Location of Transmembrane Segments of Na+/ca2+ Exchanger NCX1 Investigated with Chemical Crosslinkers

Abstract

The sodium-calcium exchanger (NCX1) is a plasma membrane protein important in regulating calcium in cardiac myocytes. The topological model is comprised of nine transmembrane segments (TMs) and a large intracellular loop, which has two Ca2+ binding domains (CBD1 and CBD2), between TMs five and six. CBD1 and CBD2 have been crystallized recently and are important in regulating the function of NCX1. On the other hand, the three dimensional structure of the full length NCX1 is unknown. To gain insights into that 3-D structure, we performed cysteine crosslinking experiments. Pairs of amino acids in different TMs were mutated to cysteine on the backbone of cysteineless NCX1. The mutated NCXs were expressed in an insect cell line and treated with cysteine-specific chemical crosslinkers followed by SDS-PAGE to determine the proximity of the introduced cysteines. The results allow us to place TMS I, IV and IX into the context of the other TMS. By combining our new results with our previous work ( J Biol Chem. 2006, 281: 22808-14; J Biol Chem. 2001, 276:194-9.) , we propose that TMs II and VII, which contain a number of hydrophilic residues, are surrounded by the remaining TMs.