Cytoplasmic Domain of the Ubiquitous Na+/H+ Exchanger NHE1 Can Confer Ca2+ Responsiveness to the Apical Isoform NHE3

Abstract

Na؉ uptake in response to ionomycin and thrombin was observed in N3N1, accompanied by an alkaline shift of pH i sensitivity (ϳ0.2 pH units). Deletion of the cytoplasmic calmodulin-binding domain within N3N1 resulted in a constitutive alkaline shift of pH i sensitivity and abolished the activation by ionomycin and thrombin. Together, these data reinforce our concept of Ca 2؉ -induced activation of NHE1. Furthermore, they provide evidence for a functional interaction of the autoinhibitory domain of NHE1 with the H ؉ -modifier site of a different isoform, NHE3.Calcium ion is an important second messenger in mammalian cells, regulating various cell functions including muscle contraction, secretion, cell cycle progression, and a large variety of nerve cell functions. In many of these processes, elevation of the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and subsequent Ca 2ϩ -dependent activation of a ubiquitous regulator protein calmodulin (CaM) 1 have been recognized as a major mechanism of signal transduction in response to hormonal stimulation or membrane depolarization (1, 2).The electroneutral plasma membrane Na ϩ /H ϩ exchanger isoform 1 (NHE1) has been shown to be one of the targets regulated by intracellular Ca 2ϩ (3-8). NHE1 (9) is a ubiquitous amiloride-sensitive transporter that regulates pH i and cell volume (10, 11), and its structure-function relationship has been studied extensively (7,(12)(13)(14)(15)(16)(17). We have recently shown that NHE1 is a CaM-binding protein containing high and low affinity CaM-binding sites in the middle of the carboxyl-terminal cytoplasmic domain (17). Based on the analysis of function of NHE1 mutant molecules that do not bind CaM, we proposed that Ca 2ϩ -induced activation of NHE1 occurs via direct binding of Ca 2ϩ /CaM to the high affinity site that has an autoinhibitory function (7). However, further experiments were required to unambiguously confirm this hypothesis because of lack of evidence for the direct effect of Ca 2ϩ /CaM on the exchange activity.When NHE1 is activated in response to various stimuli such as growth factors, calcium, and hyperosmotic stress, it is generally accepted that pH i sensitivity of Na ϩ /H ϩ exchange increases without an apparent change in V max (18 -20). This is thought to result from increased affinity of the allosteric modifier site of the exchanger for the intracellular H ϩ (21). However, recently cloned other exchanger isoforms (NHE2, NHE3, and NHE4) (22-25) differ greatly from NHE1 in their regulation. Growth factors activate the epithelial isoforms NHE2 and NHE3 by increasing V max (26,27). Phorbol ester stimulates NHE1 and NHE2 but inhibits NHE3 (26,27). Hyperosmolarity stimulates NHE1, NHE2, and NHE4 but inhibits NHE3 (28 -30). These differences appear to be attributable to sequence divergence of the cytoplasmic domains of these NHE isoforms. The amiloride-resistant NHE3 that is expressed in the apical membrane of epithelial cells in kidney or intestine is the least related isoform among four mammalian NHEs. The NHE3 cy...

Mitogen-activated Protein Kinase-dependent Activation of the Na+/H+ Exchanger Is Mediated through Phosphorylation of Amino Acids Ser770 and Ser771

“…Phosphorylation of the Na ϩ /H ϩ exchanger isoform I mediates ϳ50% of the stimulatory effect of growth factors (9). Although kinase consensus and phosphorylation sites have been identified in the cytoplasmic domain in vitro (10, 18, 19), …”

“…The cytosolic domain regulates the membrane domain with phosphorylation having been shown to occur in the distal region of the cytosolic domain, within the last 178 amino acids (9,10). We have demonstrated that the MAP kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) are implicated in growth factor activation of NHE1.…”

Na+/H+ Exchangers of Mammalian Cells

“…Nevertheless, the transmembrane regions of other isoforms can respond to conformational changes of the tail induced by CaM, since insertion of the CaM-binding domain of NHE1 conferred [Ca 2ϩ ] sensitivity to NHE3 (54).…”

Hypertonicity Activates Na+/H+ Exchange through Janus Kinase 2 and Calmodulin

“…Kinases that have been shown to directly phosphorylate NHE-1 include p90 S6 kinase (4) and the Nck-interacting kinase (5). However, deletion of the major phosphorylation sites contained within residues 636 -815 of NHE-1 only reduces its response to growth factors by about 50% (6), suggesting that mechanisms of regulation other than direct phosphorylation of NHE-1 are also important.…”