Dematin and Adducin Tether Sodium-Hydrogen Exchanger, NHE1, to Erythrocyte Membrane Cytoskeleton
Authors: Lu Yunzhe , Rivera Alicia , Chishti Athar 
Published: October 2019

Abstract
Dematin is a critical component of the membrane junctional complex in red blood cells. It tethers the spectrin cytoskeleton proteins to the membrane and its genetic deletion in mice causes dissociation of the spectrin, actin and β-adducin from the membrane resulting in the collapse of the red blood cells (RBCs). As dematin lacks a transmembrane domain, it is still unclear how this critical component of the junctional complex is anchored to the RBC membrane. Our previous studies have shown that the multi-transmembrane glucose transporter-1 (GLUT1) interacts with dematin and β-adducin in human RBCs, suggesting a potential role for GLUT1 in recruiting dematin to the membrane. However, as mouse RBCs do not express a GLUT1 homologue, an equivalent membrane receptor for dematin and/or adducin in mice remains to be determined. Using multiple in vitro and in vivo biochemical assays, here we demonstrate that the ubiquitously expressed plasma membrane Na+/H+ exchanger, NHE1 (Slc9a1), is one of the receptors for dematin and β-adducin in mature mouse red blood cells. NHE1 directly interacts with the core domain of dematin. Moreover, the dematin headpiece domain mutant S381E, which binds to the core domain with a higher affinity than the wild type, abolished the biochemical interaction between dematin and NHE1. This observation suggests that NHE1 and dematin headpiece domain compete for the same binding site(s) on the core domain. Furthermore, this finding highlights a molecular mechanism whereby an intermolecular switch of dematin regulates its interaction with NHE1 by phosphorylation. Dematin and β-adducin directly interact with NHE1 at its membrane-proximal cytoplasmic domain, which in turn regulates NHE1 activity in response to growth factor stimuli and intracellular pH alterations. Accordingly,we observed an increased cellular sodium content in erythrocytes of dematin headpiece and adducin double knockout mice (DAKO), suggesting a higher NHE1 activity in DAKO erythrocytes. Unlike GLUT1, NHE1 is expressed in both mouse and human RBCs. Thus, our results provide a novel mechanism for linking NHE1 to membrane skeleton and multiple cell signaling pathways through dematin and adducin (Figure 1). Since NHE1 is one of the major regulators of intracellular pH and hypertonic stress, our findings raise the possibility that the dematin-adducin-NHE1 complex may modulate these functions in RBCs as well as in other cell types with broad impact on the regulation of the actin cytoskeleton and cell migration.
Figure 1 Dematin and adducin link erythrocyte junctional complex to membrane via multiple receptors. A, WT RBC cytoskeleton. B, Mutant (DAKO) RBC cytoskeleton. Images show a grossly deranged membrane skeleton in DAKO as compared to wild type. Red arrows show enlarged pores and yellow arrows indicate the presence of aggregates. Bars correspond to 0.2 µM. C, Schematic diagram of dematin, adducin, and NHE1 linking the complex to multiple signaling pathways. D, Proposed new model of the erythrocyte junctional complex. Figure 1. Dematin and adducin link erythrocyte junctional complex to membrane via multiple receptors. A, WT RBC cytoskeleton. B, Mutant (DAKO) RBC cytoskeleton. Images show a grossly deranged membrane skeleton in DAKO as compared to wild type. Red arrows show enlarged pores and yellow arrows indicate the presence of aggregates. Bars correspond to 0.2 µM. C, Schematic diagram of dematin, adducin, and NHE1 linking the complex to multiple signaling pathways. D, Proposed new model of the erythrocyte junctional complex.

Disclosures
No relevant conflicts of interest to declare.