Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase α subunit

The Na+/K+-ATPase mediates electrogenic transport by exporting three Na+ ions in exchange for two K+ ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb+ ions in the crystal structures of the Na+/K+-ATPase has defined two “common” cation binding sites, I and II, which accommodate Na+ or K+ ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this “unique” Na+ binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na+/K+-ATPase α2 subunit decreases the affinity for extracellular and intracellular Na+, in agreement with previous biochemical studies. Apparently, the ΔYY deletion changes Na+ affinity at site III but leaves the common sites unaffected, whereas the more extensive ΔKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K+, the ΔYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na+ dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na+/Na+ exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na+ release/binding mechanism. In analogy to the mechanism proposed for the H+ leak currents of the wild-type Na+/K+-ATPase, we suggest that the ΔYY deletion lowers the energy barrier for the intracellular Na+ occlusion reaction, thus destabilizing the Na+-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na+/K+-ATPase α subunit represents a structural and functional relay between Na+ binding site III and the intracellular cation occlusion gate.

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Coronavirus Particle Assembly: Primary Structure Requirements of the Membrane Protein

Journal of Virology ◽

10.1128/jvi.72.8.6838-6850.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6838-6850 ◽

Cited By ~ 116

Author(s):

Cornelis A. M. de Haan ◽

Lili Kuo ◽

Paul S. Masters ◽

Harry Vennema ◽

Peter J. M. Rottier

Keyword(s):

Hepatitis Virus ◽

Critical Role ◽

Point Mutations ◽

Major Protein ◽

Dependent Manner ◽

Carboxy Terminus ◽

Concentration Dependent Manner ◽

Particle Assembly ◽

Virus Like Particles ◽

Carboxy Terminal

ABSTRACT Coronavirus-like particles morphologically similar to normal virions are assembled when genes encoding the viral membrane proteins M and E are coexpressed in eukaryotic cells. Using this envelope assembly assay, we have studied the primary sequence requirements for particle formation of the mouse hepatitis virus (MHV) M protein, the major protein of the coronavirion membrane. Our results show that each of the different domains of the protein is important. Mutations (deletions, insertions, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles. Strikingly, the extreme carboxy-terminal residue is crucial. Deletion of this single residue abolished particle assembly almost completely; most substitutions were strongly inhibitory. Site-directed mutations in the carboxy terminus of M were also incorporated into the MHV genome by targeted recombination. The results supported a critical role for this domain of M in viral assembly, although the M carboxy terminus was more tolerant of alteration in the complete virion than in virus-like particles, likely because of the stabilization of virions by additional intermolecular interactions. Interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were found to inhibit the budding of assembly-competent M in a concentration-dependent manner. However, assembly-competent M was able to rescue assembly-incompetent M when the latter was present in low amounts. These observations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assembly.

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Comparison of thermosensitive alleles of the CDC25 gene involved in the cAMP metabolism of Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/124.4.797 ◽

1990 ◽

Vol 124 (4) ◽

pp. 797-806 ◽

Cited By ~ 2

Author(s):

A Petitjean ◽

F Hilger ◽

K Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Critical Role ◽

Missense Mutations ◽

Nucleotide Exchange ◽

Carboxy Terminus ◽

Reading Frame ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Carboxy Terminal

Abstract The CDC25 gene from Saccharomyces cerevisiae is an essential component of the RAS-adenylate cyclase pathway. Genetic and biochemical evidence has led to the proposal that the gene product may act upstream of RAS, possibly as a guanine nucleotide exchange factor. We report here the cloning, sequencing and characterization of four mutations in the CDC25 gene. All four are missense mutations which reside within the carboxy-terminal quarter of the single open reading frame found within the gene. Three of the four are missense mutations in the same amino acid codon. A search of protein data bases reveals that the carboxy terminus of the putative CDC25 gene product is similar to that of LTE1, a gene required for growth at low temperature and SCD25, a suppressor of cdc25. Taken together these data indicate that the carboxy terminus of CDC25 plays a critical role in the function of the CDC25 gene product and that other proteins, such as LTE1 or SCD25, may have related activities.

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Analysis of the membrane domain of the gastric H(+)/K(+)-ATPase

Journal of Experimental Biology ◽

10.1242/jeb.203.1.161 ◽

2000 ◽

Vol 203 (1) ◽

pp. 161-170

Author(s):

K. Munson ◽

N. Lambrecht ◽

J.M. Shin ◽

G. Sachs

Keyword(s):

Binding Site ◽

Covalent Binding ◽

Peptide Sequencing ◽

Cation Binding ◽

Beta Subunit ◽

In Vitro Translation ◽

Membrane Domain ◽

Binding Region ◽

Α Subunit ◽

Cation Binding Site

A structure of the catalytic or alpha subunit of the H(+)/K(+)-ATPase, with ten transmembrane segments, and of the beta subunit, with a single such segment, was established using a combination of tryptic cleavage and peptide sequencing and in vitro translation. Sites at which covalent ligands bind to external surfaces were also defined by cleavage, separation and sequencing. Cys813 was found to be the common covalent binding site for all the substituted pyridyl methylsulfinyl benzimidazoles. The binding region of a K(+)-competitive reagent, the 1,2 α -imidazo-pyridine SCH 28080, was defined by the kinetic effects of site-specific mutations. Amino acids substitutions in membrane-spanning segments M1, M3, M4 and M6 were found to influence the apparent inhibitor constant, K(i), to varying degrees, some having a large effect, some a moderate effect and some a slight effect, whereas some mutations had no effect. We interpret changes in K(i) without effects on the apparent Michaelis constant, K(m), as affecting SCH 28080 binding only. Mutation of Cys813 significantly affected the K(i) for SCH 28080, explaining the prevention of benzimidazole inhibition by the imidazo-pyridine.A model of the α subunit was constructed with a vestibule on the luminal surface of the pump bounded by M1-M6 and containing the SCH 28080 binding region. The cation binding site is suggested to be more towards the cytoplasmic face of the enzyme's membrane domain. This model predicts the membrane peptide associations for the catalytic subunit. Biochemical and yeast two-hybrid methods place the beta subunit in association with M8, whereas similar methods place M5/6 in proximity to M9/10. These results, when combined with analysis of the two-dimensional crystals of the sarcoplasmic reticular Ca(2+) and Neurospora crassa H(+)-ATPases, provide the basis for a tentative model of the arrangement of the six core segments of the gastric H(+)/K(+)-ATPase.

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Characterization of three TRAPPC11 variants suggests a critical role for the extreme carboxy terminus of the protein

Scientific Reports ◽

10.1038/s41598-019-50415-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Miroslav P. Milev ◽

Daniela Stanga ◽

Anne Schänzer ◽

Andrés Nascimento ◽

Djenann Saint-Dic ◽

...

Keyword(s):

Membrane Trafficking ◽

Critical Role ◽

Missense Variant ◽

Compound Heterozygous ◽

Autophagic Flux ◽

Carboxy Terminus ◽

Trafficking Defects ◽

Carboxy Terminal ◽

Foie Gras

AbstractTRAPPC11 was identified as a component of the TRAPP III complex that functions in membrane trafficking and autophagy. Variants in TRAPPC11 have been reported to be associated with a broad spectrum of phenotypes but all affected individuals display muscular pathology. Identifying additional variants will further our understanding of the clinical spectrum of phenotypes and will reveal regions of the protein critical for its functions. Here we report three individuals from unrelated families that have bi-allellic TRAPPC11 variants. Subject 1 harbors a compound heterozygous variant (c.1287 + 5G > A and c.3379_3380insT). The former variant results in a partial deletion of the foie gras domain (p.Ala372_Ser429del), while the latter variant results in a frame-shift and extension at the carboxy terminus (p.Asp1127Valfs*47). Subjects 2 and 3 both harbour a homozygous missense variant (c.2938G > A; p.Gly980Arg). Fibroblasts from all three subjects displayed membrane trafficking defects manifested as delayed endoplasmic reticulum (ER)-to-Golgi transport and/or a delay in protein exit from the Golgi. All three individuals also show a defect in glycosylation of an ER-resident glycoprotein. However, only the compound heterozygous subject displayed an autophagic flux defect. Collectively, our characterization of these individuals with bi-allelic TRAPPC11 variants highlights the functional importance of the carboxy-terminal portion of the protein.

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Structure of the agonist-binding site of the nicotinic acetylcholine receptor. [3H]acetylcholine mustard identifies residues in the cation-binding subsite.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)54504-x ◽

1991 ◽

Vol 266 (34) ◽

pp. 23354-23364

Author(s):

J.B. Cohen ◽

S.D. Sharp ◽

W.S. Liu

Keyword(s):

Binding Site ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Cation Binding ◽

Agonist Binding ◽

Nicotinic Acetylcholine

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Phorbol ester-induced expression of the common, low-affinity binding site for primary prostanoids in vascular smooth muscle cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)34055-4 ◽

1990 ◽

Vol 265 (9) ◽

pp. 4871-4875

Author(s):

K Hanasaki ◽

M Kishi ◽

H Arita

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Binding Site ◽

Vascular Smooth Muscle Cells ◽

Phorbol Ester ◽

Muscle Cells ◽

Affinity Binding ◽

Induced Expression ◽

The Common

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Effect of Urea on the Divalent Cation Binding Site of Pyruvate Kinase

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)62093-9 ◽

1971 ◽

Vol 246 (13) ◽

pp. 4363-4365 ◽

Cited By ~ 5

Author(s):

Gene L. Cottam ◽

Albert S. Mildvan

Keyword(s):

Binding Site ◽

Pyruvate Kinase ◽

Divalent Cation ◽

Cation Binding ◽

Cation Binding Site

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Monoclonal antibodies identify residues 199–216 of the integrin α2 vWFA domain as a functionally important region within α2β1

Biochemical Journal ◽

10.1042/bj3500485 ◽

2000 ◽

Vol 350 (2) ◽

pp. 485-493 ◽

Cited By ~ 1

Author(s):

Danny S. TUCKWELL ◽

Lyndsay SMITH ◽

Michelle KORDA ◽

Janet A. ASKARI ◽

Sentot SANTOSO ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Binding Site ◽

Conformational Changes ◽

Cation Binding ◽

Inhibitory Antibody ◽

Collagen Binding ◽

Collagen Peptide ◽

Binding Residue ◽

Important Region ◽

Domain Mapping

Integrin α2β1 is the major receptor for collagens in the human body, and the collagen-binding site on the α2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant α2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse α2β1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr221 to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-β1 antibody Mab13, and binding of Gi9 and JA218 to α2β1 was inhibited by substituting Mn2+ for Mg2+, demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212–216, while JA208 bound between residues 199–216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

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Silencing of the Gene for the α-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

Molecular Endocrinology ◽

10.1210/mend.11.11.9971 ◽

1997 ◽

Vol 11 (11) ◽

pp. 1651-1658 ◽

Cited By ~ 1

Author(s):

Limin Liu ◽

Douglas Leaman ◽

Michel Villalta ◽

R. Michael Roberts

Keyword(s):

Transcription Factor ◽

Binding Site ◽

Gene Promoter ◽

Site Directed Mutagenesis ◽

Mobility Shift ◽

Α Subunit ◽

Choriocarcinoma Cells ◽

Human Choriocarcinoma Cells ◽

Electrophoretic Mobility Shift Assays ◽

Jar Cells

Abstract CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG β-subunit (hCGβ) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGβ gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG α-subunit (hCGα) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the −170 hCGα promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the −148 hCGα or the −99 hCGα promoter. Unexpectedly, no Oct-3/4-binding site was identified within the −170 to −148 region of the hCGα promoter, although one was found around position −115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGα gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGα mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCGα and hCGβ gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGα and hCGβ genes.

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