Abstract
The Na+/H + Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na+/H+ exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na+/H+ exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na+ transport capacity without affecting transport affinity (Km = 44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ 32P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na+ transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.
Highlights
The type 1 Na+/H+ exchanger (NHE1) is a ubiquitously distributed plasma membrane protein that is centrally involved in many physiological processes including fluid secretion, apoptosis, cell growth/proliferation, cell pH regulation and cell volume regulation
We previously demonstrated that activation of Na+/H+ Exchanger isoform 1 (NHE1)-mediated Na+ transport activity in osmotically shrunken Amphiuma tridactylum red blood cell (atRBC) is dependent upon a rate-limiting phosphorylation-dependent biochemical event [31]
To test the notion that osmotic cell shrinkage (OCS) and CLA treatment utilize the same biochemical mechanisms to increase NHE1 activity, we examined NHE1 activity, including MichaelisMenten Na+ transport kinetics in osmotically shrunken atRBCs
Summary
The type 1 Na+/H+ exchanger (NHE1) is a ubiquitously distributed plasma membrane protein that is centrally involved in many physiological processes including fluid secretion, apoptosis, cell growth/proliferation, cell pH regulation and cell volume regulation Appropriate activation of NHE1 during RVI depends upon signaling mechanisms that are very poorly understood [2,3,5,7,8,9,10,11,12,13]. Control of NHE1 transport activity involves multiple intracellular signaling molecules, including MAP/ERK kinases and Ca++/calmodulin, as well as phosphorylation (or dephosphorylation) at one or more serine (S) residues of the cytosolic C-terminus of NHE1, including (human numbering) S648, S703, S723, S726, S729, S770, S771, S785, and S796 [14,15,16,17,18,19,20,21,22]. Phosphorylation of NHE1 has never been demonstrated in response to OCS [23,27,28], suggesting that OCS increases NHE1 transport activity through a mechanism that differs from other forms of activation
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