Xiuju Li

Dimeric Structure of Human Na ! /H ! Exchanger Isoform 1 Overproduced in Saccharomyces cerevisiae *

The Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H+ in exchange for one extracellular Na+. The human NHE1 isoform is involved in heart disease and cell growth and proliferation. Although details of NHE1 regulation and transport are being revealed, there is little information available on the structure of the intact protein. In this report, we demonstrate overexpression, purification, and characterization of the human NHE1 (hNHE1) protein in Saccharomyces cerevisiae. Overproduction of the His-tagged protein followed by purification via nickel-nitrilotriacetic acid-agarose chromatography yielded 0.2 mg of pure protein/liter of cell culture. Reconstitution of hNHE1 in proteoliposomes demonstrated that the protein was active and responsive to an NHE1-specific inhibitor. Circular dichroism spectroscopy of purified hNHE1 revealed that the protein contains 41% α-helix, 23% β-sheet, and 36% random coil. Size exclusion chromatography indicated that the protein-detergent micelle was in excess of 200 kDa, consistent with an hNHE1 dimer. Electron microscopy and single particle reconstruction of negatively stained hNHE1 confirmed that the protein was a dimer, with a compact globular domain assigned to the transmembrane region and an apical ridge assigned to the cytoplasmic domain. The transmembrane domain of the hNHE1 reconstruction was clearly dimeric, where each monomer had a size and shape consistent with the predicted 12 membrane-spanning segments for hNHE1.

Structural and Functional Analysis of Transmembrane XI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na+/H+ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H+ in exchange for one extracellular Na+. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp238, Pro239, and Glu247. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe230, Gly231, Ala236, Val237, Ala244, Val245, and Glu248 were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn227, Ile233, and Leu243 lining the translocation pore.

B-Raf Associates with and Activates the NHE1 Isoform of the Na+/H+ Exchanger

The serine/threonine kinase B-Raf is the second most frequently occurring human oncogene after Ras. Mutations of B-Raf occur with the highest incidences in melanoma, and the most common mutant, V600E, renders B-Raf constitutively active. The sodium proton exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein responsible for regulating intracellular pH, cell volume, cell migration, and proliferation. A screen of protein kinases that bind to NHE1 revealed that B-Raf bound to the cytosolic regulatory tail of NHE1. Immunoprecipitation of NHE1 from HeLa and HEK cells confirmed the association of B-Raf with NHE1 in vivo. The expressed and purified C-terminal 182 amino acids of the NHE1 protein were also shown to associate with B-Raf protein in vitro. Because treatment with the kinase inhibitor sorafenib decreased NHE1 activity in HeLa and HEK cells, we examined the role of B-Raf in regulating NHE1 in malignant melanoma cells. Melanoma cells with the B-RafV600E mutation demonstrated increased resting intracellular pH that was dependent on elevated NHE1 activity. NHE1 activity after an acute acid load was also elevated in these cell lines. Moreover, inhibition of B-Raf activity by either sorafenib, PLX4720, or siRNA reduction of B-Raf levels abolished ERK phosphorylation and decreased NHE1 activity. These results demonstrate that B-Raf associates with and stimulates NHE1 activity and that B-RafV600E also increases NHE1 activity that raises intracellular pH.

Structural and functional analysis of TM XI of the nhe1 isoform of the NA+/H+ exchanger

The Na+/H+ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals by extruding an intracellular H+ in exchange for one extracellular Na+. We characterized structural and functional aspects of the critical transmembrane (TM) segment XI (residues 449-470) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM XI was mutated to cysteine in the background of the cysteine-less protein and the sensitivity to water-soluble sulfhydryl reactive compounds MTSET ((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES ((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues with at least moderate activity remaining. Of the residues tested, only proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The activity of the L465C mutant was almost completely eliminated, whereas that of the L457C and I461C mutants was partially affected. The structure of a peptide representing TM XI (residues Lys447-Lys472) was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. The structure consisted of helical regions between Asp447-Tyr454 and Phe460-Lys471 at the N and C termini of the peptide, respectively, connected by a region with poorly defined, irregular structure consisting of residues Gly455-Gly459. TM XI of NHE1 had a structural similarity to TM XI of the Escherichia coli Na+/H+ exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with residue Leu465 contributing to the pore.

Na+/H+ exchanger isoform 1 facilitates cardiomyocyte embryonic stem cell differentiation.

Embryonic stem cells provide one potential source of cardiomyocytes for cardiac transplantation; however, after differentiation of stem cells in vitro, cardiomyocytes usually account for only a minority of cells present. To gain insights into improving cardiomyocyte development from stem cells, we examined the role of the Na(+)/H(+) exchanger isoform 1 (NHE1) in cardiomyocyte differentiation. NHE1 protein and message levels were induced by treatment of CGR8 cells to form embryoid bodies and cardiomyocytes. The NHE1 protein was present on the cell surface and NHE1 inhibitor-sensitive activity was detected. Inhibition of NHE1 activity during differentiation of CGR8 cells prevented cardiomyocyte differentiation as indicated by decreased message for transcription factors Nkx2-5 and Tbx5 and decreased levels of alpha-myosin heavy chain protein. Increased expression of NHE1 from an adenoviral vector facilitated cardiomyocyte differentiation. Similar results were found with cardiomyocyte differentiation of P19 embryonal carcinoma cells. CGR8 cells were treated to induce differentiation, but when differentiation was inhibited by dispersing the EBs, myocardial development was inhibited. The results demonstrate that NHE1 activity is important in facilitating stem cell differentiation to cardiomyocyte lineage. Elevated NHE1 expression appears to be triggered as part of the process that facilitates cardiomyocyte development.

Mutation of SLC9A1, encoding the major Na+/H+ exchanger, causes ataxia–deafness Lichtenstein–Knorr syndrome

Lichtenstein-Knorr syndrome is an autosomal recessive condition that associates sensorineural hearing loss and cerebellar ataxia. Here, we report the first identification of a gene involved in Lichtenstein-Knorr syndrome. By using a combination of homozygosity mapping and whole-exome sequencing, we identified the homozygous p.Gly305Arg missense mutation in SLC9A1 that segregates with the disease in a large consanguineous family. Mutant glycine 305 is a highly conserved amino acid present in the eighth transmembrane segment of all metazoan orthologues of NHE1, the Na(+)/H(+) exchanger 1, encoded by SLC9A1. We demonstrate that the p.Gly305Arg mutation causes the near complete de-glycosylation, mis-targeting and loss of proton pumping activity of NHE1. The comparison of our family with the phenotypes of spontaneous and knockout Slc9a1 murine models demonstrates that the association between ataxia and hearing loss is caused by complete or near complete loss of function of NHE1 and altered regulation of pHi in the central nervous system.

Correlating structure, dynamics, and function in transmembrane segment VII of the Na+/H+exchanger isoform 1

We place (15)N nuclear magnetic resonance relaxation analysis and functional mutagenesis studies in the context of our previous structural and mutagenesis work to correlate structure, dynamics and function for the seventh transmembrane segment of the human Na(+)/H(+) exchanger isoform 1. Although G261-S263 was previously identified as an interruption point in the helical structure of this isolated transmembrane peptide in dodecylphosphocholine micelles, and rapid conformational exchange was implicated in the NOE measurements, the six (15)N labelled residues examined in this study all have similar dynamics on the ps-ns time scale. A mathematical model incorporating chemical exchange is the best fit for residues G261, L264, and A268. This implies that a segment of residues from G261 to A268 samples different conformations on the mus-ms time scale. Chemical exchange on an intermediate time scale is consistent with an alternating-access cycle where E262 is bent away from the cytosol during proton translocation by the exchanger. The functional importance of chemical exchange at G261-A268 is corroborated by the abrogated activity of the full-length exchanger with the bulky and restricting Ile substitutions F260I, G261I, E262I, S263I, and A268I.

Structural and functional analysis of extracellular loop 2 of the Na(+)/H(+) exchanger.

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pHi) by removing one intracellular H(+) in exchange for one extracellular Na(+). It has a large 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments and several membrane-associated segments. Extracellular regions of this domain are believed to contribute to cation coordination, transport and sensitivity to inhibitors. In this study we characterized the structure and function of extracellular loop 2. Mutation of residues Pro153, Pro154 and Phe155 demonstrated that these residues were critical for efficient NHE1 function. Mutations to Ala resulted in decreases in cation affinity and in decreases in activity of the protein, these were more marked in both Pro154 and Phe155. NMR spectroscopy was used to characterize the solution structure of a peptide NAc-Gly150-Phe155-NH(2). The peptide showed at least three different conformers in solution due to cis-trans isomerization of the Thr(152)-Pro(153) and Pro(153)-Pro(154) peptide bonds. The trans-trans conformation appeared to be in an extended conformation, whereas the cis-trans conformation showed a propensity to form a beta turn. Our results show that the EL2 region is critical to NHE1 function and that a peptide of the EL2 region can adopt different structures in solution potentially forming a beta turn that is important in function of the full protein Mutation of Pro(154) could disrupt the beta turn, affecting helix packing and the protein structure and function.

Topological analysis of the Na+/H+ exchanger.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed integral membrane protein present in mammalian cells. It is made up of a hydrophobic 500 amino acid membrane domain that transports and removes protons from within cells, and a regulatory intracellular cytosolic domain made of approximately 315 amino acids. Determining the structure of NHE1 is critical for both an understanding of the Na+/H+ exchange mechanism of transport, and in the design of new improved inhibitors for use in treatment of several diseases in which it is involved. Differing models of the NHE1 protein have been proposed. The first model suggested by two groups proposes that amino acids 1-500 form a 12 transmembrane segment spanning region in which amino acids 1-127 form two transmembrane segments, and amino acids 315-411 form a single transmembrane segment with membrane associated segments. A second model based on the structure of the Escherichia coli Na+/H+ exchanger protein proposes an overall similar topology, but suggests amino acids 1-127 are removed as a signal sequence and are not present in the mature protein. It also suggests a different topology of amino acids 315-411 to form three transmembrane segments. We used cysteine scanning accessibility and examination of glycosylation of the mature protein to characterize the NHE1 protein. Our results demonstrate that the model of NHE1 is correct which suggests that amino acids 1-127 form two transmembrane segments that remain connected to the mature protein, and the segment between amino acids 315-411 is one transmembrane segment.

Calmodulin-dependent binding to the NHE1 cytosolic tail mediates activation of the Na+/H+ exchanger by Ca2+ and endothelin

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a single proton (H(+)) in exchange for one extracellular Na(+). The human protein contains a ∼500-amino acid membrane domain and a regulatory, ∼315-amino acid cytosolic domain. NHE1 is activated by a number of hormones including endothelin (ET) and by Ca(2+). The regulatory tail possesses an inhibitory calmodulin (CaM)-binding domain, and inhibition of NHE1 is relieved by binding of a Ca(2+)-CaM complex. We examined the dynamics of ET-1 and Ca(2+) regulation of binding to NHE1 in vivo. CFP was linked to the NHE1 protein cytoplasmic COOH terminus. This was stably transfected into AP-1 cells that are devoid of their own NHE1 protein. The protein was expressed and targeted properly and retained NHE1 activity comparable to the wild-type protein. We examined the in vivo coupling of NHE1 to CaM by Förster resonance energy transfer using CaM linked to the fluorescent protein Venus. CaM interaction with NHE1 was dynamic. Removal of serum reduced CaM interaction with NHE1. Addition of the Ca(2+) ionophore ionomycin increased the interaction between CaM and NHE1. We expressed an ET receptor in AP-1 cells and also found a time-dependent association of NHE1 with CaM in vivo that was dependent on ET treatment. The results are the first demonstration of the in vivo association of NHE1 and CaM through ET-dependent signaling pathways.