Structures of cyanobacterial bicarbonate transporter SbtA and its complex with PII-like SbtB

BackgroundAutosomal recessive congenital hereditary corneal dystrophy (CHED) is a rare isolated developmental anomaly of the eye characterised by diffuse bilateral corneal clouding that may lead to visual impairment requiring corneal transplantation. CHED is known to be caused by mutations in the solute carrier family 4 member 11 (SLC4A11) gene which encodes a membrane transporter protein (sodium bicarbonate transporter-like solute carrier family 4 member 11).MethodsTo identify SLC4A11 gene mutations associated with CHED (OMIM: #217700), genomic DNA was extracted from whole blood and sequenced for all exons and intron-exon boundaries in two large Tunisian families.ResultsA novel deletion SLC4A11 mutation (p. Leu479del; c.1434_1436del) is responsible for CHED in both analysed families. This non-frameshift mutation was found in a homozygous state in affected members and heterozygous in non-affected members. In silico analysis largely support the pathogenicity of this alteration that may leads to stromal oedema by disrupting the osmolarity balance. Being localised to a region of alpha-helical secondary structure, Leu479 deletion may induce protein-compromising structural rearrangements.ConclusionTo the best of our knowledge, this is the first clinical and genetic study exploring CHED in Tunisia. The present work also expands the list of pathogenic genotypes in SLC4A11 gene and its associated clinical diagnosis giving more insights into genotype–phenotype correlations.

Download Full-text

N and C control of ABC-type bicarbonate transporter Cmp and its LysR-type transcriptional regulator CmpR in a heterocyst-forming cyanobacterium, Anabaena sp.

Environmental Microbiology ◽

10.1111/j.1462-2920.2011.02683.x ◽

2012 ◽

Vol 14 (4) ◽

pp. 1035-1048 ◽

Cited By ~ 23

Author(s):

Rocío López-Igual ◽

Silvia Picossi ◽

Javier López-Garrido ◽

Enrique Flores ◽

Antonia Herrero

Keyword(s):

Transcriptional Regulator ◽

Bicarbonate Transporter ◽

Cyanobacterium Anabaena ◽

Anabaena Sp

Download Full-text

Regulators of Slc4 bicarbonate transporter activity

Frontiers in Physiology ◽

10.3389/fphys.2015.00166 ◽

2015 ◽

Vol 6 ◽

Cited By ~ 13

Author(s):

Ian M. Thornell ◽

Mark O. Bevensee

Keyword(s):

Bicarbonate Transporter

Download Full-text

Structural basis for the allosteric regulation of the SbtA bicarbonate transporter by the PII-like protein, SbtB, from Cyanobium sp. PCC7001

10.1101/762807 ◽

2019 ◽

Author(s):

Joe A. Kaczmarski ◽

Nan-Sook Hong ◽

Bratati Mukherjee ◽

Laura T. Wey ◽

Loraine Rourke ◽

...

Keyword(s):

Metal Ion ◽

Transcriptional Control ◽

Allosteric Regulation ◽

Inhibitory Effect ◽

Photosynthetic Performance ◽

Regulatory Proteins ◽

Structural Basis ◽

Titration Calorimetry ◽

X Ray ◽

Bicarbonate Transporter

ABSTRACTCyanobacteria have evolved a suite of enzymes and inorganic carbon (Ci) transporters that improve photosynthetic performance by increasing the localized concentration of CO2 around the primary CO2-fixating enzyme, Rubisco. This CO2-concentrating mechanism (CCM) is highly regulated, responds to illumination/darkness cycles and allows cyanobacteria to thrive under limiting Ci conditions. While the transcriptional control of CCM activity is well understood, less is known about how regulatory proteins might allosterically regulate Ci transporters in response to changing conditions. Cyanobacterial sodium-dependent bicarbonate transporters (SbtAs) are inhibited by PII-like regulatory proteins (SbtBs), with the inhibitory effect being modulated by adenylnucleotides. Here, we used isothermal titration calorimetry to show that SbtB from Cyanobium sp. PCC7001 (SbtB7001) binds AMP, ADP, cAMP and ATP with micromolar-range affinities. X-ray crystal structures of apo- and nucleotide-bound SbtB7001 revealed that while AMP, ADP and cAMP have little effect on the SbtB7001 structure, binding of ATP stabilizes the otherwise flexible T-loop and that the flexible C-terminal C-loop adopts several distinct conformations. We also show that ATP binding affinity is increased ten-fold in the presence of Ca2+ and we present an X-ray crystal structure of Ca2+ATP:SbtB7001 that shows how this metal ion facilitates additional stabilizing interactions with the apex of the T-loop. We propose that the Ca2+ATP-induced conformational change observed in SbtB7001 is important for allosteric regulation of SbtA activity by SbtB and is consistent with changing adenylnucleotide levels in illumination/darkness cycles.GRAPHICAL ABSTRACT

Download Full-text

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

AJP Cell Physiology ◽

10.1152/ajpcell.00589.2001 ◽

2002 ◽

Vol 282 (6) ◽

pp. C1278-C1289 ◽

Cited By ~ 90

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.

Download Full-text

Interactions of transmembrane carbonic anhydrase, CAIX, with bicarbonate transporters

AJP Cell Physiology ◽

10.1152/ajpcell.00157.2007 ◽

2007 ◽

Vol 293 (2) ◽

pp. C738-C748 ◽

Cited By ~ 83

Author(s):

Patricio E. Morgan ◽

Silvia Pastoreková ◽

Alan K. Stuart-Tilley ◽

Seth L. Alper ◽

Joseph R. Casey

Keyword(s):

Gastric Mucosa ◽

Carbonic Anhydrase ◽

Catalytic Domain ◽

Hek293 Cells ◽

Bicarbonate Transport ◽

Physical Interaction ◽

Human Gastric Mucosa ◽

Bicarbonate Transporter ◽

Bicarbonate Transporters ◽

Physical Interactions

Association of some plasma membrane bicarbonate transporters with carbonic anhydrase enzymes forms a bicarbonate transport metabolon to facilitate metabolic CO2-HCO3−conversions and coupled HCO3−transport. The transmembrane carbonic anhydrase, CAIX, with its extracellular catalytic site, is highly expressed in parietal and other cells of gastric mucosa, suggesting a role in acid secretion. We examined in transfected HEK293 cells the functional and physical interactions between CAIX and the parietal cell Cl−/HCO3−exchanger AE2 or the putative Cl−/HCO3−exchanger SLC26A7. Coexpression of CAIX increased AE2 transport activity by 28 ± 7% and also activated transport mediated by AE1 and AE3 (32 ± 10 and 37 ± 9%, respectively). In contrast, despite a transport rate comparable to that of AE3, coexpressed CAIX did not alter transport associated with SLC26A7. The CAIX-associated increase of AE2 activity did not result from altered AE2 expression or cell surface processing. CAIX was coimmunoprecipitated with the coexpressed SLC4 polypeptides AE1, AE2, and AE3, but not with SLC26A7. GST pull-down assays with a series of domain-deleted forms of CAIX revealed that the catalytic domain of CAIX mediated interaction with AE2. AE2 and CAIX colocalized in human gastric mucosa, as indicated by coimmunofluorescence. This is the first example of a functional and physical interaction between a bicarbonate transporter and a transmembrane carbonic anhydrase. We conclude that CAIX can bind to some Cl−/HCO3−exchangers to form a bicarbonate transport metabolon.

Download Full-text

Involvement of a CbbR Homolog in Low CO2-Induced Activation of the Bicarbonate Transporter Operon in Cyanobacteria

Journal of Bacteriology ◽

10.1128/jb.183.6.1891-1898.2001 ◽

2001 ◽

Vol 183 (6) ◽

pp. 1891-1898 ◽

Cited By ~ 69

Author(s):

Tatsuo Omata ◽

Satoshi Gohta ◽

Yukari Takahashi ◽

Yoshimi Harano ◽

Shin-ichi Maeda

Keyword(s):

Specific Binding ◽

Regulatory Region ◽

Transcriptional Regulator ◽

Bisphosphate Carboxylase ◽

Purple Photosynthetic Bacteria ◽

Bicarbonate Transporter ◽

Enhanced Strain ◽

Operon Expression ◽

Pcc 7942 ◽

Pcc 6803

ABSTRACT The cmpABCD operon of Synechococcus sp. strain PCC 7942, encoding a high-affinity bicarbonate transporter, is transcribed only under CO2-limited conditions. InSynechocystis sp. strain PCC 6803, the slr0040, slr0041, slr0043, and slr0044 genes, forming an operon with a putative porin gene (slr0042), were identified as the cmpA, cmpB, cmpC, and cmpDgenes, respectively, on the basis of their strong similarities to the corresponding Synechococcus cmp genes and their induction under low CO2 conditions. Immediately upstream of and transcribed divergently from the Synechocystis cmp operon is a gene (sll0030) encoding a homolog of CbbR, a LysR family transcriptional regulator of the CO2 fixation operons of chemoautotrophic and purple photosynthetic bacteria. Inactivation of sll0030, but not of another closely relatedcbbR homolog (sll1594), abolished low CO2 induction of cmp operon expression. Gel retardation assays showed specific binding of the Sll0030 protein to the sll0030-cmpA intergenic region, suggesting that the protein activates transcription of the cmp operon by interacting with its regulatory region. A cbbR homolog similar to sll0030 and sll1594 was cloned fromSynechococcus sp. strain PCC 7942 and shown to be involved in the low CO2-induced activation of the cmpoperon. We hence designated the Synechocystis sll0030 gene and the Synechococcus cbbR homolog cmpR. In the mutants of the cbbR homologs, upregulation of ribulose-1,5-bisphosphate carboxylase/oxygenase operon expression by CO2 limitation was either unaffected (strain PCC 6803) or enhanced (strain PCC 7942), suggesting existence of other low CO2-responsive transcriptional regulator(s) in cyanobacteria.

Download Full-text

Mouse Slc4a11 expressed in Xenopus oocytes is an ideally selective H+/OH− conductance pathway that is stimulated by rises in intracellular and extracellular pH

AJP Cell Physiology ◽

10.1152/ajpcell.00259.2016 ◽

2016 ◽

Vol 311 (6) ◽

pp. C945-C959 ◽

Cited By ~ 20

Author(s):

Evan J. Myers ◽

Aniko Marshall ◽

Michael L. Jennings ◽

Mark D. Parker

Keyword(s):

Xenopus Oocytes ◽

Driving Forces ◽

Physiological Role ◽

Extracellular Ph ◽

Intrinsic Activity ◽

Corneal Endothelial Cells ◽

Bicarbonate Transporter ◽

First Time ◽

Intracellular Alkalinization ◽

Congenital Hereditary Endothelial Dystrophy

The SLC4A11 gene encodes the bicarbonate-transporter-related protein BTR1, which is mutated in syndromes characterized by vision and hearing loss. Signs of these diseases [congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome] are evident in mouse models of Slc4a11 disruption. However, the intrinsic activity of Slc4a11 remains controversial, complicating assignment of its (patho)physiological role. Most studies concur that Slc4a11 transports H+ (or the thermodynamically equivalent species OH−) rather than HCO3−, but disparities have arisen as to whether the transport is coupled to another species such as Na+ or NH3/NH4+. Here for the first time, we examine the action of mouse Slc4a11 in Xenopus oocytes. We simultaneously monitor changes in intracellular pH, membrane potential, and conductance as we alter extracellular pH, revealing the electrical and chemical driving forces that underlie the observed ion fluxes. We find that mSlc4a11 is an ideally selective H+/OH− conductive pathway, the action of which is uncoupled from the cotransport of any other ion. We also find that the activity of mSlc4a11 is independently enhanced by both extracellular and intracellular alkalinization, suggesting OH− as the most likely substrate and providing a novel explanation for the apparent NH3-dependence of Slc4a11-mediated currents reported by others. We suggest that the unique properties of Slc4a11 action underlie its value as a pH regulator in corneal endothelial cells.

Download Full-text