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...
The calmodulin (CaM)-binding domain reduces the affinity of the Na+/H+ exchanger NHE1 for intracellular H+ by exerting an autoinhibitory function in quiescent cells. We replaced this domain (aa 637-656) with homologous segments from other NHE isoforms (NHE2 and 4) or functionally similar regions from other sources (Na+/Ca2+ exchanger, CaM-dependent protein kinase II, plasma membrane Ca2+-pump, or CaM-binding peptide Trp3). The NHE-1-, NHE2-, and NHE4-segments bound CaM with Kds of 16, 130, and 27 nM, respectively. These chimeric molecules were expressed in the exchanger-deficient cell PS120. NHE1 with incorporated NHE2-segment was activated in response to Ca2+-mobilizing agents ionomycin and thrombin resulting in an alkaline shift of the intracellular pH (pHi)-dependence of 22Na+ uptake, as was the case with the intact rat NHE2. In contrast, incorporation of the NHE4-segment or other CaM-binding segments induced a constitutive alkaline shift of pHi-dependence with concomitant abolishment of Ca2+-dependent activation, indicating that these segments could not function as an autoinhibitory domain in NHE1. Detailed analyses revealed that Leu639, Lys651 and Tyr652, conserved in the NHE1- and NHE2-segments, but not in the NHE4-segment, are important for the autoinhibition. Furthermore, 125I-labeled CaM-binding peptide from NHE1 was efficiently crosslinked to the NHE1 protein, suggesting that the inhibitory domain physically interacts with part(s) of the molecule. Together, these findings support the notion that the reduction of H+ affinity in Na+/H+ exchange occurs through a mechanism involving a highly sequence-specific interaction of the inhibitory domain with its putative acceptor in NHE1.
To precisely identify the cytoplasmic subdomains that are responsible for the intracellular pH (pHi)-sensitivity, ATP depletion-induced inhibition and Ca2+ activation of the Na+/H+ exchanger (NHE1), we generated a set of deletion mutants of carboxyl-terminated cytoplasmic domain and expressed them in the exchanger-deficient cell line PS120. We evaluated pHi-sensitivity of these mutants by measuring the resting pHi in cells placed in an acidic medium (pH 6.0) and pHi-dependence of 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Detailed analysis revealed that the cytoplasmic domain of NHE1 is consists of at least four subdomains in terms of pHi-sensitivity of the unstimulated NHE1: I, aa 516-590/595; II, aa 596-635; III aa 636-659; and IV, aa 660-815. Subdomains II and IV were silent for pHi-sensitivity. Subdomain I had a pHi-maintenance function, preserving pHi-sensitivity in a physiological range, whereas subdomain III, overlapping with the high affinity calmodulin (CaM)-binding site, exhibited an autoinhibitory function. Deletion of subdomain I abolished the decrease of pHi-sensitivity induced by cell ATP depletion, indicating that domain I plays a crucial role in this phenomenon. Deletion of subdomain III rendered the inhibition by ATP depletion less efficient, suggesting the possible interaction between subdomains I and III. On the other hand, tandem elongation of subdomain II by insertion did not affect either the inhibitory function of domain III or the removal of this inhibition by ionomycin or thrombin. However, deletion of subdomain II partially abolished the inhibitory effect of subdomain III. Subdomain II thus seems to function as a mobile "flexible loop," permitting the CaM-binding subdomain III to exert its normal function. These findings, together with our previous data, support a concept that cell ATP, Ca2+, and growth factors regulate NHE1 via a mechanism involving direct or indirect interactions of specific cytoplasmic subdomains with the "H(+)-modifier site.".
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