NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

Abstract

Many transport proteins, including pumps, channels, and transporters, are regulated by phosphoinositides. This rapidly expanding list includes voltage-gated K ϩ channels, inwardly rectifying K ϩ channels, and members of the TRP channel family, ENaC, NHE1, and NBCe1 (1-8). This regulation has been explained on the basis of two contrasting mechanisms. (i) There is direct phosphoinositide interaction with specific amino acids in the transport protein, explained either on the basis of charge (8 -13) or presence of canonical lipid binding domains such as pleckstrin homology domains (1,8,14). Common to these direct interaction studies has been the demonstration that mutagenesis of specific amino acids leads to changes in molecular interactions with phosphoinositides that lead to subsequent changes in physiologic channel or transporter activity.(ii) An indirect mechanism involves an additional intermediate, either protein or lipids, that binds via a phosphoinositide-dependent mechanism (5, 11).Sodium/hydrogen exchangers (SLC9a family) are ubiquitous transporters serving many functions in the cell, including regulation of intracellular pH, cell volume, and sodium absorption (15, 16). Grinstein and co-workers (17) have demonstrated that NHE1, the ubiquitous member of the sodium hydrogen exchanger gene family, is regulated by PI(4,5)P 2 , which binds to its C terminus. Using a unique whole cell patch pipette technique, Fuster et al. (18) showed that NHE3 is rapidly stimulated in opossum kidney cells by intracellular application of PI(3,4,5)P 3 .2 However, the mechanism of this stimulation is unknown. We hypothesized that the epithelial brush border Na ϩ /H ϩ antiporter NHE3 binds phosphoinositides based on the recognition that gene families have similar structural/functional organization (19). The aim of this study was to understand the mechanism of NHE3 regulation by phosphoinositides by the following: (i) investigating whether NHE3 can directly bind phosphoinositides; (ii) identifying regions and amino acids that are necessary for this interaction, and (iii) studying the physiologic relevance of this interaction. EXPERIMENTAL PROCEDURESMaterials-Lipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, PI(3,4,5)P 3 , and PI(4,5)P 2 were from Avanti Polar Lipids. QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). EZ-link sulfo-NHS-*

Unconventional Chemiosmotic Coupling of NHA2, a Mammalian Na+/H+ Antiporter, to a Plasma Membrane H+ Gradient

“…Data were processed and corrected for autofluorescence (measured from cells not loaded with BCECF). Calibration of the BCECF fluorescence ratio versus pH i was performed in the presence of 0.5 mM of the K ϩ /H ϩ ionophore nigericin (in "K-clamp media" of varying pH values, see below) according to a protocol described previously (15)(16)(17). For study of Na ϩ /H ϩ exchange activity cells were pulsed with NH 4 Cl (40 mM NH 4 Cl in TMA-medium, for 20 min).…”

AKT and GSK-3 Are Necessary for Direct Ezrin Binding to NHE3 as Part of a C-terminal Stimulatory Complex

“…The consensus sequence for GSK-3 substrates is (S/T)XXX(S/T)-P where the N-terminal S/T usually is the target residue phosphorylated and the C-terminal S/T is the site of priming phosphorylation. Although mutation of both serines in this putative GSK-3 site, Ser 522 Although different kinases affect individual Ser in the NHE3-F1 domain Ser cluster, they are in the same putative ␣-helix at which NHE3 directly binds ezrin, which has been shown to be involved in regulation of basal and stimulated NHE3 activity (13,21). Consequently, we tested the hypothesis that the Akt and GSK-3 effects and direct ezrin binding to the NHE3-C terminus were related.…”

“…Two separate complexes have been identified, both of which involve ezrin, although with different functions. One complex, which is the topic of this study, is present nearer the N terminus and predominantly regulates basal and stimulated NHE3 activity (13,21,29). The second complex includes the major site at which the scaffolding NHERF family of multi-PDZ proteins attach to NHE3 along with two active protein kinases; CaMKII, which inhibits NHE3 activity; and CK2, which simulates NHE3 under basal conditions (14,36,37).…”

“…In addition to ezrin binding, this putative helix also contains aa shown to be necessary for NHE3 to bind the phosphoinositides phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate and a putative PKD-binding domain. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4, 5-trisphosphate have been shown to be necessary for basal and regulated NHE3 activity (21). PDK is necessary for normal NHE3 activity, which was reduced when hypomorphic PDK1 was present (50).…”

“…GSK-3 is a kinase that often acts downstream of PI3K and Akt (30), is often inactivated by Akt (31), and is constitutively active in cells. GSK-3 activity has been shown to be inhibited through phosphorylation of Ser 21 in GSK-3␣ and Ser 9 in GSK-3␤. SB-216763 has been shown to be a potent inhibitor of GSK-3, while having no significant influence on the activity of other kinases (32).…”