Single alpha-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 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.
Palty
Raz
R
Department of Physiology, Zlotowski Center for Neuroscience, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Hershfinkel
Michal
M
Yagev
Oren
O
Saar
Drorit
D
Barkalifa
Ronit
R
Khananshvili
Daniel
D
Peretz
Asher
A
Grossman
Yoram
Y
Sekler
Israel
I
D019943:Amino Acid Substitution; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D017136:Ion Transport / Q000187:drug effects / Q000235:genetics; D007700:Kinetics; D020125:Mutation, Missense; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology
C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; D000889:Anti-Arrhythmia Agents; D020033:Protein Isoforms; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D013890:Thiourea; D002118:Calcium