Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter

2002 ◽  
Vol 282 (2) ◽  
pp. C408-C416 ◽  
Author(s):  
Pejvak Sassani ◽  
Alexander Pushkin ◽  
Eitan Gross ◽  
Alla Gomer ◽  
Natalia Abuladze ◽  
...  
Keyword(s):  
Sodium Bicarbonate ◽  
Mammalian Cells ◽  
Functional Characterization ◽  
Amino Acid Level ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Bicarbonate Transporter ◽  
Sodium Bicarbonate Cotransporter ◽  
Protein Variants

Sodium-bicarbonate cotransporters are homologous membrane proteins mediating the electrogenic or electroneutral transport of sodium and bicarbonate. Of the functionally characterized sodium-bicarbonate cotransporters (NBC), NBC1proteins are known to be electrogenic. Here we report the cloning and functional characterization of NBC4c, a new splice variant of the NBC4 gene. At the amino acid level, NBC4c is 56% identical to NBC1 protein variants and 40% identical to electroneutral NBC3. When expressed in mammalian cells, NBC4c mediates electrogenic sodium-bicarbonate cotransport. The transport of sodium and bicarbonate is chloride independent and is completely inhibited by DIDS. NBC4c transcripts were detected in several tissues including brain, heart, kidney, testis, pancreas, muscle, and peripheral blood leukocytes. The data indicate that NBC4c is an electrogenic sodium-bicarbonate cotransporter. The finding that both NBC1 and NBC4c proteins function as electrogenic sodium-bicarbonate cotransporters will aid in determining the structural motifs responsible for this unique functional property, which distinguishes these transporters from other members of the bicarbonate transporter superfamily.

scholarly journals Functional characterization of neotropical snakes peripheral blood leukocytes subsets: Linking flow cytometry cell features, microscopy images and serum corticosterone levels

2017 ◽  
Vol 74 ◽  
pp. 144-153 ◽  
Author(s):  
Marcelo Pires Nogueira de Carvalho ◽  
Nicolle Gilda Teixeira Queiroz-Hazarbassanov ◽  
Cristina de Oliveira Massoco ◽  
Sávio Stefanini Sant’Anna ◽  
Mariana Mathias Lourenço ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Functional Characterization ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Serum Corticosterone ◽  
Microscopy Images

scholarly journals Structural and Functional Characterization of SARS-CoV-2 RBD Domains Produced in Mammalian Cells

2021 ◽  
Author(s):  
Christoph Gstöttner ◽  
Tao Zhang ◽  
Anja Resemann ◽  
Sophia Ruben ◽  
Stuart Pengelley ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Functional Characterization

Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p

1998 ◽  
Vol 111 (10) ◽  
pp. 1341-1349 ◽  
Author(s):  
M. Imoto ◽  
I. Tachibana ◽  
R. Urrutia
Keyword(s):  
Endoplasmic Reticulum ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Functional Characterization ◽  
Luciferase Reporter ◽  
Pleckstrin Homology ◽  
Rich Region ◽  
Gtpase Domain ◽  
Proline Rich Region

Dynamin proteins containing a GTPase domain, a pleckstrin homology motif and a proline-rich tail participate in receptor-mediated endocytosis in organisms ranging from insects to vertebrates. In addition, dynamin-related GTPases, such as the yeast Golgi protein Vps1p, which lack both the pleckstrin homology motif and the proline-rich region, participate in vesicular transport within the secretory pathway in lower eukaryotes. However, no data is available on the existence of Vps1p-like proteins in mammalian cells. In this study, we report the identification and characterization of a novel gene encoding a human dynamin-related protein, DRP1, displaying high similarity to the Golgi dynamin-like protein Vps1p from yeast and to a Caenorhabditis elegans protein deposited in the databank. These proteins are highly conserved in their N-terminal tripartite GTPase domain but lack the pleckstrin homology motif and proline-rich region. Northern blot analysis reveals that the DRP1 mRNA is detected at high levels in human muscle, heart, kidney and brain. Immunolocalization studies in Chinese hamster ovary (CHO) cells using an epitope-tagged form of DRP1 and confocal microscopy show that this protein is concentrated in a perinuclear region that labels with the endoplasmic reticulum marker DiOC6(3) and the Golgi marker C5-DMB-Cer. In addition, the localization of DRP1 is highly similar to the localization of the endoplasmic reticulum and cis-Golgi GTPase Rab1A, but not to the staining for the trans-Golgi GTPase Rab6. Furthermore, overexpression of a cDNA encoding a GTP binding site mutant of DRP1 (DRP1(K38E)) in CHO cells decreases the amount of a secreted luciferase reporter protein, whereas the overexpression of wild-type DRP1 increases the secretion of this marker. Together, these results constitute the first structural and functional characterization of a mammalian protein similar to the yeast dynamin-related GTPase Vps1p and indicate that the participation of these proteins in secretion has been conserved throughout evolution.

Immunolocalization of electrogenic sodium-bicarbonate cotransporters pNBC1 and kNBC1 in the rat eye

2001 ◽  
Vol 281 (5) ◽  
pp. F920-F935 ◽  
Author(s):  
Dean Bok ◽  
Matthew J. Schibler ◽  
Alexander Pushkin ◽  
Pejvak Sassani ◽  
Natalia Abuladze ◽  
...  
Keyword(s):  
Protein Expression ◽  
Sodium Bicarbonate ◽  
Molecular Mechanisms ◽  
Coding Region ◽  
Sodium Bicarbonate Cotransporter ◽  
Band Keratopathy ◽  
Proximal Renal Tubular Acidosis ◽  
Renal Tubular ◽  
Protein Variants ◽  
Renal Phenotype

The human NBC1 gene encodes two electrogenic sodium-bicarbonate cotransport proteins, pNBC1 and kNBC1, which are candidate proteins for mediating electrogenic sodium-bicarbonate cotransport in ocular cells. Mutations in the coding region of the human NBC1 gene in exons common to both pNBC1 and kNBC1 result in a syndrome with a severe ocular and renal phenotype (blindness, band keratopathy, glaucoma, cataracts, and proximal renal tubular acidosis). In the present study, we determined the pattern of electrogenic sodium-bicarbonate cotransporter protein expression in rat eye. For this purpose, pNBC1- and kNBC1-specific antibodies were generated and used to detect these NBC1 protein variants by immunoblotting and immunocytochemistry. pNBC1 is expressed in cornea, conjunctiva, lens, ciliary body, and retina, whereas the expression of kNBC1 is restricted to the conjunctiva. These results provide the first evidence for extrarenal kNBC1 protein expression. The data in this study will serve as a basis for understanding the molecular mechanisms responsible for abnormalities in ocular electrogenic sodium-bicarbonate cotransport in patients with mutations in the NBC1 gene.

Functional characterization of human NBC4 as an electrogenic Na+-HCO 3 − cotransporter (NBCe2)

2002 ◽  
Vol 282 (6) ◽  
pp. C1278-C1289 ◽  
Author(s):  
Leila V. Virkki ◽  
Darren A. Wilson ◽  
Richard D. Vaughan-Jones ◽  
Walter F. Boron
Keyword(s):  
Human Heart ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Regulatory Function ◽  
Disulfonic Acid ◽  
Xenopus Laevis Oocytes ◽  
Slow Recovery ◽  
Bicarbonate Transporter ◽  
Electrode Voltage

We have functionally characterized Na+-driven bicarbonate transporter (NBC)4, originally cloned from human heart by Pushkin et al. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215–218, 2000). Of the four NBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no. AF293337 ) in Xenopus laevis oocytes and assayed membrane potential ( V m) and pH regulatory function with microelectrodes. Exposing an NBC4c-expressing oocyte to a solution containing 5% CO2 and 33 mM HCO[Formula: see text]elicited a large hyperpolarization, indicating that the transporter is electrogenic. The initial CO2-induced decrease in intracellular pH (pHi) was followed by a slow recovery that was reversed by removing external Na+. Two-electrode voltage clamp of NBC4c-expressing oocytes revealed large HCO[Formula: see text]- and Na+-dependent currents. When we voltage clamped V m far from NBC4c's estimated reversal potential ( E rev), the pHirecovery rate increased substantially. Both the currents and pHi recovery were blocked by 200 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). We estimated the transporter's HCO[Formula: see text]:Na+ stoichiometry by measuring E rev at different extracellular Na+ concentration ([Na+]o) values. A plot of E rev against log[Na+]o was linear, with a slope of 54.8 mV/log[Na+]o. This observation, as well as the absolute E rev values, are consistent with a 2:1 stoichiometry. In conclusion, the behavior of NBC4c, which we propose to call NBCe2-c, is similar to that of NBCe1, the first electrogenic NBC.

scholarly journals Isolation, Synthesis, and Antimicrobial Activities of Naturally Occurring θ-Defensin Isoforms from Baboon Leukocytes

2008 ◽  
Vol 76 (12) ◽  
pp. 5883-5891 ◽  
Author(s):  
Angie E. Garcia ◽  
George Ösapay ◽  
Patti A. Tran ◽  
Jun Yuan ◽  
Michael E. Selsted
Keyword(s):  
Bone Marrow ◽  
Single Species ◽  
Antimicrobial Activities ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Naturally Occurring ◽  
Test Organisms ◽  
High Degree

ABSTRACT θ-Defensins are macrocyclic antimicrobial peptides that were previously isolated from leukocytes of a single species, the rhesus macaque. We now report the characterization of baboon θ-defensins (BTDs) expressed in bone marrow and peripheral blood leukocytes. Four cDNAs encoding θ-defensin precursors were characterized, allowing for the prediction of 10 theoretical θ-defensins (BTD-1 to BTD-10) produced by binary, head-to-tail splicing of nonapeptides excised from paired precursors. Five of the predicted θ-defensins were purified from baboon leukocytes, and synthetic versions of each were prepared. Anti-θ-defensin antibody localized the peptides in circulating neutrophils and monocytes and in immature and mature myeloid elements in bone marrow. Each of the BTDs possessed antimicrobial activity against bacterial and fungal test organisms in vitro. Peptide activities varied markedly despite a high degree of sequence conservation among the θ-defensins tested. Thus, baboons express numerous θ-defensins which appear to differentially contribute to host defense against diverse pathogens.

scholarly journals Functional characterization of ecdysone receptor gene switches in mammalian cells

FEBS Journal ◽  
2006 ◽  
Vol 273 (24) ◽  
pp. 5550-5563 ◽  
Author(s):  
Siva K. Panguluri ◽  
Prasanna Kumar ◽  
Subba R. Palli
Keyword(s):  
Mammalian Cells ◽  
Receptor Gene ◽  
Functional Characterization ◽  
Ecdysone Receptor ◽  
Gene Switches

scholarly journals The Characterization of peripheral blood leukocytes during the development of adjuvant arthritis in rats.

Ensho ◽  
1992 ◽  
Vol 12 (1) ◽  
pp. 55-61
Author(s):  
Yoshihide Segawa ◽  
Masakatsu Nozaki ◽  
Kaito Tsurumi ◽  
Shunro Kohbata
Keyword(s):  
Peripheral Blood ◽  
Adjuvant Arthritis ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes
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