Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites.
Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na+i- and Ca2+i-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that microM levels of Ca2+i inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. Mutations within the putative regulatory Ca2+i binding site of CALX1. 1, like corresponding alterations in NCX1.1, led to reduced ability (i.e. D516V and D550I) or inability (i.e. G555P) of Ca2+i to inhibit Na+-Ca2+ exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Delta310-313) of Na+i-dependent inactivation. These results indicate that the respective regulatory roles of the Ca2+i binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca2+i. We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca2+i regulatory phenotypes was feasible. With one chimera (i.e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca2+i. This result indicates that the regulatory Ca2+i binding site of NCX1.1 retains function in a CALX1. 1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca2+i binding signal.
Dyck
C
C
Institute of Cardiovascular Sciences, Department of Physiology, Faculty of Medicine, University of Manitoba, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba R2H 2A6, Canada.
Maxwell
K
K
Buchko
J
J
Trac
M
M
Omelchenko
A
A
Hnatowich
M
M
Hryshko
L V
LV
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D004330:Drosophila / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium