Single α-domain constructs of the Na⁺/Ca²⁺ exchanger, NCLX, oligomerize to form a functional exchanger

Spliced isoforms of the Na⁺/Ca²⁺ exchanger, NCLX, truncated at the α-repeat region have been identified. The activity and functional organization of such proteins are, however, poorly understood. In the present work, we have studied Na⁺/Ca²⁺ exchange mediated by single α-repeat constructs (α1 and α2) of NCLX. Sodium-dependent calcium transport was fluorescently detected in both the reversal and forward modes; calcium-dependent outward currents were also recorded using a whole cell patch configuration in HEK293 cells heterologously expressing either the α1 or α2 single-domain proteins. In contrast, calcium transport and reversal currents were not detected when cells were transfected with a vector or with an α2 mutant (α2-S273T). Thus, our data indicate that the single α-domain constructs mediate electrogenic Na⁺/Ca²⁺ exchange. The α1 domain, but not the α2, exhibited partial sensitivity to the NCX inhibitor, KB-R7943, while Li⁺-dependent Ca²⁺ efflux was detected in cells expressing either the α1 or α2 construct. The functional organization of the single α-domain constructs was assessed using a dominant-negative approach. Coexpression of the α1 or α2 constructs with the nonfunctional α2-S273T mutant had a synergistic inhibitory effect on Na⁺/Ca²⁺ transport. Dose-dependence analysis of the inhibition of α2 construct activity by the α2-S273T mutant indicated that the functional unit is either a dimer or a trimer. Immunoprecipitation analysis indicated that the α2 construct indeed interacts with the α-S273T mutant. Taken together, our data indicate that although single α1 or α2 domain constructs are independently capable of Na ⁺/Ca²⁺ exchange, oligomerization is required for their activity. Such organization may give rise to transport activity with distinct kinetic parameters and physiological roles.