Mutations of Arg440 and Gly455/Gly456 oppositely change pH sensing of Na+/H+ exchanger 1

Abstract

To identify important amino acid residues involved in intracellular pH (pHi) sensing of Na+/H+ exchanger 1, we produced single-residue substitution mutants in the region of the exchanger encompassing the putative 11th transmembrane segment (TM11) and its adjacent intracellular (intracellular loop (IL) 5) and extracellular loops (extracellular loop 6). Substitution of Arg440 in IL5 with other residues except positively charged Lys caused a large shift in pHi dependence of 22Na+ uptake to an acidic side, whereas substitution of Gly455 or Gly456 within the highly conserved glycine-rich sequence of TM11 shifted pHi dependence to an alkaline side. The observed alkaline shift was larger with substitution of Gly455 with residues with increasing sizes, suggesting the involvement of the steric effect. Interestingly, mutation of Arg440 (R440D) abolished the ATP depletion-induced acidic shift in pHi dependence of 22Na+ uptake as well as the cytoplasmic alkalinization induced by various extracellular stimuli, whereas with that of Gly455 (G455Q) these functions were preserved. These mutant exchangers did not alter apparent affinities for extracellular transport substrates Na+ and H+ and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride. These results suggest that positive charge at Arg440 is required for normal pHi sensing, whereas mutation-induced perturbation of the TM11 structure may be involved in the effects of Gly mutations. Thus, both Arg440 in IL5 and Gly residues in the conserved segment of TM11 appear to constitute important elements for proper functioning of the putative "pHi sensor" of Na+/H+ exchanger 1.