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

Abstract

Calmodulin (CaM) engages in Ca2+-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 ([Ca2+]) and by phosphorylation of S648 in the first CaM-binding a-helix. In cells, Ca2+-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.

Chemical shift assignments of calmodulin under standard conditions at neutral pH

“…The vast majority of these structures involve Ca 2+ -bound forms of the CaM N-lobe and C-lobe that each bind to opposite sides of a target helix in a collapsed structure as seen in the structures of CaM bound to the myosin light chain kinase (MLCK) (PDB: 1CDL), CaM kinase II (PDB: 1CDM), CaM kinase kinase (PDB: 1CKK), ryanodine receptor 1 (PDB: 2BCX), ryanodine receptor 2 (PDB: 7KL5), CaV1.2 IQ-motif (2F3Y) and many other target proteins. By contrast, the two lobes of CaM are also known to each bind to separate target binding sites in a bipartite fashion as seen for CaM bound to the estrogen receptor-α (Zhang et al 2012 ) and the Na + /H + exchanger (NHE1) (Sjogaard-Frich et al 2021 ). Lastly, CaM binding to the anthrax adenylate cyclase exotoxin can lead to a complete remodeling of the target complex (Drum et al 2002 ).…”

Cytoskeletal activation of NHE1 regulates mechanosensitive cell volume adaptation and proliferation

“…Calmodulin (CaM) is an ubiquitous Ca 2+ receptor that has been shown to bind to NHE1 ( 28, 29, 50 ). To examine whether Ca 2+ -CaM regulates NHE1 activity, we treated cells with a Ca 2+ -CaM antagonist, W7.…”

“…A central question that arises is whether actomyosin directly influences cell volume via contractile force or indirectly regulates cell volume via signals to ion transporters. Ca 2+ related signaling cascades initiated by mechanosensitive ion channels is a promising candidate that bridge actomyosin contractility with ion transport (1,(28)(29)(30). Currently, how cell volume regulation is achieved through actomyosin and Ca 2+ mediated signaling is unknown.…”

PI-(3,5)P2-mediated oligomerization of the endosomal sodium/proton exchanger NHE9

“…2+ calmodulin binding to a site distal to the peripheral helix(Sjogaard-Frich et al, 2021).Our structure of NHE9*CC implies that the C-terminal tail may act in an autoinhibitory manner in all the NHEs in the absence of binding partners. It, thus, seems likely that the relocation of an auto-inhibitory C-terminal tail by binding partners could represent a general mode for positive allosteric regulation in all NHEs.…”