Dynamic Na+/H+ Exchanger 1 (NHE1):Calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation
- University of Copenhagen, Denmark
- Rigshospitalet, Denmark
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.
Data availability
Source data files are provided for Figure 1, 3, 5 and 6. Resonance assignments of the ternary complex of CaM and two H1 have been deposited in the Biological Magnetic Resonance Bank (BMRB) under ID code 34521. The atomic coordinates for the ternary complex of CaM and two H1 have been deposited in the Protein Data Bank under the ID code 6zbi.
-
1H, 13C and 15N chemical shift assignments of the 2:1 complex between NHE1 and calmodulinBiological Magnetic Resonance Data Bank, 34521.
-
Ternary complex of Calmodulin bound to 2 molecules of NHE1RCSB Protein Data Bank, 6ZBI.
Article and author information
Author details
Birthe B Kragelund
Stine Falsig Pedersen
Funding
Danish Research Councils (4181-00344)
- Birthe B Kragelund
Novo Nordisk Fonden (NNF15OC0016670)
- Birthe B Kragelund
Novo Nordisk Fonden (NNF18OC0034070)
- Stine Falsig Pedersen
Novo Nordisk Fonden (NNF19OC0057241)
- Stine Falsig Pedersen
Novo Nordisk Fonden (NNF18OC0032996)
- Birthe B Kragelund
Villum Fonden
- Birthe B Kragelund
Carlsbergfondet (CF20-0491)
- Stine Falsig Pedersen
Novo Nordisk
- Lise M Sjøgaard-Frich
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Sjøgaard-Frich et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Download links
Further reading
-
- Cell Biology
Niemann–Pick disease type C (NPC) is a devastating lysosomal storage disease characterized by abnormal cholesterol accumulation in lysosomes. Currently, there is no treatment for NPC. Transcription factor EB (TFEB), a member of the microphthalmia transcription factors (MiTF), has emerged as a master regulator of lysosomal function and promoted the clearance of substrates stored in cells. However, it is not known whether TFEB plays a role in cholesterol clearance in NPC disease. Here, we show that transgenic overexpression of TFEB, but not TFE3 (another member of MiTF family) facilitates cholesterol clearance in various NPC1 cell models. Pharmacological activation of TFEB by sulforaphane (SFN), a previously identified natural small-molecule TFEB agonist by us, can dramatically ameliorate cholesterol accumulation in human and mouse NPC1 cell models. In NPC1 cells, SFN induces TFEB nuclear translocation via a ROS-Ca2+-calcineurin-dependent but MTOR-independent pathway and upregulates the expression of TFEB-downstream genes, promoting lysosomal exocytosis and biogenesis. While genetic inhibition of TFEB abolishes the cholesterol clearance and exocytosis effect by SFN. In the NPC1 mouse model, SFN dephosphorylates/activates TFEB in the brain and exhibits potent efficacy of rescuing the loss of Purkinje cells and body weight. Hence, pharmacological upregulating lysosome machinery via targeting TFEB represents a promising approach to treat NPC and related lysosomal storage diseases, and provides the possibility of TFEB agonists, that is, SFN as potential NPC therapeutic candidates.
-
- Cell Biology
Dynamic interactions between gut mucosal cells and the external environment are essential to maintain gut homeostasis. Enterochromaffin (EC) cells transduce both chemical and mechanical signals and produce 5-hydroxytryptamine to mediate disparate physiological responses. However, the molecular and cellular basis for functional diversity of ECs remains to be adequately defined. Here, we integrated single-cell transcriptomics with spatial image analysis to identify 14 EC clusters that are topographically organized along the gut. Subtypes predicted to be sensitive to the chemical environment and mechanical forces were identified that express distinct transcription factors and hormones. A Piezo2+ population in the distal colon was endowed with a distinctive neuronal signature. Using a combination of genetic, chemogenetic, and pharmacological approaches, we demonstrated Piezo2+ ECs are required for normal colon motility. Our study constructs a molecular map for ECs and offers a framework for deconvoluting EC cells with pleiotropic functions.
