| The mammalian Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein that exchanges one intracellular H+ for one extracellular Na+. NHE1 is involved in the damage that occurs to the myocardium during ischemia and reperfusion, and inhibition of NHE1 reduces Na+/Ca2+ exchanger-mitigated Ca2+ overload. We studied the function and structure of NHE1, specifically focusing on residues in transmembrane (TM) segment IV. To accomplish this, we characterized the expression, surface targeting, and activity of NHE1 mutants in stably transfected cells that lack an endogenous Na +/H+ exchanger. We used site-directed mutagenesis to study the importance of three highly conserved proline residues, mutating Pro167 and Pro168 to alanine, glycine, and cysteine, and Pro178 to alanine. All mutations of Pro167 and Pro 168 caused significantly reduced activity, while mutation of Pro178 had no effect on activity. We next used cysteine-scanning mutagenesis in combination with reaction with sulfhydryl reactive reagents to identify residues in TM IV that line the ion transport pore. In this case we individually mutated each residue in TM IV to cysteine in a cysteineless NHE1 protein, and we found that residues in TM IV are exceptionally sensitive to mutation, with all of the single cysteine mutants displaying partially impaired activity. We treated the most active single-cysteine mutants with sulfhydryl reactive reagents and found that Phe161Cys was the only mutant that had significantly reduced activity following treatment, indicating that Phe161 likely lines the ion transport pore of NHE1. Based on this result and the NMR structure of a TM IV peptide, we mutated Phe161 to alanine, leucine, and lysine, and Asp159 and Asp172 to glutamic acid, asparagine, and glutamine. After correction for expression and plasma membrane targeting the only mutant that had decreased activity was Asp159G1u. We suggest that in the Asp159G1u mutant the increased length of the side chain causes the acidic functional group to protrude into the pore, thereby reducing transport. Overall, our results support the importance of TM IV in NHE1 structure and function, with Pro167 and Pro168 being essential for function, and with Phe161 and possibly Asp159 lining the ion transport pore of NHE1. |
