We studied the role of the interaction of calcineurin homologous protein 1 (CHP1) with the Na(+)/H(+) exchanger 1 (NHE1), particularly its EF-hand Ca(2+) binding motifs, in the intracellular pH (pH(i))-dependent regulation of NHE1. We found that (45)Ca(2+) binds to two EF-hand motifs (EF3 and 4) of the recombinant CHP1 proteins with high affinity (apparent K(d) = approximately 90 nM). Complex formation between CHP1 and the CHP1 binding domain of NHE1 resulted in a marked increase in the Ca(2+) binding affinity (K(d) = approximately 2 nM) by promoting a conformational change of the EF-hands toward the tightly Ca(2+)-bound form. This suggests that CHP1 always contains two Ca(2+) ions when associated with NHE1 in cells. Interestingly, overexpression of GFP-tagged CHP1 with mutations in EF3 or EF4 significantly reduced the exchange activity in the neutral pH(i) range and partly impaired the activation of NHE1 in response to various stimuli, such as growth factors and osmotic stress. Furthermore, we found that, in addition to reducing the activity (V(max)), a CHP1 binding-defective NHE1 mutant had a marked reduction in pH(i) sensitivity ( approximately 0.7 pH unit acidic shift), which consequently abolished various regulatory responses of NHE1. These observations suggest that the association of NHE1 with CHP1 is crucial for maintenance of the pH(i) sensitivity of NHE1 and that tightly bound Ca(2+) ions may serve as important structural elements in the "pH(i) sensor" of NHE1.

We examined the effect of intracellular acidification on the reverse mode of Na efflux, whereas mutation at Gly 455 (G455Q) caused a significant alkaline shift. Because these mutations and ATP depletion cause correspondingly similar effects on the forward mode of Na ؉ /H ؉ exchange, it is most likely that they alter exchange activity by modulating affinity of the internal modifier site for protons. The data provide substantial evidence that a proton modifier site(s) distinct from the transport site controls activities of at least three NHE isoforms through cooperative interaction with multiple protons.

To identify important amino acid residues involved in intracellular pH (pH i ) 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 Arg 440 in IL5 with other residues except positively charged Lys caused a large shift in pH i dependence of 22 Na ؉ uptake to an acidic side, whereas substitution of Gly 455 or Gly 456 within the highly conserved glycine-rich sequence of TM11 shifted pH i dependence to an alkaline side. The observed alkaline shift was larger with substitution of Gly 455 with residues with increasing sizes, suggesting the involvement of the steric effect. Interestingly, mutation of Arg 440 (R440D) abolished the ATP depletion-induced acidic shift in pH i dependence of 22 Na ؉ uptake as well as the cytoplasmic alkalinization induced by various extracellular stimuli, whereas with that of Gly 455 (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 Arg 440 is required for normal pH i sensing, whereas mutation-induced perturbation of the TM11 structure may be involved in the effects of Gly mutations. Thus, both Arg 440 in IL5 and Gly residues in the conserved segment of TM11 appear to constitute important elements for proper functioning of the putative "pH i sensor" of Na ؉ /H ؉ exchanger 1.

The plasma membrane Na þ /H þ exchangers (NHE) require calcineurin B homologous protein (CHP) as an obligatory binding partner for ion transport. Here, we report the first crystal structure of CHP (CHP2 isoform) in complex with its binding domain in NHE1. We show that the cytoplasmic a-helix of NHE1 is inserted into the hydrophobic cleft formed by N-and C-lobes of CHP2 and that the size and shape of this crevice together with hydrogen bond formation at multiple positions assure a high degree of specificity for interaction with NHE members. Structure-based mutagenesis revealed the importance of hydrophobic interactions between CHP/NHE1 for the function of NHE1. Furthermore, the crystal structure shows the existence of a protruding CHP-unique region, and deletion of this region in CHP2 inhibited the NHE1 activity by inducing the acidic shift of intracellular pH dependence, while preserving interaction with NHE1. These findings suggest that CHP serves as an obligatory subunit that is required both for supporting the basic activity and regulating the pH-sensing of NHE1 via interactions between distinct parts of these proteins.

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