Regulation of AE2 anion exchanger by intracellular  pH: critical regions of the NH<sub>2</sub>-terminal cytoplasmic domain

Stewart, Andrew K.; Chernova, Marina N.; Kunes, Yune Z.; Alper, Seth L.

doi:10.1152/ajpcell.2001.281.4.c1344

Cited by 69 publications

(142 citation statements)

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“…Full-length cDNAs were cloned into PCR-cloning vector pCRII (Invitrogen), and plasmid cDNA inserts were sequenced in entirety to ensure absence of PCR-introduced mutations, then subcloned into oocyte expression vector pXT7 (13,50) and mammalian expression vector pcDNA3 (Invitrogen). For immunodetection at the oocyte surface, each cDNA in pXT7 was also hemagglutinin (HA)-tagged at its NH 2-terminus through flexible linker sequence GASG.…”

Section: Methodsmentioning

confidence: 99%

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SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

Stewart

Shmukler

Vandorpe

et al. 2011

American Journal of Physiology-Cell Physiology

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The secretin-stimulated human pancreatic duct secretes HCO3−-rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO3− secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl−/HCO3− exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ∼140 mM HCO3− or more, mouse and rat ducts secrete ∼40–70 mM HCO3−. Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO3− secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl−/Cl− exchange and electroneutral Cl−/HCO3− exchange. gpSlc26a6 in Xenopus oocytes mediated Cl−/Cl− exchange and bidirectional exchange of Cl− for oxalate and sulfate, but Cl−/HCO3− exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl−, oxalate, and sulfate transport but no detectable Cl−/HCO3− exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of 36Cl− influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO3− secretion in species that share a high HCO3− secretory output.

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Section: Methodsmentioning

confidence: 99%

“…Measurement of oocyte pH i. Oocyte intracellular pH (pHi) was measured using pH microelectrodes as described previously (50). pH electrodes were pulled on a vertical electrode puller (model 730, Kopf Instruments) from borosilicate glass tubing (M1B150F-6, WPI) prewashed with nitric acid and ethanol.…”

Section: Methodsmentioning

confidence: 99%

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

Stewart

Shmukler

Vandorpe

et al. 2011

American Journal of Physiology-Cell Physiology

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“…15 and 16), consistent with its proposed role in pH i recovery from alkaline loads. Recombinant AE2 when expressed in tissue culture cells (17, 18) or in Xenopus oocytes (12,13,19) is highly sensitive to changes in both pH o and pH i . Similarly, recombinant cardiac AE3 expressed in Xenopus oocytes is responsive to changes in pH i (19).…”

mentioning

confidence: 99%

“…Recombinant AE2 when expressed in tissue culture cells (17, 18) or in Xenopus oocytes (12,13,19) is highly sensitive to changes in both pH o and pH i . Similarly, recombinant cardiac AE3 expressed in Xenopus oocytes is responsive to changes in pH i (19).Structure-function experiments have indicated that an operationally defined AE2 "pH sensor site" that enhances pH o sensitivity of anion transport is localized to the transmembrane domain (13). This study also demonstrated that the extracellular proton sensitivity (pH o(50) value) of AE2-mediated 36 Cl Ϫ influx (ϳ7.0 under conditions of unclamped pH i ) was acidshifted by up to 0.7 units following various truncations of the cytoplasmic NH 2 -terminal domain.…”

mentioning

confidence: 99%

“…This result suggested the presence, between aa 99 and 510 of the 705-aa AE2 NH 2 -terminal cytoplasmic domain, of a "pH modifier site" that modulates anion transport activity of the transmembrane domain. More recently, 36 Cl Ϫ efflux assays in AE2-expressing oocytes revealed that pH sensitivity of the transporter requires the integrity of two non-contiguous regions of the NH 2 -terminal cytoplasmic domain: the highly conserved aa 336 -347 (20) and the less well conserved region of aa 391-510 (19). The con-served residues aa 336 -347 play a similar role in pH-dependent regulation of the AE3 polypeptide and are likely to be important in other SLC4 transporters, as well (20).…”

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Acute pH-dependent Regulation of AE2-mediated Anion Exchange Involves Discrete Local Surfaces of the NH2-terminal Cytoplasmic Domain

Stewart

Kerr

Chernova

et al. 2004

Journal of Biological Chemistry

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We have previously defined in the NH 2 -terminal cytoplasmic domain of the mouse AE2/SLC4A2 anion exchanger a critical role for the highly conserved amino acids (aa) 336 -347 in determining wild-type pH sensitivity of anion transport. We have now engineered hexaAla ((A) 6 ) and individual amino acid substitutions to investigate the importance to pH-dependent regulation of AE2 activity of the larger surrounding region of aa 312-578. 4,4 -Diisothiocyanostilbene-2,2 -disulfonic acid (DIDS)-sensitive 36 Cl ؊ efflux from AE2-expressing Xenopus oocytes was monitored during changes in pH i or pH o in HEPES-buffered and in 5% CO 2 /HCO 3 ؊ -buffered conditions. Wild-type AE2-mediated 36 Cl ؊ efflux was profoundly inhibited at low pH o , with a pH o(50) value ‫؍‬ 6.75 ؎ 0.05 and was stimulated up to 10-fold by intracellular alkalinization. Individual mutation of several amino acid residues at non-contiguous sites preceding or following the conserved sequence aa 336 -347 attenuated pH i and/or pH o sensitivity of 36 Cl ؊ efflux. The largest attenuation of pH sensitivity occurred with the AE2 mutant (A) 6 357-362. This effect was phenocopied by AE2 H360E, suggesting a crucial role for His 360 . Homology modeling of the three-dimensional structure of the AE2 NH 2 -terminal cytoplasmic domain (based on the structure of the corresponding region of human AE1) predicts that those residues shown by mutagenesis to be functionally important define at least one localized surface region necessary for regulation of AE2 activity by pH.The anion exchangers AE1, AE2, and AE3 are polypeptide products of the SLC4 bicarbonate transporter gene superfamily that mediate Na ϩ -independent Cl Ϫ /HCO 3 Ϫ exchange. Anion exchangers contribute to the regulation of intracellular pH (pH i ), cell volume, tonicity, and intracellular Cl (Cl Ϫ ) in metazoan cells (1-5). The AE polypeptides share a highly conserved hydrophobic, polytopic, transmembrane domain of Ͼ500 amino acids (aa), 1 with a short COOH-terminal cytoplasmic tail capable of binding carbonic anhydrase II (6, 7). This transmembrane domain is preceded by a less extensively conserved hydrophilic NH 2 -terminal cytoplasmic domain of 400 -700 aa (3). The polytopic transmembrane domain can mediate anion exchange in the absence of nearly the entire NH 2 -terminal cytoplasmic domain (8, 9). Whereas the NH 2 -terminal cytoplasmic domain of erythroid AE1 is important for its binding to multiple cytoskeletal proteins, glycolytic enzymes, and hemoglobin (10), functions of the cytoplasmic NH 2 -terminal domains of AE2 and AE3 remain less extensively investigated.The polypeptide products of the various SLC4 AE anion exchanger genes differ in their acute regulation by pH. Native AE1-mediated Cl Ϫ /HCO 3 Ϫ exchange in erythrocytes (11) and heterologous AE1-mediated Cl Ϫ /Cl Ϫ exchange in Xenopus oocytes (12, 13) both display a broad pH versus activity profile, consistent with the primary role of erythroid AE1 in facilitating CO 2 /HCO 3 Ϫ exchange between the respiring tissues and lungs (14). In contras...

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Na⁺/H⁺Exchangers

Orlowski

Grinstein

2011

Comprehensive Physiology

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Tightly coupled exchange of Na(+) for H(+) occurs across the surface membrane of virtually all living cells. For years, the underlying molecular entity was unknown and the full physiological significance of the exchange process was not appreciated, but much knowledge has been gained in the last two decades. We now realize that, unlike most of the other transporters that specialize in supporting one specific function, Na(+)/H(+) exchangers (NHE) participate in a remarkable assortment of physiological processes, ranging from pH homeostasis and epithelial salt transport, to systemic and cellular volume regulation. In parallel, we have learned a great deal about the biochemistry and molecular biology of Na(+)/H(+) exchange. Indeed, it has now become apparent that exchange is mediated not by one, but by a diverse family of related yet distinct carriers (antiporters) sometimes present in different cell types and located in various intracellular compartments. Each one of these has unique structural features that dictate its functional role and mode of regulation. The biological relevance of Na(+)/H(+) exchange is emphasized by its evolutionary conservation; analogous exchangers are present from bacteria to man. Because of its wide distribution and versatile function, Na(+)/H(+) exchange has attracted an enormous amount of interest and therefore generated a vast literature. The vastness and complexity of the field has been compounded by the multiplicity of NHE isoforms. For reasons of space and in the spirit of this series, this overview is restricted to the family of mammalian NHEs.

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Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH₂-terminal cytoplasmic domain

Cited by 69 publications

References 30 publications

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

Acute pH-dependent Regulation of AE2-mediated Anion Exchange Involves Discrete Local Surfaces of the NH2-terminal Cytoplasmic Domain

Na⁺/H⁺Exchangers

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Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH2-terminal cytoplasmic domain

Cited by 69 publications

References 30 publications

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

Acute pH-dependent Regulation of AE2-mediated Anion Exchange Involves Discrete Local Surfaces of the NH2-terminal Cytoplasmic Domain

Na+/H+Exchangers

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Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH₂-terminal cytoplasmic domain

Na⁺/H⁺Exchangers