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Transmembrane segment packing of the Na(+)/Ca(2+) exchanger investigated with chemical cross-linkers.

ABSTRACT: 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.

SUBMITTER: Ren X 

PROVIDER: S-EPMC3005958 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Transmembrane segment packing of the Na(+)/Ca(2+) exchanger investigated with chemical cross-linkers.

Ren Xiaoyan X   Nicoll Debora A DA   Xu Lida L   Qu Zhilin Z   Philipson Kenneth D KD  

Biochemistry 20100909 39

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 che  ...[more]

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