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Walter F. Boron - One of the best experts on this subject based on the ideXlab platform.
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effects of optional structural elements including two alternative amino termini and a new splicing cassette iv on the function of the sodium bicarbonate cotransporter nbcn1 SLC4A7
The Journal of Physiology, 2013Co-Authors: Ying Liu, Mark D. Parker, Xue Qin, Deng Ke Wang, Yi Min Guo, Harindarpal S Gill, Nathan Morris, Liming Chen, Walter F. BoronAbstract:Key points • The human SLC4A7 gene and the mouse SLC4A7 gene each have alternative promoters that can yield two groups of NBCn1 variants, one in which the extreme N terminus begins with MEAD (representing the first four residues of the N-terminal domain (Nt)) and the other in which it begins with MERF. • The mouse SLC4A7 gene contains, and the human SLC4A7 gene is predicted to contain, a novel exon that encodes an alternatively spliced cassette IV of 20 aa in the cytoplasmic Nt domain of NBCn1. This new cassette IV is in a position homologous to that of a previously described cassette in the Nt of NBCn2. • From combinations of known optional structural elements (OSEs), SLC4A7 is theoretically able to produce 32 major variants, of which 16 have now been identified, 10 for the first time in the present study. • With heterologous expression in Xenopus oocytes, the OSEs have strong effects on surface abundance and intrinsic HCO3− transport activity. Cassettes II, III and the novel cassette IV have stimulatory effects on the intrinsic HCO3− transport activity of NBCn1. Abstract The SLC4A7 gene encodes the electroneutral sodium/HCO3 cotransporter NBCn1, which plays important physiological and pathophysiological roles in many cell types. Previous work identified six NBCn1 variants differing in the sequence of the extreme N terminus – MEAD in rat only, MERF in human only – as well as in the optional inclusion of cassettes I, II, and III. Earlier work also left open the question of whether optional structural elements (OSEs) affect surface abundance or intrinsic (per-molecule) transport activity. Here, we demonstrate for the first time that SLC4A7 from one species can express both MEAD- and MERF-NBCn1. We also identify a novel cassette IV of 20 aa, and extend by 10 the number of full-length NBCn1 variants. The alternative N termini and four cassettes could theoretically produce 32 major variants. Moreover, we identify a group of cDNAs predicted to encode just the cytosolic N-terminal domain (Nt) of NBCn1. A combination of electrophysiology and biotinylation shows that the OSEs can affect surface abundance and intrinsic HCO3− transport activity of NBCn1, as expressed in Xenopus oocytes. Specifically, MEAD tends to increase whereas novel cassette IV reduces surface abundance. Cassettes II, III and novel cassette IV all appear to increase the intrinsic activity of NBCn1.
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the slc4 family of bicarbonate hco3 transporters
Molecular Aspects of Medicine, 2013Co-Authors: Michael F Romero, Mark D. Parker, Anping Chen, Walter F. BoronAbstract:The SLC4 family consists of 10 genes (SLC4A1-5; SLC4A7-11). All encode integral membrane proteins with very similar hydropathy plots-consistent with 10-14 transmembrane segments. Nine SLC4 members encode proteins that transport HCO3(-) (or a related species, such as CO3(2-)) across the plasma membrane. Functionally, eight of these proteins fall into two major groups: three Cl-HCO3 exchangers (AE1-3) and five Na(+)-coupled HCO3(-) transporters (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE). Two of the Na(+)-coupled transporters (NBCe1, NBCe2) are electrogenic; the other three Na(+)-coupled HCO3(-) transporters and all three AEs are electroneutral. In addition, two other SLC4 members (AE4, SLC4A9 and BTR1, SLC4A11) do not yet have a firmly established function. Most, though not all, SLC4 members are functionally inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). SLC4 proteins play important roles many modes of acid-base homeostasis: the carriage of CO2 by erythrocytes, the transport of H(+) or HCO3(-) by several epithelia, as well as the regulation of cell volume and intracellular pH.
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substrate specificity of the electrogenic sodium bicarbonate cotransporter nbce1 a slc4a4 variant a from humans and rabbits
American Journal of Physiology-renal Physiology, 2013Co-Authors: Seong-ki Lee, Walter F. Boron, Mark D. ParkerAbstract:In the basolateral membrane of proximal-tubule cells, NBCe1-A (SLC4A4, variant A), operating with an apparent Na+:HCO3− stoichiometry of 1:3, contributes to the reclamation of HCO3− from the glomer...
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cloning and characterization of novel human slc4a8 gene products encoding na driven cl hco3 exchanger variants ndcbe a c and d
Physiological Genomics, 2008Co-Authors: Mark D. Parker, Patrice Bouyer, Christopher M. Daly, Walter F. BoronAbstract:The reported sequences of the human and mouse Na+-driven Cl−/HCO3− exchangers (NDCBEs) differ greatly in their extreme cytosolic COOH termini (Ct). In human NDCBE (NDCBE-B), a 17-amino acid (aa) sequence replaces 66 aa at the equivalent position in mouse NDCBE (NDCBE-A). We performed 5′- and 3′-rapid amplification of cDNA ends (RACE) on human brain cDNA, followed by PCR of full-length cDNAs to determine whether the human SLC4A8 gene was capable of producing the mouselike Ct sequence. Our study confirmed the presence in human cDNA of mouse NDCBE-like transcripts (human NDCBE-A) and also disclosed the existence of three further novel NDCBE transcripts that we have called NDCBE-C, NDCBE-D, and NDCBE-D′. The novel NDCBE-C/D/D′ transcripts initiate at a novel “exon 0” positioned ∼35 kb upstream of the first exon of NDCBE-A/B. NDCBE-C/D/D′ protein products are predicted to be truncated by 54 aa in the cytosolic NH2 terminus (Nt) compared with NDCBE-A/B. Our data, combined with a new in silico analysis of partial transcripts reported by others in the region of the human SLC4A8 gene, increase the known extent of the SLC4A8 gene by 49 kb, to 124 kb. A functional comparison of NDCBE-A/B/C/D expressed in Xenopus oocytes demonstrates that the Nt variation does not affect the basal functional expression of NDCBE, but those with the shorter Ct have a 25–50% reduced functional expression compared with those with the longer Ct. By comparison with an artificially truncated NDCBE that contains neither 17-aa nor 66-aa Ct cassette, we determined that the functional difference is unrelated to the 66-aa cassette of NDCBE-A/C, but is instead due to an inhibitory effect of the 17-aa cassette of NDCBE-B/D.
Ebbe Boedtkjer - One of the best experts on this subject based on the ideXlab platform.
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sodium bicarbonate cotransporter nbcn1 SLC4A7 affects locomotor activity and hearing in mice
Behavioural Brain Research, 2021Co-Authors: Inyeong Choi, Christian Aalkjaer, Vibeke Secher Dam, Kristian Beedholm, Seongho Bae, Donald J Noble, Sandra M Garraway, Ebbe BoedtkjerAbstract:Despite a widespread expression pattern in the central nervous system, the role of the sodium bicarbonate cotransporter NBCn1/SLC4A7 has not been investigated for locomotor activity, emotion and cognition. Here, we addressed the behavioral consequences of NBCn1 knockout and evaluated hearing and vision that are reportedly impaired in an earlier line of NBCn1 knockout mice and may contribute to behavioral changes. In a circular open field, the knockout mice traveled a shorter distance, especially in the periphery of the chamber, than wildtype littermates. The knockout mice also traveled a shorter total distance in a home cage-like open field. Rearing and grooming behaviors were reduced. The knockout and control mice displayed similar time spent and number of open and closed arms in the elevated plus maze test, indicating negligible change in anxiety. In the Morris water maze test, both groups of mice learned the location of an escape platform within comparable time on the training trials and showed similar platform identification on the probe trial. The knockout mice maintained normal visual responses in the optokinetic drum and produced evoked potentials in response to light stimuli. However, these mice failed to produce auditory evoked potentials. qPCR revealed a robust expression of an alternatively transcribed NBCn1 variant in the knockout mouse retina. These results indicate that NBCn1 deletion leads to reduced locomotor activity in mice by affecting their exploratory behaviors or emotionality. The deletion also causes hearing loss, but its effect on vision varies between different lines of knockout mice.
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lbos 02 04 blood pressure associated polymorphisms in SLC4A7 sodium bicarbonate co transporter nbcn1 are linked to gene expression and intracellular ph regulation
Journal of Hypertension, 2016Co-Authors: Ebbe Boedtkjer, Mark J CaulfieldAbstract:Chromosome 3p24.1 containing the SLC4A7 gene has been identified by genome-wide association studies as one of the genomic loci that influence blood pressure. SLC4A7 encodes electroneutral Na+/HCO3– co-transporter (NBCn1) which regulates intracellular pH (pHi) in various tissues including vascular smooth muscle and endothelium, with knockout models demonstrating an altered blood pressure phenotype. We conducted a functional study of blood pressure-associated single nucleotide polymorphisms at the SLC4A7 locus in primary cultures of human vascular smooth muscle and endothelial cells. Primary cultures of human umbilical vein endothelial cells and artery smooth muscle cells of different SLC4A7 genotypes were used. DNA-protein interactions were identified by formaldehyde-associated isolation of regulatory elements (FAIRE) and electrophoretic mobility shift assays (EMSA). Gene expression was quantified by allelic imbalance analyses and protein immunoblots. NBCn1 protein function was evaluated using the baseline pHi and rate of dimethyl-amiloride-insensitive, Na+/HCO3–-dependent pHi recovery following ammonium-prepulse-induced intracellular acidification. A10 cells overexpressing 326E or 326K SLC4A7 variants were used to verify primary culture results. FAIRE identified the BP risk allele as associated with preferentially open chromatin, and EMSA identified allele-dependent DNA-protein interactions. Consistent with this, the risk allele was associated with increased NBCn1 mRNA, protein expression, availability at the cell membrane and subsequently higher Na+/HCO3–-dependent, dimethlyamiloride-insensitive net base flux and steady-state pHi compared to protective allele homozygotes. However, in the presence of Na+/H+ exchange activity, the genotypic effect of NBCn1 on net acid extrusion and steady-state pHi was detected only in vascular smooth muscle cells but not endothelial cells. There was no discernable effect of the missense polymorphism resulting in the amino acid substitution E326K. These findings provide an insight into the molecular mechanism underlying the association of variation at the SLC4A7 locus and blood pressure, revealing an important genotypic influence on SLC4A7 expression and pHi regulation in vascular smooth muscle cells.
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disrupting na hco 3 cotransporter nbcn1 SLC4A7 delays murine breast cancer development
Oncogene, 2016Co-Authors: Soojung Lee, Stine F Pedersen, Trine V Axelsen, Anne Poder Andersen, Pernille Vahl, Ebbe BoedtkjerAbstract:Increased metabolism and insufficient blood supply cause acidic waste product accumulation in solid cancers. During carcinogenesis, cellular acid extrusion is upregulated but the underlying molecular mechanisms and their consequences for cancer growth and progression have not been established. Genome-wide association studies have indicated a possible link between the Na+,HCO3–-cotransporter NBCn1 (SLC4A7) and breast cancer. We tested the functional consequences of NBCn1 knockout (KO) for breast cancer development. NBCn1 protein expression increased 2.5-fold during breast carcinogenesis and was responsible for the increased net acid extrusion and alkaline intracellular pH of breast cancer compared with normal breast tissue. Genetic disruption of NBCn1 delayed breast cancer development: tumor latency was ~50% increased while tumor growth rate was ~65% reduced in NBCn1 KO compared with wild-type (WT) mice. Breast cancer histopathology in NBCn1 KO mice differed from that in WT mice and included less aggressive tumor types. The extracellular tumor microenvironment in NBCn1 KO mice contained higher concentrations of glucose and lower concentrations of lactate than that in WT mice. Independently of NBCn1 genotype, the cleaved fraction of poly(ADP-ribose) polymerase (PARP)-1 and expression of monocarboxylate transporter (MCT)1 increased while phosphorylation of Akt and ERK1 decreased as functions of tumor volume. Cell proliferation, evaluated from Ki-67 and phospho-histone H3 staining, was ~60% lower in breast cancer of NBCn1 KO than that of WT mice when corrected for variations in tumor size. We conclude that NBCn1 facilitates acid extrusion from breast cancer tissue, maintains the alkaline intracellular environment and promotes aggressive cancer development and growth.
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antibody independent localization of the electroneutral na hco3 cotransporter nbcn1 SLC4A7 in mice
American Journal of Physiology-cell Physiology, 2008Co-Authors: Ebbe Boedtkjer, Jeppe Praetorius, Ernstmartin Fuchtbauer, Christian AalkjaerAbstract:The expression pattern of the electroneutral Na+-HCO3−cotransporter NBCn1 (SLC4A7) was investigated by β-galactosidase staining of mice with a LacZ insertion into the NBCn1 gene. This method is of ...
Ira Kurtz - One of the best experts on this subject based on the ideXlab platform.
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the apical na hco3 cotransporter SLC4A7 nbcn1 does not contribute to bicarbonate transport by mouse salivary gland ducts
Journal of Cellular Physiology, 2019Co-Authors: Ningyan Yang, Ira Kurtz, Taro Mukaibo, James E MelvinAbstract:The HCO3 - secretion mechanism in salivary glands is unclear but is thought to rely on the co-ordinated activity of multiple ion transport proteins including members of the Slc4 family of bicarbonate transporters. SLC4A7 was immunolocalized to the apical membrane of mouse submandibular duct cells. In contrast, SLC4A7 was not detected in acinar cells, and correspondingly, SLC4A7 disruption did not affect fluid secretion in response to cholinergic or β-adrenergic stimulation in the submandibular gland (SMG). Much of the Na + -dependent intracellular pH (pH i ) regulation in SMG duct cells was insensitive to 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, S0859, and to the removal of extracellular HCO 3 - . Consistent with these latter observations, the SLC4A7 null mutation had no impact on HCO 3 - secretion nor on pH i regulation in duct cells. Taken together, our results revealed that SLC4A7 targets to the apical membrane of mouse SMG duct cells where it contributes little if any to pH i regulation or stimulated HCO 3 - secretion.
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Prevention of the Disrupted Enamel Phenotype in Slc4a4-Null Mice Using Explant Organ Culture Maintained in a Living Host Kidney Capsule
PloS one, 2014Co-Authors: Xin Wen, Ira Kurtz, Michael L PaineAbstract:Slc4a4-null mice are a model of proximal renal tubular acidosis (pRTA). Slc4a4 encodes the electrogenic sodium base transporter NBCe1 that is involved in transcellular base transport and pH regulation during amelogenesis. Patients with mutations in the SLC4A4 gene and Slc4a4-null mice present with dysplastic enamel, amongst other pathologies. Loss of NBCe1 function leads to local abnormalities in enamel matrix pH regulation. Loss of NBCe1 function also results in systemic acidemic blood pH. Whether local changes in enamel pH and/or a decrease in systemic pH are the cause of the abnormal enamel phenotype is currently unknown. In the present study we addressed this question by explanting fetal wild-type and Slc4a4-null mandibles into healthy host kidney capsules to study enamel formation in the absence of systemic acidemia. Mandibular E11.5 explants from NBCe1-/- mice, maintained in host kidney capsules for 70 days, resulted in teeth with enamel and dentin with morphological and mineralization properties similar to cultured NBCe1+/+ mandibles grown under identical conditions. Ameloblasts express a number of proteins involved in dynamic changes in H+/base transport during amelogenesis. Despite the capacity of ameloblasts to dynamically modulate the local pH of the enamel matrix, at least in the NBCe1-/- mice, the systemic pH also appears to contribute to the enamel phenotype. Extrapolating these data to humans, our findings suggest that in patients with NBCe1 mutations, correction of the systemic metabolic acidosis at a sufficiently early time point may lead to amelioration of enamel abnormalities.
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time course of auditory impairment in mice lacking the electroneutral sodium bicarbonate cotransporter nbc3 SLC4A7
Developmental Brain Research, 2005Co-Authors: Ivan A Lopez, Dora Acuna, Gary C Galbraith, Dean Bok, Akira Ishiyama, Weixin Liu, Ira KurtzAbstract:Abstract Mice with a targeted disruption of the gene encoding the stilbene-insensitive electroneutral sodium bicarbonate cotransporter (NBC3; SLC4A7 ) exhibit cochlear and retinal degeneration. To establish the progressive nature of sensory cells loss in SLC4A7 −/− deficient mice, we studied the morphology of cochleas of SLC4A7 −/− and SLC4A7 +/+ mice from postnatal day two (P2) to ninety (P90). Cell death was evaluated in SLC4A7 −/− cochleas using the TUNEL technique and caspase-3 immunoreactivity. The time course of NBC3 expression in the cochlea was assessed by immunohistochemistry using an antibody against NBC3. Between P2 and P8, SLC4A7 −/− mice cochlea exhibit normal morphology. There was a normal complement of inner and outer hair cells from the hook to the apical region. At P15, SLC4A7 −/− mice cochlea inner and outer hair cells were still present at the hook region, and vacuoles were seen underneath Hensen's cells. At P21, inner and outer hair cells were degenerated in this region. Between P30 and P90, there was a pronounced loss of hair cells and spiral ganglia neurons. Morphological analysis of the spiral ligament showed a progressive loss of type II and IV fibrocytes beginning at day 21. Transmission electron microscopy observations at P30 and P90 revealed that type II and IV fibrocytes showed shrinkage and vacuolization. In addition, hair cells were deteriorated with evidence of shrinkage and picnotic nuclei. TUNEL staining showed apoptotic cells at P8 in the organ of Corti at the basal region of the cochlea. At P15, caspase-3 immunoreactivity was present in supporting cells of the organ of Corti. NBC3 mild immunoreactivity was detected in the organ of Corti at P11. There was an increase in the expression of NBC3 in the spiral ligament between P17 and P19. From P21 to P90, NBC3 expression was confined to the spiral ligament and inner and outer sulcus cells. The vestibular sensory epithelia from SLC4A7 −/− mice were normal from P2 to P90. Damage of the sensory epithelia at the high frequency zone of the cochlea suggests that NBC3 may play an important physiological role in this region.
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cloning tissue distribution genomic organization and functional characterization of nbc3 a new member of the sodium bicarbonate cotransporter family
Journal of Biological Chemistry, 1999Co-Authors: Alexander Pushkin, Natalia Abuladze, Ivan Lee, James Hwang, Debra Newman, Ira KurtzAbstract:Previous functional studies have demonstrated that muscle intracellular pH regulation is mediated by sodium-coupled bicarbonate transport, Na+/H+ exchange, and Cl-/bicarbonate exchange. We report the cloning, sequence analysis, tissue distribution, genomic organization, and functional analysis of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC3, from human skeletal muscle. mNBC3 encodes a 1214-residue polypeptide with 12 putative membrane-spanning domains. The approximately 7.8-kilobase transcript is expressed uniquely in skeletal muscle and heart. The NBC3 gene (SLC4A7) spans approximately 80 kb and is composed of 25 coding exons and 24 introns that are flanked by typical splice donor and acceptor sequences. Expression of mNBC3 cRNA in Xenopus laevis oocytes demonstrated that the protein encodes a novel stilbene-insensitive 5-(N-ethyl-N-isopropyl)-amiloride-inhibitable sodium bicarbonate cotransporter.
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cloning tissue distribution genomic organization and functional characterization of nbc3 a new member of the sodium bicarbonate cotransporter family
Journal of Biological Chemistry, 1999Co-Authors: Alexander Pushkin, Natalia Abuladze, James Hwang, Debra Newman, Ira KurtzAbstract:Abstract Previous functional studies have demonstrated that muscle intracellular pH regulation is mediated by sodium-coupled bicarbonate transport, Na+/H+ exchange, and Cl−/bicarbonate exchange. We report the cloning, sequence analysis, tissue distribution, genomic organization, and functional analysis of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC3, from human skeletal muscle. mNBC3 encodes a 1214-residue polypeptide with 12 putative membrane-spanning domains. The ∼ 7.8-kilobase transcript is expressed uniquely in skeletal muscle and heart. The NBC3 gene (SLC4A7) spans ∼80 kb and is composed of 25 coding exons and 24 introns that are flanked by typical splice donor and acceptor sequences. Expression of mNBC3 cRNA inXenopus laevis oocytes demonstrated that the protein encodes a novel stilbene-insensitive 5-(N-ethyl-N-isopropyl)-amiloride-inhibitable sodium bicarbonate cotransporter.
Robin A Felder - One of the best experts on this subject based on the ideXlab platform.
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sodium bicarbonate cotransporter nbce2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells
PLOS ONE, 2018Co-Authors: John J Gildea, Robert M Carey, Helen E Mcgrath, Brandon A Kemp, Julia M Carlson, Hanh T Tran, Dora Bigler Wang, Christophe J Langouetastrie, Pedro A Jose, Robin A FelderAbstract:Rationale Salt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure. Objective To characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5. Methods and results The expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte nuclear factor type 4A (HNF4A) to DNA was increased in hRPTCs carrying HV SLC4A5 rs7571842 but not rs10177833. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was abolished by HNF4A antagonists. Conclusion NBCe2 activity is stimulated by an increase in intracellular sodium and is hyper-responsive in hRPTCs carrying HV SLC4A5 rs7571842 through an aberrant HNF4A-mediated mechanism.
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abstract p242 role of human renal proximal tubule sodium bicarbonate cotransporter nbce2 slc4a5 in salt sensitivity of blood pressure
Hypertension, 2015Co-Authors: John J Gildea, Robert M Carey, Helen E Mcgrath, Brandon A Kemp, Julia M Carlson, Dora Bigler Wang, Christophe J Langouetastrie, Pedro A Jose, Tran T Hanh, Robin A FelderAbstract:The sodium bicarbonate cotransporter NBCe2 (encoded by SLC4A5) partially regulates renal tubular sodium bicarbonate transport. Hypothesis: since SLC4A5 single nucleotide polymorphisms (SNPs, rs10177833 and rs7571842) are associated with salt sensitivity of blood pressure, the gene product, NBCe2, would be involved with the etiology of human salt sensitivity. NBCe2 was localized in freshly fixed renal tissue and in primary and immortalized RPT cell (RPTC) cultures from tissue or isolated from urine. Basal expression of NBCe2 mRNA and protein was not different between RPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, total transcellular sodium transport, NHE3 protein expression, and Cl-/HCO3- exchanger activity were higher in SLC4A5 HV than WT RPTCs (WT: 8.6±1.2 mM NaCl n=6, 5207.1±386.4 RFU n=36, 0.265±0.006 pH unit/min, n=33 respectively; VS HV: 14.75±0.7 mM NaCl n=4, 6946.2±500.4 RFU n=48, 0.314±0.018 pH unit/min n=35 respectively, p<0.01). Aberrant sodium transport was even more evident after increasing intracellular sodium, which resulted in increased NBCe2 mRNA, NBCe2 protein and bicarbonate transport in HV RPTCs compared to WT (WT 146% ± 24% , 109% ± 4.7%, 89% ± 4.5%, respectively, VS HV 214% ± 23%, 128% ± 5.7%, 141% ± 4.8%, respectively N=8-12, p<0.05). RPTCs carrying HV variants showed increased binding of HNF4A to SLC4A5 DNA, which was blocked by two HNF4A antagonists. Assays in RPTCs isolated from urine showed increased bicarbonate-dependent pH recovery in RPTCs from salt-sensitive subjects who are HV for SLC4A5. NBCe2 under high sodium is hyper-responsive in RPTCs carrying SLC4A5 HV through an aberrant HNF4A-mediated mechanism.
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abstract 356 a role for renal proximal tubule sodium bicarbonate cotransporter slc4a5 in salt sensitivity of blood pressure
Hypertension, 2012Co-Authors: Julia M Carlson, John J Gildea, Helen E Mcgrath, Robin A FelderAbstract:SLC4A5 is a sodium-bicarbonate co-transporter involved with sodium homeostasis. Based on unpublished data, two SLC4A5 single nucleotide polymorphisms (SNPs rs1017783 and rs7571842) have been highly associated with an individual’s salt-sensitivity status. Since the renal proximal tubule (RPT) regulates a large percentage of renal sodium transport, we investigated whether SLC4A5 was present in this nephron segment. Using confocal immunofluorescence microscopy, we found expression of SLC4A5 in human RPT cell plasma membrane and intracellular membrane vesicles. We then examined the physiologic implications of the SLC4A5 SNPs in human RPT cells. Using immunoblotting and RT-PCR, we found no significant differences in basal SLC4A5 expression in RPT cells between individuals that are homozygous variant at both SNPs and individuals that are wild-type (WT) for both alleles. Stimulation of the dopaminergic system with 1μM fenoldopam, or the renin-angiotensin system with 10 nM angiotensin II or 10 nM angiotensin III (n=18 per treatment) over 3 and 24 hours did not significantly alter SLC4A5 protein or 24 hour mRNA expression. These data indicate that SLC4A5 is not directly regulated by either the renal dopaminergic or renin-angiotensin system. However, 24 hour stimulation with the sodium ionophore monensin (MON, 1μM) significantly increased overall mRNA expression of SLC4A5 by 182±0.098% over vehicle (VEH) (ΔCq VEH=0.283±0.035; n=18, p
Mark D. Parker - One of the best experts on this subject based on the ideXlab platform.
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effects of optional structural elements including two alternative amino termini and a new splicing cassette iv on the function of the sodium bicarbonate cotransporter nbcn1 SLC4A7
The Journal of Physiology, 2013Co-Authors: Ying Liu, Mark D. Parker, Xue Qin, Deng Ke Wang, Yi Min Guo, Harindarpal S Gill, Nathan Morris, Liming Chen, Walter F. BoronAbstract:Key points • The human SLC4A7 gene and the mouse SLC4A7 gene each have alternative promoters that can yield two groups of NBCn1 variants, one in which the extreme N terminus begins with MEAD (representing the first four residues of the N-terminal domain (Nt)) and the other in which it begins with MERF. • The mouse SLC4A7 gene contains, and the human SLC4A7 gene is predicted to contain, a novel exon that encodes an alternatively spliced cassette IV of 20 aa in the cytoplasmic Nt domain of NBCn1. This new cassette IV is in a position homologous to that of a previously described cassette in the Nt of NBCn2. • From combinations of known optional structural elements (OSEs), SLC4A7 is theoretically able to produce 32 major variants, of which 16 have now been identified, 10 for the first time in the present study. • With heterologous expression in Xenopus oocytes, the OSEs have strong effects on surface abundance and intrinsic HCO3− transport activity. Cassettes II, III and the novel cassette IV have stimulatory effects on the intrinsic HCO3− transport activity of NBCn1. Abstract The SLC4A7 gene encodes the electroneutral sodium/HCO3 cotransporter NBCn1, which plays important physiological and pathophysiological roles in many cell types. Previous work identified six NBCn1 variants differing in the sequence of the extreme N terminus – MEAD in rat only, MERF in human only – as well as in the optional inclusion of cassettes I, II, and III. Earlier work also left open the question of whether optional structural elements (OSEs) affect surface abundance or intrinsic (per-molecule) transport activity. Here, we demonstrate for the first time that SLC4A7 from one species can express both MEAD- and MERF-NBCn1. We also identify a novel cassette IV of 20 aa, and extend by 10 the number of full-length NBCn1 variants. The alternative N termini and four cassettes could theoretically produce 32 major variants. Moreover, we identify a group of cDNAs predicted to encode just the cytosolic N-terminal domain (Nt) of NBCn1. A combination of electrophysiology and biotinylation shows that the OSEs can affect surface abundance and intrinsic HCO3− transport activity of NBCn1, as expressed in Xenopus oocytes. Specifically, MEAD tends to increase whereas novel cassette IV reduces surface abundance. Cassettes II, III and novel cassette IV all appear to increase the intrinsic activity of NBCn1.
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the slc4 family of bicarbonate hco3 transporters
Molecular Aspects of Medicine, 2013Co-Authors: Michael F Romero, Mark D. Parker, Anping Chen, Walter F. BoronAbstract:The SLC4 family consists of 10 genes (SLC4A1-5; SLC4A7-11). All encode integral membrane proteins with very similar hydropathy plots-consistent with 10-14 transmembrane segments. Nine SLC4 members encode proteins that transport HCO3(-) (or a related species, such as CO3(2-)) across the plasma membrane. Functionally, eight of these proteins fall into two major groups: three Cl-HCO3 exchangers (AE1-3) and five Na(+)-coupled HCO3(-) transporters (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE). Two of the Na(+)-coupled transporters (NBCe1, NBCe2) are electrogenic; the other three Na(+)-coupled HCO3(-) transporters and all three AEs are electroneutral. In addition, two other SLC4 members (AE4, SLC4A9 and BTR1, SLC4A11) do not yet have a firmly established function. Most, though not all, SLC4 members are functionally inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). SLC4 proteins play important roles many modes of acid-base homeostasis: the carriage of CO2 by erythrocytes, the transport of H(+) or HCO3(-) by several epithelia, as well as the regulation of cell volume and intracellular pH.
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substrate specificity of the electrogenic sodium bicarbonate cotransporter nbce1 a slc4a4 variant a from humans and rabbits
American Journal of Physiology-renal Physiology, 2013Co-Authors: Seong-ki Lee, Walter F. Boron, Mark D. ParkerAbstract:In the basolateral membrane of proximal-tubule cells, NBCe1-A (SLC4A4, variant A), operating with an apparent Na+:HCO3− stoichiometry of 1:3, contributes to the reclamation of HCO3− from the glomer...
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cloning and characterization of novel human slc4a8 gene products encoding na driven cl hco3 exchanger variants ndcbe a c and d
Physiological Genomics, 2008Co-Authors: Mark D. Parker, Patrice Bouyer, Christopher M. Daly, Walter F. BoronAbstract:The reported sequences of the human and mouse Na+-driven Cl−/HCO3− exchangers (NDCBEs) differ greatly in their extreme cytosolic COOH termini (Ct). In human NDCBE (NDCBE-B), a 17-amino acid (aa) sequence replaces 66 aa at the equivalent position in mouse NDCBE (NDCBE-A). We performed 5′- and 3′-rapid amplification of cDNA ends (RACE) on human brain cDNA, followed by PCR of full-length cDNAs to determine whether the human SLC4A8 gene was capable of producing the mouselike Ct sequence. Our study confirmed the presence in human cDNA of mouse NDCBE-like transcripts (human NDCBE-A) and also disclosed the existence of three further novel NDCBE transcripts that we have called NDCBE-C, NDCBE-D, and NDCBE-D′. The novel NDCBE-C/D/D′ transcripts initiate at a novel “exon 0” positioned ∼35 kb upstream of the first exon of NDCBE-A/B. NDCBE-C/D/D′ protein products are predicted to be truncated by 54 aa in the cytosolic NH2 terminus (Nt) compared with NDCBE-A/B. Our data, combined with a new in silico analysis of partial transcripts reported by others in the region of the human SLC4A8 gene, increase the known extent of the SLC4A8 gene by 49 kb, to 124 kb. A functional comparison of NDCBE-A/B/C/D expressed in Xenopus oocytes demonstrates that the Nt variation does not affect the basal functional expression of NDCBE, but those with the shorter Ct have a 25–50% reduced functional expression compared with those with the longer Ct. By comparison with an artificially truncated NDCBE that contains neither 17-aa nor 66-aa Ct cassette, we determined that the functional difference is unrelated to the 66-aa cassette of NDCBE-A/C, but is instead due to an inhibitory effect of the 17-aa cassette of NDCBE-B/D.