Structure and function of the NHE1 isoform of the Na+/H+ exchanger

2002 ◽  
Vol 80 (5) ◽  
pp. 499-508 ◽  
Author(s):  
Emily Slepkov ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Integral Membrane Protein ◽  
Amino Acid Residues ◽  
Transmembrane Helices ◽  
Cytoskeletal Organization ◽  
Transmembrane Segments ◽  
And Function ◽  
Extracellular Sodium

The Na+/H+ exchanger is a ubiquitous, integral membrane protein involved in pH regulation. It removes intracellular acid, exchanging a proton for an extracellular sodium ion. There are seven known isoforms of this protein that are the products of distinct genes. The first isoform discovered (NHE1) is ubiquitously distributed throughout the plasma membrane of virtually all tissues. It plays many different physiological roles in mammals, including important functions in regulation of intracellular pH, in heart disease, and in cytoskeletal organization. The first 500 amino acids of the protein are believed to consist of 12 transmembrane helices, a membrane-associated segment, and two reentrant loops. A C-terminal regulatory domain of approximately 315 amino acids regulates the protein and mediates cyto skel etal interactions. Studies are underway to determine the amino acid residues important in NHE1 function. At present, it is clear that transmembrane segment IV is important in NHE1 function and that transmembrane segments VII and IX are also involved in transport. Further experiments are required to elucidate the mechanism of transport and regulation of this multifunctional protein.Key words: cation transport, intracellular pH, membrane proteins, Na+/H+ exchanger.

scholarly journals Structural and functional analysis of the Na+/H+ exchanger

2007 ◽  
Vol 401 (3) ◽  
pp. 623-633 ◽  
Author(s):  
Emily R. Slepkov ◽  
Jan K. Rainey ◽  
Brian D. Sykes ◽  
Larry Fliegel
Keyword(s):  
Intracellular Ph ◽  
Sequence Similarity ◽  
Structural Data ◽  
Structural Similarity ◽  
Integral Membrane Protein ◽  
Volume Control ◽  
Amino Acid Residues ◽  
Physiological Processes ◽  
High Resolution Structure ◽  
Extracellular Sodium

The mammalian NHE (Na+/H+ exchanger) is a ubiquitously expressed integral membrane protein that regulates intracellular pH by removing a proton in exchange for an extracellular sodium ion. Of the nine known isoforms of the mammalian NHEs, the first isoform discovered (NHE1) is the most thoroughly characterized. NHE1 is involved in numerous physiological processes in mammals, including regulation of intracellular pH, cell-volume control, cytoskeletal organization, heart disease and cancer. NHE comprises two domains: an N-terminal membrane domain that functions to transport ions, and a C-terminal cytoplasmic regulatory domain that regulates the activity and mediates cytoskeletal interactions. Although the exact mechanism of transport by NHE1 remains elusive, recent studies have identified amino acid residues that are important for NHE function. In addition, progress has been made regarding the elucidation of the structure of NHEs. Specifically, the structure of a single TM (transmembrane) segment from NHE1 has been solved, and the high-resolution structure of the bacterial Na+/H+ antiporter NhaA has recently been elucidated. In this review we discuss what is known about both functional and structural aspects of NHE1. We relate the known structural data for NHE1 to the NhaA structure, where TM IV of NHE1 shows surprising structural similarity with TM IV of NhaA, despite little primary sequence similarity. Further experiments that will be required to fully understand the mechanism of transport and regulation of the NHE1 protein are discussed.

Functional role of arginine 425 in the mammalian Na+/H+ exchanger

2014 ◽  
Vol 92 (6) ◽  
pp. 541-546 ◽  
Author(s):  
Xiuju Li ◽  
Yike Ma ◽  
Larry Fliegel
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Transmembrane Helices ◽  
Basic Residue ◽  
Mutant Proteins ◽  
Transmembrane Segments ◽  
Nhe1 Activity ◽  
And Function ◽  
Extracellular Sodium

Na+/H+ exchanger isoform 1 (NHE1) is the principal plasma membrane Na+/H+ exchanger of mammalian cells and functions by exchanging one intracellular proton for one extracellular sodium ion. Critical transmembrane segments of Na+/H+ exchangers have discontinuous transmembrane helices, which result in a dipole within the membrane. Amino acid R425 has been suggested to play an important role in neutralizing one such helix dipole. To investigate this hypothesis, R425 was mutated to alanine, glutamine, histidine, or lysine and the mutant NHE1 proteins were expressed and characterized in NHE1-deficient cells. The R425A and R425E mutants exhibited complete loss of expression of mature, fully glycosylated NHE1, reduced expression overall, and greatly reduced cell surface targeting. The cell surface targeting, expression, and activity of the R425H and R425K mutant proteins were also impaired, though residual NHE1 activity remained. When reduced targeting and expression were accounted for, the R425H and R425K mutant proteins had activity similar to that of the wild-type protein. The results suggest that R425 is critical for NHE1 expression, targeting, and activity and that replacement with another basic residue can rescue activity. The findings are consistent with a role for R425 in both neutralizing a helix dipole and maintaining NHE1 structure and function.

scholarly journals Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids

2016 ◽  
Vol 113 (38) ◽  
pp. 10559-10564 ◽  
Author(s):  
Karin Öjemalm ◽  
Takashi Higuchi ◽  
Patricia Lara ◽  
Erik Lindahl ◽  
Hiroaki Suga ◽  
...  
Keyword(s):  
Amino Acids ◽  
Membrane Protein ◽  
Side Chain ◽  
Transmembrane Helices ◽  
Transmembrane Segments ◽  
Charged Amino Acids ◽  
Protein Biogenesis ◽  
Quantitative Basis ◽  
Apparent Free Energy ◽  
Integration Efficiency

Cotranslational translocon-mediated insertion of membrane proteins into the endoplasmic reticulum is a key process in membrane protein biogenesis. Although the mechanism is understood in outline, quantitative data on the energetics of the process is scarce. Here, we have measured the effect on membrane integration efficiency of nonproteinogenic analogs of the positively charged amino acids arginine and lysine incorporated into model transmembrane segments. We provide estimates of the influence on the apparent free energy of membrane integration (ΔGapp) of “snorkeling” of charged amino acids toward the lipid–water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone hydrogen bonds (H-bonds) on ΔGapp. These results help establish a quantitative basis for our understanding of membrane protein assembly in eukaryotic cells.

Functional characterization of the transmembrane segment VII of the NHE1 isoform of the Na+/H+ exchangerThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

2007 ◽  
Vol 85 (3-4) ◽  
pp. 319-325 ◽  
Author(s):  
Jie Ding ◽  
Raymond W.P. Ng ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
70Th Birthday ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Transmembrane Segment ◽  
Transmembrane Segments ◽  
Pore Lining ◽  
Positively Charged ◽  
Selection Of

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH. It extrudes 1 intracellular H+ in exchange for 1 extracellular Na+. It has 2 large domains, an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the critical transmembrane segment TM VII. Residues Leu 255, Leu 258, Glu 262, Leu 265, Asn 266, Asp 267, Val 269, Val 272, and Leu 273 were all mutated to cysteine residues in the cysteineless NHE1 isoform. Mutation of amino acids E262, N266, and D267 caused severe defects in activity and targeting of the intact full length protein. The balance of the active mutants were examined for sensitivity to the sulfhydryl reactive reagents, positively charged MTSET ((2- (trimethylammonium)ethyl)methanethiosulfonate) and negatively charged MTSES ((2-sulfonatoethyl)methanethiosulfonate). Leu 255 and Leu 258 were sensitive to MTSET but not to MTSES. The results suggest that these amino acids are pore-lining residues. We present a model of TM VII that shows that residues Leu 255, Leu 258, Glu 262, Asn 266, and Asp 267 lie near the same face of TM VII, lining the ion transduction pore.

scholarly journals C-Terminal Truncations of the Saccharomyces cerevisiae PMA1 H+-ATPase Have Major Impacts on Protein Conformation, Trafficking, Quality Control, and Function

2013 ◽  
Vol 13 (1) ◽  
pp. 43-52 ◽  
Author(s):  
A. Brett Mason ◽  
Kenneth E. Allen ◽  
Carolyn W. Slayman
Keyword(s):  
Amino Acids ◽  
Quality Control ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Amino Acid Residues ◽  
Membrane Expression ◽  
C Terminus ◽  
Conditional Expression ◽  
Membrane Spanning ◽  
And Function

ABSTRACTThe C-terminal tail of yeast plasma membrane (PM) H+-ATPase extends approximately 38 amino acids beyond the final membrane-spanning segment (TM10) of the protein and is known to be required for successful trafficking, stability, and regulation of enzyme activity. To carry out a detailed functional survey of the entire length of the tail, we generated 15 stepwise truncation mutants. Eleven of them, lacking up to 30 amino acids from the extreme terminus, were able to support cell growth, even though there were detectable changes in plasma membrane expression, protein stability, and ATPase activity. Three functionally distinct regions of the C terminus could be defined. (i) Truncations upstream of Lys889, removing more than 30 amino acid residues, yielded no viable mutants, and conditional expression of such constructs supported the conclusion that the stretch from Ala881(at the end of TM10) to Gly888is required for stable folding and PM targeting. (ii) The stretch between Lys889and Lys916, a region known to be subject to kinase-mediated posttranslational modification, was shown here to be ubiquitinated in carbon-starved cells as part of cellular quality control and to be essential for normal ATPase folding and stability, as well as for autoinhibition of ATPase activity during glucose starvation. (iii) Finally, removal of even one or two residues (Glu917and Thr918) from the extreme C terminus led to visibly reduced expression of the ATPase at the plasma membrane. Thus, the C terminus is much more than a simple appendage and profoundly influences the structure, biogenesis, and function of the yeast H+-ATPase.

Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH2-terminal cytoplasmic domain

2001 ◽  
Vol 281 (4) ◽  
pp. C1344-C1354 ◽  
Author(s):  
A. K. Stewart ◽  
M. N. Chernova ◽  
Y. Z. Kunes ◽  
S. L. Alper
Keyword(s):  
Amino Acids ◽  
Intracellular Ph ◽  
Xenopus Oocytes ◽  
Cytoplasmic Domain ◽  
Amino Acid Residues ◽  
Constant Ph ◽  
Activity Curve ◽  
Critical Regions ◽  
Intracellular Alkalinization

The role of intracellular pH (pHi) in regulation of AE2 function in Xenopus oocytes remains unclear. We therefore compared AE2-mediated 36Cl− efflux from Xenopus oocytes during imposed variation of extracellular pH (pHo) or variation of pHi at constant pHo. Wild-type AE2-mediated 36Cl−efflux displayed a steep pHo vs. activity curve, with pHo(50) = 6.91 ± 0.04. Sequential NH2-terminal deletion of amino acid residues in two regions, between amino acids 328 and 347 or between amino acids 391 and 510, shifted pHo(50) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocyte pHi at constant pHo and were shown to be neither substrates nor inhibitors of AE2-mediated Cl−transport. At constant pHo, AE2 was inhibited by intracellular acidification and activated by intracellular alkalinization. Our data define structure-function relationships within the AE2 NH2-terminal cytoplasmic domain, which demonstrates distinct structural requirements for AE2 regulation by intracellular and extracellular protons.

Herbicide Resistance and Growth of D1 Ala251 Mutants in Chlamydomonas

1997 ◽  
Vol 52 (9-10) ◽  
pp. 654-664 ◽  
Author(s):  
Britta Förster ◽  
Peter B. Heifetz ◽  
Anita Lardans ◽  
John E. Boynton ◽  
Nicholas W. Gillham
Keyword(s):  
Amino Acids ◽  
Herbicide Resistance ◽  
Structural Integrity ◽  
D1 Protein ◽  
Transmembrane Helices ◽  
Low Light ◽  
Photoautotrophic Growth ◽  
Wildtype Strain ◽  
And Function ◽  
Doubling Times

We elucidated the effects of substituting seven amino acids for Ala at residue 251 of the Chlamydomonas reinhardtii D1 protein on herbicide resistance and photoautotrophic growth. Ala251 has been suggested to play a key role in the structural integrity and function of the stromal loop between transmembrane helices IV and V of D1 and has previously been shown to affect resistance to “classical” PSII specific herbicides. Sensitive and rapid microtiter assays were employed to compare herbicide resistance and photoautotrophic growth in the various mutants. Substitution of Ala251 by Ile, Leu or Val conferred resistance to the PSII herbicides atrazine, bromacil and metribuzin but not to DCMU, and impaired photoautotrophic growth in high and low light. Compared to an otherwise isogenic wildtype strain, the lie and Val mutants exhibited nearly identical levels of herbicide resistance and reduced growth while the Leu mutant had even slower growth and higher levels of herbicide resistance. In contrast Cys, Pro, Ser and Gly mutants were phenotypically indistinguishable from wildtype in terms of herbicide sensitivity and photoautotrophic doubling times. Collectively the seven Ala251 mutations differed markedly from an Ala mutant (dr-1) at the well characterized Ser264 D1 residue in terms of herbicide resistance and photoautotrophic growth

Sodium-coupled neurotransmitter transport: structure, function and regulation.

1994 ◽  
Vol 196 (1) ◽  
pp. 237-249 ◽  
Author(s):  
B I Kanner
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glial Cells ◽  
Plasma Membranes ◽  
Nerve Terminals ◽  
Amino Group ◽  
Amino Acid Residues ◽  
Transmembrane Helices ◽  
Neurotransmitter Transport ◽  
Sodium And Potassium

The removal of neurotransmitters by their transporters--located in the plasma membranes of nerve terminals and glial cells--plays an important role in the termination of synaptic transmission. In the last 3 years, many neurotransmitter transporters have been cloned. Structurally and functionally they can be divided into two groups: glutamate transporters, of which to date three have been cloned, couple the flow of glutamate to that of sodium and potassium. The second group of transporters includes those for GABA, glycine, taurine, norepinephrine, dopamine and serotonin. They are sodium- and chloride-dependent, but do not require potassium for function. One of these, the GABAA transporter, encoded by GAT-1, is perhaps the best characterized. It has been purified and reconstituted and has a molecular mass of around 80 kDa, of which 10-15 kDa is sugar. Amino and carboxyl termini (around 50 amino acids each) are not required for function. The transporter is protected against proteolysis at multiple sites by GABA, provided that the two cosubstrates--sodium and chloride--are present. Several amino acid residues that are critical for function have been identified in the GABA transporter. These include arginine-69 and tryptophan-222 located in the first and fourth putative transmembrane helices, respectively. The first is possibly involved in the binding of chloride. The tryptophan appears to serve as a binding site for the amino group of GABA.

Mechanisms of intracellular pH regulation during postischemic reperfusion of diabetic rat hearts

Diabetes ◽  
1995 ◽  
Vol 44 (2) ◽  
pp. 196-202 ◽  
Author(s):  
N. Khandoudi ◽  
M. Bernard ◽  
P. Cozzone ◽  
D. Feuvray
Keyword(s):  
Intracellular Ph ◽  
Ph Regulation ◽  
Diabetic Rat ◽  
Intracellular Ph Regulation ◽  
Postischemic Reperfusion ◽  
Rat Hearts
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