TY - JOUR
T1 - Single α-domain constructs of the Na+/Ca2+ exchanger, NCLX, oligomerize to form a functional exchanger
AU - Palty, Raz
AU - Hershfinkel, Michal
AU - Yagev, Oren
AU - Saar, Drorit
AU - Barkalifa, Ronit
AU - Khananshvili, Daniel
AU - Peretz, Asher
AU - Grossman, Yoram
AU - Sekler, Israel
PY - 2006/10/3
Y1 - 2006/10/3
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33749358041&partnerID=8YFLogxK
U2 - 10.1021/bi060633b
DO - 10.1021/bi060633b
M3 - Article
C2 - 17002286
AN - SCOPUS:33749358041
SN - 0006-2960
VL - 45
SP - 11856
EP - 11866
JO - Biochemistry
JF - Biochemistry
IS - 39
ER -