Interactions of the fatty acid-binding protein ReP1-NCXSQ with lipid membranes. Influence of the membrane electric field on binding and orientation

The regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ) is a 15 kDa soluble, intracellular protein that regulates the activity of the Na⁺/Ca ²⁺ exchanger in the squid axon. It is a member of the cellular retinoic acid-binding proteins family and the fatty acid-binding proteins superfamily. It is composed of ten beta strands defining an inner cavity and a domain of two short alpha helix segments. In this work, we studied the binding and orientation of ReP1-NCXSQ in anionic and zwitterionic lipid membranes using molecular dynamics (MD) simulations. Binding to lipid membranes was also measured by filtration binding assay. ReP1-NCXSQ acquired an orientation in the anionic membranes with the positive end of the macrodipole pointing to the lipid membrane. Potential of mean force calculations, in agreement with experimental measurements, showed that the binding to the anionic interfaces in low ionic strength was stronger than the binding to anionic interfaces in high ionic strength or to zwitterionic membranes. The results of MD showed that the electrostatic binding can be mediated not only by defined patches or domains of basic residues but also by a global asymmetric distribution of charges. A combination of dipole–electric field interaction and local interactions determined the orientation of ReP1-NCXSQ in the interface.     

Metabolic regulation of the squid nerve Na⁺/Ca²⁺ exchanger: recent kinetic, biochemical and structural developments

The Na?/Ca2? exchangers are structural membrane proteins, essential for the extrusion of Ca2? from most animal cells. Apart from the transport sites, they have several interacting ionic and metabolic sites located at the intracellular loop of the exchanger protein. One of these, the intracellular Ca2? regulatory sites, are essential and must be occupied by Ca2? to allow any type of ion (Na? or Ca2?) translocation. Intracellular protons and Na? are inhibitory by reducing the affinity of the regulatory sites for Ca2?; MgATP stimulates by antagonizing H? and Na?. We have proposed a kinetic scheme to explain all ionic and metabolic regulation of the squid nerve Na?/Ca2? exchanger. This model uniquely accounts for most of the new kinetic data provided here; however, none of the existing models can explain the trans effects of the Ca(i)2?-regulatory sites on external cation transport sites; i.e. all models are incomplete. MgATP up-regulation of the squid Na?/Ca2? exchanger requires a cytosolic protein, which has been recently identified as a member of the lipocalin super family of Lipid Binding Proteins (LBP or FABP) of 132 amino acids (ReP1-NCXSQ, access to GenBank EU981897). This protein was cloned, expressed and purified. To be active, ReP1-NCXSQ must be phosphorylated from MgATP by a kinase present in the plasma membrane. Phosphorylated ReP1-NCXSQ can stimulate the exchanger in the absence of ATP. Experiments with proteoliposomes proved that this up-regulation can take place just with the lipid membrane and the exchanger protein. The structure of ReP1-NCXSQ predicted from the amino acid sequence has been confirmed by X-ray crystal analysis; it has a "barrel" formed by ten beta sheets and two alpha helices, with a lipid coordinated by hydrogen bonds with Arg 126 and Tyr 128.     

Squid nerve Na+/Ca2+ exchanger expressed in Saccharomyces cerevisiae: Up-regulation by a phosphorylated cytosolic protein (ReP1–NCXSQ) is identical to that of native exchanger in situ

This work shows, for the first time, a properly metabolically regulated squid nerve Na⁺/Ca²⁺ exchanger (NCXSQ1) heterologous expressed in Saccharomyces cerevisiae. The exchanger was fused to the enhanced green fluorescence protein (eGFP) on its C-terminus and had two tags, a Strep-tag II and 6 histidines, added to the N-terminal region (ST–6HB–NCXSQ1–eGFP). The eGFP fluorescence signal co-localized with that of the plasma membrane marker FM1-43 in whole cells that displayed an uptake of Ca²⁺ with the expected characteristics of the reverse Na⁺/Ca²⁺ exchange mode. Similar to squid nerve membrane vesicles, inside-out yeast plasma membrane vesicles (ISOV) showed a Ca²⁺_i regulation of the forward mode that was modulated by previously phosphorylated regulatory cytosolic protein (ReP1–NCXSQ). On the other hand, a close association between NCXSQ1 and ReP1–NCXSQ, estimated by co-immunoprecipitation, was independent of ReP1–NCXSQ phosphorylation. An additional crucial observation was that in proteoliposomes containing only the ST–6HB–NCXSQ1–eGFP protein, Na⁺/Ca²⁺ exchange was stimulated by phosphorylated ReP1–NCXSQ; i.e., this up-regulation needs no other requirement besides the lipid membrane and the exchanger protein. Finally, this work provides a potential approach to obtain enough purified NCXSQ1 for structural and biochemical studies which have been delayed due to the lack of sufficient material.     

Identification of an HMG-like protein involved in regulation of Na+/H+ exchanger expression

In this study we characterized regulation of the Na⁺/H⁺ exchanger promoter in several tissue types. A conserved poly (dA:dT) region was important in regulation of the promoter. Nuclear extracts from rat myocardium and from mouse proximal tubule cells protected the poly (dA:dT) region of the NHE1 promoter. A protein from nuclear extracts also bound to the poly (dA:dT) element in gel mobility shift binding assays. The binding was specific and was removed by mutations in the poly (dA:dT) region. Characterization of the binding to the poly (dA:dT) region in gel mobility shift assays showed that it was reduced by high concentrations of the divalent cations Mg⁺⁺ and Mn⁺⁺. The inhibition by divalent cations was reduced by decreasing the pH of the binding assay. N-terminal sequencing of the poly (dA:dT) binding protein showed that it was a member of the HMG (high mobility group) family of nuclear proteins which are important in cell growth and proliferation. The results are the first direct detection of a protein that regulates the NHE1 promoter.     

Effect of limited trypsin digestion on the renal Na+−H+ exchanger and its regulation by cAMP-Dependent protein kinase

Summary The Na⁺–H⁺ exchanger from solubilized rabbit renal brush border membranes is inhibited by cAMP-dependent protein kinase (PKA) mediated protein phosphorylation. To characterize this inhibitory response and its sensitivity to limited proteolysis, the activity of the transporter was assayed after reconstitution of the proteins into artificial lipid vesicles. Limited trypsin digestion increased the basal rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake in reconstituted proteoliposomes and blocked the inhibitory response to PKA-mediated protein phosphorylation. To determine if the inhibitory response to PKA-mediated protein phosphorylation could be restored to the trypsin-treated solubilized proteins, nontrypsinized solubilized brush border membrane proteins were separated by column chromatography. The addition of small molecular weight polypeptides, fractionated on Superose-12 FPLC (V _e=0.7), to trypsinized solubilized brush border membrane proteins restored the inhibitory response to PKA-mediated protein phosphorylation. Similarly, the addition of the 0.1m NaCl fraction from an anion exchange column, Mono Q-FPLC, also restored the inhibitory response to PKA. Both protein fractions contained a common 42–43 kDa protein which was preferentially phosphorylated by PKA.These results indicate that limited trypsin digestion dissociates the activity of the renal Na⁺–H⁺ exchanger from its regulation by PKA. It is suggested that trypsin cleaves an inhibitory component of the transporter and that this component is the site of PKA-mediated regulation. Phosphoprotein analysis of fractions that restored PKA regulation raises the possibility that a polypeptide of 42–43 kDa is involved in the inhibition of the renal Na⁺–H⁺ exchanger by PKA-mediated, protein phosphorylation.     

Optimal metabolic regulation of the mammalian heart Na(+)/Ca(2+) exchanger requires a spacial arrangements with a PtdIns(4)-5kinase

In inside-out bovine heart sarcolemmal vesicles, p-chloromercuribenzenesulfonate (PCMBS) and n-ethylmaleimide (NEM) fully inhibited MgATP up-regulation of the Na⁺/Ca²⁺ exchanger (NCX1) and abolished the MgATP-dependent PtdIns-4,5P2 increase in the NCX1-PtdIns-4,5P2 complex; in addition, these compounds markedly reduced the activity of the PtdIns(4)-5kinase. After PCMBS or NEM treatment, addition of dithiothreitol (DTT) restored a large fraction of the MgATP stimulation of the exchange fluxes and almost fully restored PtdIns(4)-5kinase activity; however, in contrast to PCMBS, the effects of NEM did not seem related to the alkylation of protein SH groups. By itself DTT had no effect on the synthesis of PtdIns-4,5P2 but affected MgATP stimulation of NCX1: moderate inhibition at 1 mM MgATP and 1 μM Ca²⁺ and full inhibition at 0.25 mM MgATP and 0.2 μM Ca²⁺. In addition, DDT prevented coimmunoprecipitation of NCX1 and PtdIns(4)-5kinase. These results indicate that, for a proper MgATP up-regulation of NCX1, the enzyme responsible for PtdIns-4,5P2 synthesis must be (i) functionally competent and (ii) set in the NCX1 microenvironment closely associated to the exchanger. This kind of supramolecular structure is needed to optimize binding of the newly synthesized PtdIns-4,5P2 to its target region in the exchanger protein.     

Cooperative interaction between Ca2+ binding sites in the hydrophilic loop of the Na+-Ca2+ exchanger

A high affinity Ca²⁺-binding domain which is located in a middle portion of the large intracellular loop of the Na⁺-Ca²⁺ exchanger contains two highly acidic sequences, each characterized by three consecutive aspartic acid residues (Levitsky DO, Nicoll DA, and Philipson KD (1994) J Biol Chem 269: 22847–22852). This portion of the protein provides secondary Ca²⁺ regulation of the exchanger activity. To determine number of Ca²⁺ binding sites participating in formation of the high affinity domain, we isolated polypeptides of different lengths encompassing the domain and measured ⁴⁵Ca²⁺ binding. The fusion proteins containing the high affinity domain were obtained in a Ca²⁺-bound form and as evidenced by shifts in there mobility in SDS-polyacrylamide gels after EGTA treatment. The Ca²⁺ binding curves obtained after equilibrium dialysis reached saturation at 1 M free Ca²⁺, Kd value being approx. 0.4 M. The Ca²⁺ binding occured in a highly cooperative manner. Upon saturation, the amount of Ca²⁺ ion bound varied from 1.3–2.1 mot per mot protein. Proteins with an aspartate in each acidic sequence mutated lacked the positive cooperativity, had lower Ca²⁺ affinity and bound two to three times less Ca²⁺. Na⁺-Ca²⁺ exchangers of tissues other than heart though different from the cardiac exchanger by molecular weight most likely possess a similar Ca²⁺ binding site. It is concluded that, by analogy with Ca²⁺ binding proteins of EF-type, the high Ca²⁺-affinity domain of the Na⁺-Ca²⁺ exchanger is comprised of at least two binding sites interacting cooperatively.     

Reconstitution of cAMP-Dependent protein kinase regulated renal Na+−H+ exchanger

Summary Studies were performed to determine if the Na⁺–H⁺ exchanger, solubilized from renal brush border membranes from the rabbit and assayed in reconstituted artificial proteoliposomes, could be regulated by cAMP-dependent protein kinase. Octyl glucoside solubilized renal apical membrane proteins from the rabbit kidney were phosphorylated by incubation with ATP and highly purified catalytic subunit of cAMP-dependent kinase.²²Na⁺ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. cAMP-dependent protein kinase resulted in sustained protein phosphorylation and a concentration-dependent decrease in the amiloride-sensitive component of pH gradient-stimulated sodium uptake. The inhibitory effect of cAMP-dependent protein kinase demonstrated an absolute requirement for ATP and was blocked by the specific protein inhibitor of this kinase. cAMP-dependent protein kinase also inhibited²²Na⁺ uptake in the absence of a pH gradient (pH_in 6.0. pH_out 6.0) and the inhibitory effect was blocked by the specific inhibitor of the kinase. Solubilized membrane proteins exhibited little endogenous protein kinase or protein phosphatase activity.These studies indicate that Na⁺–H⁺ exchange activity of proteoliposomes reconstituted with proteins from renal brush border membranes is inhibited by phosphorylation of selected proteins by cAMP-dependent protein kinase. These findings also indicate that the regulatory components of the Na⁺–H⁺ exchanger remain active during the process of solubilization and reconstitution of renal apical membrane proteins.     

Immunological crossreactivity between the retinal Na+-Ca2+,K+ and the cardiac Na+-Ca2+ exchanger proteins

We have used a series of monoclonal antibodies (mAbs) to determine the degree of microscopic structural homology between the retinal Na⁺-Ca²⁺, K⁺ and the cardiac Na⁺-Ca²⁺ exchange proteins. Sets of mAbs were raised separately to partially purified preparations of either the retinal or the recombinant myocardial exchanger. Each panel of mAbs was then screened for crossreactivity with the respective heterologous exchanger using enzyme-linked immunoassay and immunoblotting techniques. Out of 43 anti-retinal exchanger mAbs, we found 3 detecting the cardiac exchanger on immunoblots, while 4 out of 36 anti-cardiac exchanger mAbs reacted with the retinal exchanger. The strength of the crossreactions was generally weak and suggested that only low affinity epitopes were available on the heterologous proteins. For two crossreacting anti-retinal mAbs the apparent binding affinities to the cardiac exchanger were lower by more than two orders of magnitude. The overall low degree of epitope sharing among the two sets of mAbs confirms that in spite of their obvious functional and topological similarities, microscopic structural homologies between the two proteins are scarce.     

Identification and biochemical characterization of the SLC9A7 interactome

Organellar and cytosolic pH homeostasis is central to most cellular processes, including vesicular trafficking, post-translational modification/processing of proteins, and receptor-ligand interactions. SLC9A7 (NHE7) was identified as a unique (Na⁺, K⁺)/H⁺ exchanger that dynamically cycles between the trans-Golgi network (TGN), endosomes and the plasma membrane. Here we have used mass spectrometry to explore the affinity-captured interactome of NHE7, leading to the identification of cytoskeletal proteins, cell adhesion molecules, membrane transporters, and signaling molecules. Among these binding proteins, calcium-calmodulin, but not apo-calmodulin, binds to NHE7 and regulates the organellar transporter activity. Vimentin was co-immunoprecipitated with endogenous NHE7 protein in human breast cancer MDA-MB-231 cells. A sizable population of NHE7 relocalized to focal complexes in migrating cells and showed colocalization with vimentin and actin in focal complexes. Among the NHE7-binding proteins identified, CD44, a cell surface glycoprotein receptor for hyaluronate and other ligands, showed regulated interaction with NHE7. Pretreatment of the cells with phorbol ester facilitated the NHE7-CD44 interaction and the lipid raft association of CD44. When lipid rafts were chemically disrupted, the NHE7-CD44 interaction was markedly reduced. These results suggest potential dual roles of NHE7 in intracellular compartments and subdomains of cell-surface membranes.     

Mechanism and significance of P4 ATPase-catalyzed lipid transport: Lessons from a Na/K-pump

Members of the P₄ subfamily of P-type ATPases are believed to catalyze phospholipid transport across membrane bilayers, a process influencing a host of cellular functions. Atomic structures and functional analysis of P-type ATPases that pump small cations and metal ions revealed a transport mechanism that appears to be conserved throughout the family. A challenging problem is to understand how this mechanism is adapted in P₄ ATPases to flip phospholipids. P₄ ATPases form oligomeric complexes with members of the CDC50 protein family. While formation of these complexes is required for P₄ ATPase export from the endoplasmic reticulum, little is known about the functional role of the CDC50 subunits. The Na⁺/K⁺-ATPase and closely-related H⁺/K⁺-ATPase are the only other P-type pumps that are oligomeric, comprising mandatory β-subunits that are strikingly reminiscent of CDC50 proteins. Besides serving a role in the functional maturation of the catalytic α-subunit, the β-subunit also contributes specifically to intrinsic transport properties of the Na⁺/K⁺ pump. As β-subunits and CDC50 proteins likely adopted similar structures to accomplish analogous tasks, current knowledge of the Na⁺/K⁺-ATPase provides a useful guide for understanding the inner workings of the P₄ ATPase class of lipid pumps.     

Key role of a PtdIns-4,5P2 micro domain in ionic regulation of the mammalian heart Na/Ca exchanger

Phosphatidylinositol biphosphate (PtdIns-4,5P₂) plays a key role in the regulation of the mammalian heart Na⁺/Ca²⁺ exchanger (NCX1) by protecting the intracellular Ca²⁺ regulatory site against H⁺_i and (H⁺_i + Na⁺_i) synergic inhibition. MgATP and MgATP-γ-S up-regulation of NCX1 takes place via the production of this phosphoinositide. In microsomes containing PtdIns-4,5P₂ incubated in the absence of MgATP and at normal [Na⁺]_i, alkalinization increases the affinity for Ca²⁺_i to the values seen in the presence of the nucleotide at normal pH; under this condition, addition of MgATP does not increase the affinity for Ca²⁺_i any further. On the other hand, prevention of Na⁺_i inhibition by alkalinization in the absence of MgATP does not take place when the microsomes are depleted of PtdIns-4,5P₂. Experiments on NCX1–PtdIns-4,5P₂ cross-coimmunoprecipitation show that the relevant PtdIns-4,5P₂ is not the overall membrane component but specifically that tightly attached to NCX1. Consequently, the highest affinity of the Ca²⁺_i regulatory site is seen in the deprotonated and PtdIns-4,5P₂-bound NCX1. Confirming these results, a PtdIns-5-kinase also cross-coimmunoprecipitates with NCX1 without losing its functional competence. These observations indicate, for the first time, the existence of a PtdIns-5-kinase in the NCX1 microdomain.     

Characterization of a Histidine Rich Cluster of Amino Acids in the Cytoplasmic Domain of the Na+/H+ Exchanger

We examined the function of a highly conserved Histidine rich sequence ofamino acids found in the carboxyl-terminal of the Na⁺/H⁺exchanger (NHE1). A fusion protein containing the sequenceHYGHHH (540–545) and the balance of the carboxyl terminalof the protein did not bind calcium but bound to an immobilizedmetal affinity column and could be used to partially purify theexchanger protein. Mutation of the sequence to either HYGAAA orHYGRRR did not affect activity of the intact protein. Mutationto HHHHHH did not affect proton activation of the Na+/H+exchanger or localization but caused a decreased maximal velocitysuggesting that this conserved sequence is important in maximalactivity of the Na⁺/H⁺ exchanger.     

The ancient cell death suppressor BAX inhibitor-1

Bax inhibitor-1 (BI-1) was initially identified for its ability to inhibit BAX-induced apoptosis in yeast cells and is the founding member of a family of highly hydrophobic proteins localized in diverse cellular membranes. It is evolutionarily conserved and orthologues from plants can substitute for mammalian BI-1 in regard to its anti-apoptotic function suggesting a high degree of functional conservation. BI-1 interacts with BCL-2 and BCL-XL and, similar to these two anti-apoptotic proteins, the effect of BI-1 on cell death involves changes in the amount of Ca²⁺ releasable from intracellular stores. However, BI-1 is also a negative regulator of the endoplasmic reticulum stress sensor IRE1 α, it interacts with G-actin and increases actin polymerization, enhances cancer metastasis by altering glucose metabolism and activating the sodium-hydrogen exchanger, and reduces the production of reactive oxygen species through direct interaction with NADPH-P450 reductase. In this contribution, we summarize what is known about the expression, intracellular localization and structure of BI-1 and specifically illuminate its effects on the intracellular Ca²⁺ homeostasis and how this might relate to its other functions. We also present a thorough phylogenetic analysis of BI-1 proteins from major phyla together with paralogues from all BI-1 family members.     

IRBIT: A regulator of ion channels and ion transporters

IRBIT (also called AHCYL1) was originally identified as a binding protein of the intracellular Ca^2 + channel inositol 1,4,5-trisphosphate (IP₃) receptor and functions as an inhibitory regulator of this receptor. Unexpectedly, many functions have subsequently been identified for IRBIT including the activation of multiple ion channels and ion transporters, such as the Na⁺/HCO₃⁻ co-transporter NBCe1-B, the Na⁺/H⁺ exchanger NHE3, the Cl⁻ channel cystic fibrosis transmembrane conductance regulator (CFTR), and the Cl⁻/HCO₃⁻ exchanger Slc26a6. The characteristic serine-rich region in IRBIT plays a critical role in the functions of this protein. In this review, we describe the evolution, domain structure, expression pattern, and physiological roles of IRBIT and discuss the potential molecular mechanisms underlying the coordinated regulation of these diverse ion channels/transporters through IRBIT. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Structural and Functional Analysis of Transmembrane Segment IV of the Salt Tolerance Protein Sod2

Sod2 is the plasma membrane Na⁺/H⁺ exchanger of the fission yeast Schizosaccharomyces pombe. It provides salt tolerance by removing excess intracellular sodium (or lithium) in exchange for protons. We examined the role of amino acid residues of transmembrane segment IV (TM IV) (¹²⁶FPQINFLGSLLIAGCITSTDPVLSALI¹⁵²) in activity by using alanine scanning mutagenesis and examining salt tolerance in sod2-deficient S. pombe. Two amino acids were critical for function. Mutations T144A and V147A resulted in defective proteins that did not confer salt tolerance when reintroduced into S. pombe. Sod2 protein with other alanine mutations in TM IV had little or no effect. T144D and T144K mutant proteins were inactive; however, a T144S protein was functional and provided lithium, but not sodium, tolerance and transport. Analysis of sensitivity to trypsin indicated that the mutations caused a conformational change in the Sod2 protein. We expressed and purified TM IV (amino acids 125–154). NMR analysis yielded a model with two helical regions (amino acids 128–142 and 147–154) separated by an unwound region (amino acids 143–146). Molecular modeling of the entire Sod2 protein suggested that TM IV has a structure similar to that deduced by NMR analysis and an overall structure similar to that of Escherichia coli NhaA. TM IV of Sod2 has similarities to TM V of the Zygosaccharomyces rouxii Na⁺/H⁺ exchanger and TM VI of isoform 1 of mammalian Na⁺/H⁺ exchanger. TM IV of Sod2 is critical to transport and may be involved in cation binding or conformational changes of the protein.     

The Role of Arrestin α-Helix I in Receptor Binding

Arrestins rapidly bind phosphorylated activated forms of their cognate G protein-coupled receptors, thereby preventing G protein coupling and often switching signaling to other pathways. Amphipathic α-helix I (residues 100-111) has been implicated in receptor binding, but the mechanism of its action has not been determined yet. Here we show that several mutations in the helix itself and in adjacent hydrophobic residues in the body of the N-domain reduce arrestin1 binding to light-activated phosphorylated rhodopsin (P-Rh^?). On the background of phosphorylation-independent mutants that bind with high affinity to both P-Rh^? and light-activated unphosphorylated rhodopsin, these mutations reduce the stability of the arrestin complex with P-Rh^?, but not with light-activated unphosphorylated rhodopsin. Using site-directed spin labeling, we found that the local structure around α-helix I changes upon binding to rhodopsin. However, the intramolecular distances between α-helix I and adjacent β-strand I (or the rest of the N-domain), measured using double electron-electron resonance, do not change, ruling out relocation of the helix due to receptor binding. Collectively, these data demonstrate that α-helix I plays an indirect role in receptor binding, likely keeping β-strand I, which carries several phosphate-binding residues, in a position favorable for its interaction with receptor-attached phosphates.     

NHERF2/NHERF3 Protein Heterodimerization and Macrocomplex Formation Are Required for the Inhibition of NHE3 Activity by Carbachol

NHERF1, NHERF2, and NHERF3 belong to the NHERF (Na⁺/H⁺ exchanger regulatory factor) family of PSD-95/Discs-large/ZO-1 (PDZ) scaffolding proteins. Individually, each NHERF protein has been shown to be involved in the regulation of multiple receptors or transporters including Na⁺/H⁺ exchanger 3 (NHE3). Although NHERF dimerizations have been reported, results have been inconsistent, and the physiological function of NHERF dimerizations is still unknown. The current study semiquantitatively compared the interaction strength among all possible homodimerizations and heterodimerizations of these three NHERF proteins by pulldown and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 heterodimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 heterodimerization was confirmed by FRET and FRAP (fluorescence recovery after photobleach). NHERF2/NHERF3 heterodimerization is mediated by PDZ domains of NHERF2 and the C-terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in heterodimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 heterodimerization in the regulation of NHE3 activity. In addition, both PDZ domains of NHERF2 could be simultaneously occupied by NHERF3 and another ligand such as NHE3, α-actinin-4, and PKCα, promoting formation of NHE3 macrocomplexes. This study suggests that NHERF2/NHERF3 heterodimerization mediates the formation of NHE3 macrocomplexes, which are required for the inhibition of NHE3 activity by carbachol.     

Cloning of a novel human NHEDC1 (Na+/H+ exchanger like domain containing 1) gene expressed specifically in testis

The Na⁺/H⁺ exchangers (NHEs) catalyze the transport of Na⁺ in exchange for H⁺ across membranes in organisms and are required for numerous physiological processes. Here we report the cloning and characterization of a novel human NHEDC1 (Na⁺/H⁺ exchanger like domain containing 1) gene, which was mapped to human chromosome 4p24. This cDNA is 1859 bp in length, encoding a putative protein of 515 amino acids. The NHEDC1 proteins are highly conserved in mammals including human, mouse, rat, and Macaca fascicularis. One remarkable characteristic of human NHEDC1 gene is that it is exclusively expressed in the testis by RT-PCR analysis. Western blot analysis showed that the molecular weight of NHEDC1 is about 56 KDa. Guangming Ye and Cong Chen contributed equally to this work.