Physiological role and regulation of the Na+/H+ exchanger

2006 ◽  
Vol 84 (11) ◽  
pp. 1081-1095 ◽  
Author(s):  
Mackenzie E. Malo ◽  
Larry Fliegel
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Cell Movement ◽  
Physiological Role ◽  
Membrane Domain ◽  
Post Translational Modifications ◽  
Homologous Protein ◽  
The Family ◽  
Extracellular Sodium

In mammalian eukaryotic cells, the Na+/H+ exchanger is a family of membrane proteins that regulates ions fluxes across membranes. Plasma membrane isoforms of this protein extrude 1 intracellular proton in exchange for 1 extracellular sodium. The family of Na+/H+ exchangers (NHEs) consists of 9 known isoforms, NHE1–NHE9. The NHE1 isoform was the first discovered, is the best characterized, and exists on the plasma membrane of all mammalian cells. It contains an N-terminal 500 amino acid membrane domain that transports ions, plus a 315 amino acid C-terminal, the intracellular regulatory domain. The Na+/H+ exchanger is regulated by both post-translational modifications including protein kinase-mediated phosphorylation, plus by a number of regulatory-binding proteins including phosphatidylinositol-4,5-bisphosphate, calcineurin homologous protein, ezrin, radixin and moesin, calmodulin, carbonic anhydrase II, and tescalcin. The Na+/H+ exchanger is involved in a variety of complex physiological and pathological events that include regulation of intracellular pH, cell movement, heart disease, and cancer. This review summarizes recent advances in the understanding of the physiological role and regulation of this protein.

scholarly journals Novel isoform of syntaxin 1 is expressed in mammalian cells

1997 ◽  
Vol 321 (1) ◽  
pp. 151-156 ◽  
Author(s):  
Mittur N. JAGADISH ◽  
Judy T. TELLAM ◽  
S. Lance MACAULAY ◽  
Keith H. GOUGH ◽  
David E. JAMES ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Cdna Clone ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Terminal Region ◽  
Receptor Protein ◽  
Fat Cell ◽  
Syntaxin 1A ◽  
Truncated Protein

Syntaxin 1A has been identified previously as a neural-cell-specific, membrane-anchored receptor protein required for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Syntaxin 1A consists of 288 amino acid residues including a 265-residue N-terminal region exposed to the cytoplasm and a C-terminal hydrophobic stretch of 23 residues believed to anchor syntaxin to the plasma membrane. Using a human fat-cell library we have isolated a novel cDNA clone of syntaxin 1A containing an insert of 91 bp in codon 226. This insert and subsequent frame shift generated a cDNA that codes for a truncated protein of 260 residues without the C-terminal transmembrane domain characteristic of the syntaxin family. Analysis of the deduced amino acid sequence of the new cDNA clone, termed syntaxin 1C, showed that it was identical for the first 226 residues with the previously described neural syntaxin 1A, and diverged thereafter. The truncated protein lacked the botulinum neurotoxin C cleavage site (Lys253-Ala254), a feature of the syntaxin 1A protein, because of the novel C-terminal domain of 34 residues. The new C-terminal region contained a single cysteine residue and was moderately rich in proline, with three repeats of a PXP motif. The insert occurred within the region encoding the coiled-coil motifs required for interactions with synaptobrevin, α-SNAP (SNAP being soluble N-ethylmaleimide-sensitive factor attachment protein) and n-Sec1/Munc-18 (n-Sec1 being the rat brain homologue of yeast Sec1p and Munc-18 the mammalian homologue of Caenorhabditis elegans unc-18, but five residues outside the domain previously mapped as being required for binding SNAP-25. Interaction studies in vitro suggested that unlike syntaxin 1A, which binds to both Munc-18a and -18b, syntaxin 1C binds only to Munc-18b. The new isoform syntaxin 1C, which might be generated by alternative splicing of the syntaxin 1 gene, was expressed in several human tissues, including brain. Immunoprecipitation and immunoblotting with the monoclonal antibody HPC-1 and a polyclonal antibody raised against a peptide corresponding to the unique C-terminal 35 residues of syntaxin 1C failed to detect syntaxin 1C at the protein level in extracts of muscle, fat or brain.

scholarly journals Amino Acids 785, 787 of the Na+/H+ Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation

2021 ◽  
Vol 22 (21) ◽  
pp. 11349
Author(s):  
Xiuju Li ◽  
Tommy Tu ◽  
Sicheng Quan ◽  
Francisco J. Quintero ◽  
Richard Fahlman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Apparent Molecular Weight ◽  
Slight Reduction ◽  
Membrane Domain ◽  
Protein Activity ◽  
Extracellular Sodium ◽  
Vitro Protein

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser785 and Ser787, that were similar in location and context to two amino acids of the Arabidopsis Na+/H+ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783–790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.

scholarly journals A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jesusa Capera ◽  
Mireia Pérez-Verdaguer ◽  
Roberta Peruzzo ◽  
María Navarro-Pérez ◽  
Juan Martínez-Pinna ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Survival ◽  
Mammalian Cells ◽  
Physiological Role ◽  
Membrane Microdomains ◽  
Functional Link ◽  
Caveolin 1 ◽  
Molecular Determinants ◽  
Apoptotic Effects

The voltage-gated potassium channel Kv1.3 plays an apparent dual physiological role by participating in activation and proliferation of leukocytes as well as promoting apoptosis in several types of tumor cells. Therefore, Kv1.3 is considered a potential pharmacological target for immunodeficiency and cancer. Different cellular locations of Kv1.3, at the plasma membrane or the mitochondria, could be responsible for such duality. While plasma membrane Kv1.3 facilitates proliferation, the mitochondrial channel modulates apoptotic signaling. Several molecular determinants of Kv1.3 drive the channel to the cell surface, but no information is available about its mitochondrial targeting. Caveolins, which are able to modulate cell survival, participate in the plasma membrane targeting of Kv1.3. The channel, via a caveolin-binding domain (CDB), associates with caveolin 1 (Cav1), which localizes Kv1.3 to lipid raft membrane microdomains. The aim of our study was to understand the role of such interactions not only for channel targeting but also for cell survival in mammalian cells. By using a caveolin association-deficient channel (Kv1.3 CDBless), we demonstrate here that while the Kv1.3–Cav1 interaction is responsible for the channel localization in the plasma membrane, a lack of such interaction accumulates Kv1.3 in the mitochondria. Kv1.3 CDBless severely affects mitochondrial physiology and cell survival, indicating that a functional link of Kv1.3 with Cav1 within the mitochondria modulates the pro-apoptotic effects of the channel. Therefore, the balance exerted by these two complementary mechanisms fine-tune the physiological role of Kv1.3 during cell survival or apoptosis. Our data highlight an unexpected role for the mitochondrial caveolin–Kv1.3 axis during cell survival and apoptosis.

Yeast response and tolerance to polyamine toxicity involving the drug : H+ antiporter Qdr3 and the transcription factors Yap1 and Gcn4

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 945-956 ◽  
Author(s):  
Miguel C. Teixeira ◽  
Tânia R. Cabrito ◽  
Zaitunnissa M. Hanif ◽  
Rita C. Vargas ◽  
Sandra Tenreiro ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Physiological Role ◽  
Oxidative Stress Response ◽  
Yeast Cells ◽  
Intracellular Accumulation ◽  
Induced Stress ◽  
Stressed Cells ◽  
Main Regulator

The yeast QDR3 gene encodes a plasma membrane drug : H+ antiporter of the DHA1 family that was described as conferring resistance against the drugs quinidine, cisplatin and bleomycin and the herbicide barban, similar to its close homologue QDR2. In this work, a new physiological role for Qdr3 in polyamine homeostasis is proposed. QDR3 is shown to confer resistance to the polyamines spermine and spermidine, but, unlike Qdr2, also a determinant of resistance to polyamines, Qdr3 has no apparent role in K+ homeostasis. QDR3 transcription is upregulated in yeast cells exposed to spermine or spermidine dependent on the transcription factors Gcn4, which controls amino acid homeostasis, and Yap1, the main regulator of oxidative stress response. Yap1 was found to be a major determinant of polyamine stress resistance in yeast and is accumulated in the nucleus of yeast cells exposed to spermidine-induced stress. QDR3 transcript levels were also found to increase under nitrogen or amino acid limitation; this regulation is also dependent on Gcn4. Consistent with the concept that Qdr3 plays a role in polyamine homeostasis, QDR3 expression was found to decrease the intracellular accumulation of [3H]spermidine, playing a role in the maintenance of the plasma membrane potential in spermidine-stressed cells.

Plasma membrane clustering of system y+ (CAT-1) amino acid transporter as detected by immunohistochemistry

1994 ◽  
Vol 266 (5) ◽  
pp. E817-E824 ◽  
Author(s):  
M. H. Woodard ◽  
W. A. Dunn ◽  
R. O. Laine ◽  
M. Malandro ◽  
R. McMahon ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Human Fibroblasts ◽  
Amino Acid Transporter ◽  
Microtubule Assembly ◽  
Random Pattern ◽  
Transporter Protein ◽  
Acid Transporter

Transport of cationic amino acids in fully differentiated mammalian cells is mediated primarily by system y1+ [cationic amino acid transporter (CAT)-1 gene product]. Antibodies, prepared against synthetic peptide sequences predicted to be extracellular loops of the CAT-1 transporter protein, detected the transporter on the surface of cultured cells. In human fibroblasts, porcine pulmonary artery endothelial cells, and cultured rat hepatoma cells, the CAT-1 transporter protein was clustered in an apparent random pattern throughout the plasma membrane. In contrast, labeling of the fibroblasts with antibodies against the epidermal growth factor receptor or the GLUT-1 glucose transporter demonstrated a uniform staining pattern covering the entire cell surface. The CAT-1 antibody labeling was specific, as demonstrated by peptide inhibition and the lack of staining by preimmune serum. Furthermore, hepatocytes did not exhibit specific antibody binding consistent with the lack of system y1+ activity. Disruption of the microtubule assembly resulted in a reversible loss of the CAT-1 transporter clusters and a more generalized labeling of the cell body. The data demonstrate the existence of microdomains within the plasma membrane that contain the CAT-1 transporter protein.

scholarly journals Characterization of the apicomplexan amino acid transporter (ApiAT) family inToxoplasma gondii

2018 ◽  
Author(s):  
Kathryn E. R. Parker ◽  
Stephen J. Fairweather ◽  
Esther Rajendran ◽  
Martin Blume ◽  
Malcolm J. McConville ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Toxoplasma Gondii ◽  
Amino Acid Transporters ◽  
Apicomplexan Parasites ◽  
Large Neutral Amino Acids ◽  
Neutral Amino Acids ◽  
The Family

AbstractApicomplexan parasites are auxotrophic for a range of amino acids which must be salvaged from their host cells, either through direct uptake or degradation of host proteins. Here, we describe a family of plasma membrane-localized amino acid transporters, termed the Apicomplexan Amino acid Transporters (ApiATs), that are ubiquitous in apicomplexan parasites. Functional characterization of the ApiATs ofToxoplasma gondiiindicate that several of these transporters are important for intracellular growth of the tachyzoite stage of the parasite, which is responsible for acute infections. We demonstrate that the ApiAT proteinTgApiAT5-3 is an exchanger for aromatic and large neutral amino acids, with particular importance for L-tyrosine scavenging and amino acid homeostasis, and thatTgApiAT5-3 is critical for parasite virulence. Our data indicate thatT. gondiiexpresses additional proteins involved in the uptake of aromatic amino acids, and we present a model for the uptake and homeostasis of these amino acids. Our findings identify a family of amino acid transporters in apicomplexans, and highlight the importance of amino acid scavenging for the biology of this important phylum of intracellular parasites.Author SummaryThe Apicomplexa comprise a large number of parasitic protozoa that have obligate intracellular lifestyles and cause significant human and animal diseases, including malaria, cryptosporidiosis, toxoplasmosis, coccidiosis in poultry, and various cattle fevers. Apicomplexans must scavenge essential nutrients from their hosts in order to proliferate and cause disease, including a range of amino acids. The direct uptake of these nutrients is presumed to be mediated by transporter proteins located in the plasma membrane of intracellular stages, although the identities of these proteins are poorly defined. Using a combination of bioinformatic, genetic, cell biological, and physiological approaches, we have characterized a family of plasma membrane-localized transporter proteins that we have called the Apicomplexan Amino acid Transporters (ApiATs). The family is found in apicomplexans and their closest free-living relatives. We show thatTgApiAT5-3, a member of the family in the apicomplexanToxoplasma gondii, is an exchanger for aromatic and large neutral amino acids. In particular, it is critical for uptake of tyrosine, and for parasite virulence in a mouse infection model. We conclude that ApiATs are a family of plasma membrane transporters that play crucial roles in amino acid scavenging by apicomplexan parasites.

scholarly journals Amino Acids 563–566 of the Na+/H+ Exchanger Isoform 1 C-Terminal Cytosolic Tail Prevent Protein Degradation and Stabilize Protein Expression and Activity

2020 ◽  
Vol 21 (5) ◽  
pp. 1737 ◽  
Author(s):  
Xiuju Li ◽  
Debajyoti Dutta ◽  
Martin Jung ◽  
Richard Zimmermann ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Expression ◽  
Protein Degradation ◽  
Stop Codon ◽  
Protein A ◽  
Membrane Domain ◽  
Protein Levels ◽  
Extracellular Sodium ◽  
Reduced Activity

Isoform one of the mammalian Na+/H+ exchanger is a plasma membrane protein that is ubiquitously present in humans. It regulates intracellular pH through the removal of one intracellular proton in exchange for a single extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, C-terminal tail. We examined amino acids of the C-terminal tail that are important in the targeting and activity of the protein. A previous study demonstrated that stop codon polymorphisms can result in decreased activity, expression, targeting and enhanced protein degradation. Here, we determine elements that are critical in these anomalies. A series of progressive deletions of the C-terminal tail demonstrated a progressive decrease in activity and targeting, though these remained until a final drop off with the deletion of amino acids 563–566. The deletion of the 562LIAGERS568 sequence or the alteration to the 562LAAAARS568 sequence caused the decreased protein expression, aberrant targeting, reduced activity and enhanced degradation of the Na+/H+ exchanger (NHE1) protein. The 562LIAGERS568 sequence bound to other regions of the C-terminal cytosolic domain. We suggest this region is necessary for the activity, targeting, stability, and expression of the NHE1 protein. The results define a new sequence that is important in maintenance of NHE1 protein levels and activity.

Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family

2000 ◽  
Vol 347 (2) ◽  
pp. 419-429 ◽  
Author(s):  
Galia GHADDAR ◽  
Andréa Frota RUCHON ◽  
Mélanie CARPENTIER ◽  
Mieczyslaw MARCINKIEWICZ ◽  
Nabil G. SEIDAH ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Transmembrane Domain ◽  
Amino Acid Sequences ◽  
Mouse Testis ◽  
Peptidase Activity ◽  
Converting Enzyme ◽  
C Terminus ◽  
The Family

Because of their roles in controlling the activity of several bio-active peptides, members of the neprilysin family of zinc metallopeptidases have been identified as putative targets for the design of therapeutic agents. Presently, six members have been reported, these are: neprilysin, endothelin-converting enzyme (ECE)-1 and ECE-2, the Kell blood group protein, PHEX (product of the phosphate-regulating gene with homologies to endopeptidase on the X chromosome) and X-converting enzyme (XCE). In order to identify new members of this important family of peptidases, we designed a reverse transcriptase-PCR strategy based on conserved amino acid sequences of neprilysin, ECE-1 and PHEX. We now report the cloning from mouse testis of a novel neprilysin-like peptidase that we called NL1. NL1 is a glycoprotein that, among the members of the family, shows the strongest sequence identity with neprilysin. However, in contrast with neprilysin and other members of the family which are type II integral membrane proteins, NL1 was secreted when expressed in cultured mammalian cells, likely due to cleavage by a subtilisin-like convertase at a furin-like site located 22 amino acid residues in the C-terminus of the transmembrane domain. The recombinant enzyme exhibited neprilysin-like peptidase activity and was efficiently inhibited by phosphoramidon and thiorphan, two inhibitors of neprilysin. Northern blot analysis and in situ hybridization showed that NL1 mRNA was found predominantly in testis, specifically in round and elongated spermatids. This distribution of NL1 mRNA suggests that it could be involved in sperm formation or other processes related to fertility.

scholarly journals The Heavy Chain 4F2hc Modulates the Substrate Affinity and Specificity of the Light Chains LAT1 and LAT2

2020 ◽  
Vol 21 (20) ◽  
pp. 7573
Author(s):  
Satish Kantipudi ◽  
Jean-Marc Jeckelmann ◽  
Zöhre Ucurum ◽  
Patrick D. Bosshart ◽  
Dimitrios Fotiadis
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Heavy Chain ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Expression System ◽  
Functional Characterization ◽  
Light Chains ◽  
Substrate Affinity ◽  
Amino Acid Transporters

The human L-type amino acid transporters LAT1 and LAT2 mediate the transport of amino acids and amino acid derivatives across plasma membranes in a sodium-independent, obligatory antiport mode. In mammalian cells, LAT1 and LAT2 associate with the type-II membrane N-glycoprotein 4F2hc to form heteromeric amino acid transporters (HATs). The glycosylated ancillary protein 4F2hc is known to be important for successful trafficking of the unglycosylated transporters to the plasma membrane. The heavy (i.e., 4F2hc) and light (i.e., LAT1 and LAT2) chains belong to the solute carrier (SLC) families SLC3 and SLC7, and are covalently linked by a conserved disulfide bridge. Overexpression, absence, or malfunction of certain HATs is associated with human diseases and HATs are therefore considered therapeutic targets. Here, we present a comparative, functional characterization of the HATs 4F2hc-LAT1 and 4F2hc-LAT2, and their light chains LAT1 and LAT2. For this purpose, the HATs and the light chains were expressed in the methylotrophic yeast Pichia pastoris and a radiolabel transport assay was established. Importantly and in contrast to mammalian cells, P. pastoris has proven useful as eukaryotic expression system to successfully express human LAT1 and LAT2 in the plasma membrane without the requirement of co-expressed trafficking chaperone 4F2hc. Our results show a novel function of the heavy chain 4F2hc that impacts transport by modulating the substrate affinity and specificity of corresponding LATs. In addition, the presented data confirm that the light chains LAT1 and LAT2 constitute the substrate-transporting subunits of the HATs, and that light chains are also functional in the absence of the ancillary protein 4F2hc.

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