Functional analysis of polar residues important for activity of Na⁺/H⁺ exchangers

Na⁺/H⁺ exchangers are a family of ubiquitous membrane proteins. In mammals the NHE1 isoform of the protein is widely distributed through all tissues and regulates cytosolic pH by removing an intracellular H⁺ in exchange for an extracellular Na⁺. In fission yeast, excess levels of intracellular Na⁺ are detrimental to these cells, and the Na⁺/H⁺ exchanger, sod2, plays a major role in the regulation of internal sodium concentration. We examined the functional role of conserved, polar, amino acid residues occurring in membrane-associated segments of the Na⁺/H⁺ exchanger proteins. For the mammalian Na⁺/H⁺ exchanger, mutant proteins of transmembrane segments VI and VII and the membrane-associated segment from amino acids 387 to 406 were assessed by characterization of intracellular pH changes in stably transfected cells that lacked an endogenous Na⁺/H⁺ exchanger. All of the mutant proteins were expressed and were targeted properly to the plasma membrane. Mutation of amino acid residues Glu262, Asp267, and E391 affected the Na⁺/H⁺ exchanger. Conservative substitutions with alternative acidic residues restored Na⁺/H⁺ exchanger activity. The Glu262Asp mutant had a decreased affinity for Li⁺. For the yeast Na⁺/H⁺ exchanger, similar experiments examined the effect of mutagenesis of several conserved polar amino acids. Of all Histidine residues, only His 367 was significant for activity. The Asp266,267 pair were mutated simultaneously, and sod2 function was found to be significantly impaired. Results also indicated that residues Asp145 and Asp241 are important for proper function of sod2. The results support the hypothesis that side chain oxygen atoms in a few, critically placed amino acids are important in various kinds of Na⁺/H⁺ exchanger activity.