers were coexpressed in Xenopus oocytes from which plasma membrane sheets were isolated. Upon addition of Ca2+, the coexpressed pair NCX-266CFP + NCX-266YFP showed an increase in FRET in a dose-dependent manner. Similar FRET changes were observed after mutating the Ca2+ coordination site in CBD2 (E516L). Exchanger E516L is not Ca2+ regulated. In contrast, mutating the Ca2+ coordination site in CBD1 (D421A, E451A and D500V) abolished FRET changes. These residues likely disrupt binding of Ca2+ to CBD1. Nevertheless, Ca2+ regulation of NCX is retained though with a substantial decrease in apparent affinity for Ca2+. These results indicate that the Ca2+-induced conformational changes of NCX dimers arise exclusively from the movement of CBD1. Peptides of Ca2+ binding domains, flanked by CFP and YFP, recapitulated the full length exchanger results: CBD1 showed movement upon Ca2+ addition while CBD2 did not. A peptide spanning CBD1-CBD2 displayed Ca2+-dependent movement, which was abolished by mutating the Ca2+ coordination site in CBD1. Our results indicate the following: 1. Exchanger conformational changes are associated with the occupancy of a high affinity Ca2+ binding site exclusively within CBD1. 2. FRET studies confirm that the Na+-Ca2+ exchanger exists as a dimer.