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Biochemistry. 2006 Oct 3;45(39):11856-66.

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

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1
Department of Physiology, Zlotowski Center for Neuroscience, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.

Abstract

Spliced isoforms of the Na+/Ca2+ exchanger, NCLX, truncated at the alpha-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 alpha-repeat constructs (alpha1 and alpha2) 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 alpha1 or alpha2 single-domain proteins. In contrast, calcium transport and reversal currents were not detected when cells were transfected with a vector or with an alpha2 mutant (alpha2-S273T). Thus, our data indicate that the single alpha-domain constructs mediate electrogenic Na+/Ca2+ exchange. The alpha1 domain, but not the alpha2, exhibited partial sensitivity to the NCX inhibitor, KB-R7943, while Li+-dependent Ca2+ efflux was detected in cells expressing either the alpha1 or alpha2 construct. The functional organization of the single alpha-domain constructs was assessed using a dominant-negative approach. Coexpression of the alpha1 or alpha2 constructs with the nonfunctional alpha2-S273T mutant had a synergistic inhibitory effect on Na+/Ca2+ transport. Dose-dependence analysis of the inhibition of alpha2 construct activity by the alpha2-S273T mutant indicated that the functional unit is either a dimer or a trimer. Immunoprecipitation analysis indicated that the alpha2 construct indeed interacts with the alpha2-S273T mutant. Taken together, our data indicate that although single alpha1 or alpha2 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.

PMID:
17002286
DOI:
10.1021/bi060633b
[Indexed for MEDLINE]
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