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Abstract

The ubiquitous mammalian Na⁺/H⁺ exchanger NHE1 has critical functions in regulating intracellular pH, salt concentration, and cellular volume. The regulatory C-terminal domain of NHE1 is linked to the ion-translocating N-terminal membrane domain and acts as a scaffold for signaling complexes. A major interaction partner is calmodulin (CaM), which binds to two neighboring regions of NHE1 in a strongly Ca²⁺-dependent manner. Upon CaM binding, NHE1 is activated by a shift in sensitivity toward alkaline intracellular pH. Here we report the 2.23 Å crystal structure of the NHE1 CaM binding region (NHE1CaMBR) in complex with CaM and Ca²⁺. The C- and N-lobes of CaM bind the first and second helix of NHE1CaMBR, respectively. Both the NHE1 helices and the Ca²⁺-bound CaM are elongated, as confirmed by small angle x-ray scattering analysis. Our x-ray structure sheds new light on the molecular mechanisms of the phosphorylation-dependent regulation of NHE1 and enables us to propose a model of how Ca²⁺ regulates NHE1 activity.