Na⁺/H⁺Exchanger 1 Is Regulated via Its Lipid-Interacting Domain, Which Functions as a Molecular Switch: A Pharmacological Approach Using Indolocarbazole Compounds

Abstract

The plasma membrane Na 1 /H 1 exchanger 1 (NHE1) is rapidly activated in response to various stimuli. The membraneproximal cytoplasmic region (∼60 residues), termed the lipidinteracting domain (LID), is an important regulatory domain of NHE1. Here, we used a pharmacological approach to further characterize the role of LID in the regulation of NHE1. Pharmacological analysis using staurosporine-like indolocarbazole and bisindolylmaleimide compounds suggested that the phorbol ester-and receptor agonist-induced activation of NHE1 occurs through a protein kinase C-independent mechanism. In particular, only indolocarbazole compounds that inhibited NHE1 activation were able to interact with the LID, suggesting that the inhibition of NHE1 activation is achieved through the direct action of these compounds on the LID. Furthermore, in addition to phorbol esters and a receptor agonist, okadaic acid and hyperosmotic stress, which are known to activate NHE1 through unknown mechanisms, were found to promote membrane association of the LID concomitant with NHE1 activation; these effects were inhibited by staurosporine, as well as by a mutation in the LID. Binding experiments using the fluorescent ATP analog trinitrophenyl ATP revealed that ATP and the NHE1 activator phosphatidylinositol 4,5-bisphosphate bind competitively to the LID. These findings suggest that modulation of NHE1 activity by various activators and inhibitors occurs through the direct binding of these molecules to the LID, which alters the association of the LID with the plasma membrane. IntroductionThe ubiquitous Na 1 /H 1 exchanger isoform 1 (NHE1) catalyzes acid extrusion across the cell membrane by electroneutral ion exchange coupled to Na 1 influx and serves as a key regulator of intracellular pH (pH i ), Na 1 concentration, and cell volume in virtually all tissues (Wakabayashi et al., 1997;Orlowski and Grinstein, 2004). NHE1 elicits relatively high exchange activity under unstimulated physiologic conditions (basal state) and is further activated in response to various stimuli, including hormones, growth factors, and mechanical stress (activated state). This activation is attributable to a change in its affinity for intracellular H 1 ; thus, it can be easily detected in the neutral pH i range as a stimuli-induced cytoplasmic alkalinization in the absence of bicarbonate (Wakabayashi et al., 1997). Although NHE1 activation plays a physiologically important role in optimizing the intracellular ionic environment, it is also thought to promote the pathogenesis of disease, such as heart failure and cancer (Cardone et al., 2005;Karmazyn et al., 2008;Wakabayashi et al., 2013). In fact, treatment with NHE1 inhibitors reduced the pathologic phenotypes of animal models with acute and chronic heart diseases (Karmazyn et al., 2008).The molecular mechanism of the hormonal activation of NHE1 has been the focus of many investigations. The stimulation of Gq-coupled receptors induces the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] via the a...

A Histidine Cluster in the Cytoplasmic Domain of the Na-H Exchanger NHE1 Confers pH-sensitive Phospholipid Binding and Regulates Transporter Activity

“…If the set point or pH dependence of a proton modifier site is changed by ligand-dependent NHE1 phosphorylation, it remains to be determined whether the pK a values of histidine residues regulating PI(4,5)P 2 binding might be upshifted. Wakabayashi et al (36,37) proposed that growth factors and hormones increasing diacylglycerol activate NHE1 through a lipid-interacting domain (LID) encompassing amino acids 542-598 in human NHE1 that includes the PI(4,5)P 2 binding region. They found that the LID domain binds phorbol esters and used FRET reporters to show increased association of the NHE1 C terminus with the plasma membrane in cells treated with phorbol esters.…”

Na+/H+ exchange in the tumour microenvironment: does NHE1 drive breast cancer carcinogenesis?

“…PKC is thought to be involved in the hormonal activation of NHE1, but is not known to phosphorylate the exchanger. Instead, it is proposed that diacylglycerol and phorbol esters bind directly to the lipid interacting domain, inducing conformational changes in this region by increasing its association with membrane lipids (Shimada-Shimizu et al, 2014, Wakabayashi et al, 2010.…”

Na+–H+ exchanger-1 (NHE1) regulation in kidney proximal tubule

“…The initial juxtamembrane portion of the carboxy terminus contains two polybasic motifs (at residues 513–520 and 556–564 of NHE1) that bind inner leaflet phosphoinositides such as phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] [73–77], consistent with NHE1 compartmentalization to lipid rafts [78–80], which may be enriched for phosphoinositides [81]. The cytoskeletal adaptor proteins ezrin/radixin/moesin (ERM) bind the same NHE1 residues [82–84], but this interaction, as well as downstream ramifications, appear to be independent of Na + –H + translocation, whereas PI(4,5)P2 binding to the same sites is required for Na + –H + exchange [73, 82, 83].…”