Dynamic Na+/H+ exchanger 1 (NHE1) - calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation

Dynamic Na+/H+ 1 (NHE1) - stoichiometry structure regulate Ca2+-dependent NHE1 activation. Abstract Calmodulin (CaM) engages in Ca 2+ -dependent interactions with numerous proteins, including a still incompletely understood physical and functional interaction with the human Na + / H + -exchanger NHE1. Using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, and fibroblasts stably expressing wildtype and mutant NHE1, we discovered multiple accessible states of this functionally important complex existing in different NHE1:CaM stoichiometries and structures. We determined the NMR solution structure of a ternary complex in which CaM links two NHE1 cytosolic tails. In vitro , stoichiometries and affinities could be tuned by variations in NHE1:CaM ratio and calcium ([Ca 2+ ]) and by phosphorylation of S648 in the first CaM-binding a -helix. In cells, Ca 2+ -CaM-induced NHE1 activity was reduced by mimicking S648 phosphorylation and by mutation of the first CaM-binding a -helix, whereas it was unaffected by inhibition of Akt, one of several kinases phosphorylating S648. Our results demonstrate a diversity of NHE1:CaM interaction modes and suggest that CaM may contribute to NHE1 dimerization and thereby augment NHE1 regulation. We propose that a similar structural diversity is of relevance to many other CaM complexes. first CaM-binding helix of NHE1, we employed a combination of solution state-nuclear magnetic resonance (NMR) isothermal titration calorimetry (ITC), NHE1 mutations, proximity ligation assays (PLA), and real-time analyses of NHE1-mediated acid extrusion after stable expression of wildtype (WT) and variant NHE1 in mammalian fibroblasts. Our findings reveal that, in solution, CaM interacts with NHE1 by exploiting different binding modes and stoichiometries, thereby forming several possible structures. Accordingly, in cells, Ca 2+ -induced NHE1 activation, but not NHE1:CaM proximity, was modulated by mutations preventing (S648A) or mimicking (S648D) phosphorylation, as well as reduced by charge-reversal (CR) mutations of the first CaM-binding site. This was not dependent on Akt kinase, inhibition of which did not affect Ca 2+ -induced activation of NHE1 under these conditions. Consistent with this notion, S648 phosphorylation could by Ser/Thr kinases. We propose that the interaction CaM and NHE1 is highly dynamic, involving both CaM-binding helices of NHE1, and with a functional role of S648 that is not exclusively downstream Akt. We suggest that the high degree of diversity observed for NHE1:CaM complexes may be relevant to other CaM complexes, particularly those involving membrane proteins. and two NHE1 peptides, pointing to a possible role of CaM in stabilizing NHE1 dimers via binding to two C-terminal regulatory tails. Our results provide novel insight into the dynamics and regulation of NHE1:CaM interaction, and we suggest that an additional NHE1 regulatory layer tuned by Ca 2+ -CaM availability may contribute to NHE1 dimerization. As CaM binding to membrane proteins is widespread, similar regulatory mechanisms and structural diversity may be relevant for tuning oligomerization and function in many other membrane proteins.