Transmembrane segment packing of the Na(+)/Ca(2+) exchanger investigated with chemical cross-linkers.
The Na(+)/Ca(2+) exchanger (NCX1) is a plasma membrane protein important in regulating Ca(2+) in cardiac myocytes. The topological model is comprised of nine transmembrane segments (TMSs). To gain insights into the TMS packing arrangement of NCX1, we performed cysteine cross-linking experiments. Pairs of amino acids in different TMSs were mutated to cysteine on the backbone of a cysteineless NCX1. The mutated exchangers were expressed in an insect cell line and treated with cysteine-specific chemical cross-linkers followed by SDS-PAGE to determine the proximity of the introduced cysteines. Previously, we showed that TMSs 2, 3, 7, and 8 are near one another and that residues in TMSs 1 and 2 are close to TMS 6. In this report, we use the same approach to provide evidence for the arrangement of the remaining three TMSs (4, 5, and 9). We present a computer-generated two-dimensional model of transmembrane packing that minimizes the lengths of all cross-links.
Ren
Xiaoyan
X
Department of Physiology, Cardiovascular Research Laboratories, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095-1760, USA.
Nicoll
Debora A
DA
Xu
Lida
L
Qu
Zhilin
Z
Philipson
Kenneth D
KD
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D007313:Insecta; D017354:Point Mutation; D017433:Protein Structure, Secondary; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics
D003432:Cross-Linking Reagents; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D003545:Cysteine