Biomedical subjects

Joseph R Casey

Publications and source records attributed to Joseph R Casey.

26 records · Page 2Linked to original sources

PubMed2002

Novel topology in C-terminal region of the human plasma membrane anion exchanger, AE1.

Human AE1 performs electroneutral exchange of Cl(-) for HCO(3)(-) across the erythrocyte membrane. We examined the topology of the AE1 C-terminal region using cysteine-scanning mutagenesis and sulfhydryl-specific chemistry. Eighty individual cysteine residues, introduced into an otherwise cysteine-less mutant between Phe(806) and Cys(885), were expressed by transient transfection of HEK293 cells. Topology of the region was determined by comparing cysteine labeling with the membrane-permeant cysteine-directed reagent biotin maleimide, with or without prior labeling with the membrane-impermeant reagents, bromotrimethylammoniumbimane bromide (qBBr) and lucifer yellow iodoacetamide (LYIA). Phe(806)-Leu(835), Ser(852)-Ala(855), and Ile(872)-Cys(885) were labeled by biotin maleimide, suggesting their location in an aqueous environment. In contrast, Phe(836)-Lys(851) and Ser(856)-Arg(871) were not labeled by biotin maleimide and therefore localize to the plane of the bilayer, as transmembrane segments (TM). Labeling by qBBr revealed that Pro(815)-Lys(829) and Ser(852)-Ala(855) are accessible to the extracellular medium. Pro(815)-Lys(829) mutants were also labeled with LYIA. Mutants Ile(872)-Cys(885) were inaccessible to the extracellular medium and thus localized to the intracellular surface of AE1. Functional assays revealed that one face of each of two AE1 TMs was sensitive to mutation. Based on these results, we propose a topology model for the C-terminal region of the membrane domain of human AE1.

Amino Acid Sequence↗

PubMed2002

Carbonic anhydrase II binds to and enhances activity of the Na+/H+ exchanger.

We examined the ability of carbonic anhydrase II to bind to and affect the transport efficiency of the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. The C-terminal region of NHE1 was expressed in Escherichia coli fused with an N-terminal glutathionine S-transferase or with a C-terminal polyhistidine tag. Using a microtiter plate binding assay we showed that the C-terminal region of NHE1 binds carbonic anhydrase II (CAII) and binding was stimulated by low pH and blocked by antibodies against the C-terminal of NHE1. The binding to NHE1 was confirmed by demonstrating protein-protein interaction using affinity blotting with CAII and immobilized NHE1 fusion proteins. CAII co-immunoprecipitated with NHE1 from CHO cells suggesting the proteins form a complex in vivo. In cells expressing CAII and NHE1, the H(+) transport rate was almost 2-fold greater than in cells expressing NHE1 alone. The CAII inhibitor acetazolamide significantly decreased the H(+) transport rate of NHE1 and transfection with a dominant negative CAII inhibited NHE1 activity. Phosphorylation of the C-terminal of NHE1 greatly increased the binding of CAII. Our study suggests that NHE1 transport efficiency is influenced by CAII, likely through a direct interaction at the C-terminal region. Regulation of NHE1 activity by phosphorylation could involve modulation of CAII binding.

Animals↗

PubMed2002

The extracellular component of a transport metabolon. Extracellular loop 4 of the human AE1 Cl-/HCO3- exchanger binds carbonic anhydrase IV.

Cytosolic carbonic anhydrase II (CAII) and the cytoplasmic C-terminal tails of chloride/bicarbonate anion exchange (AE) proteins associate to form a bicarbonate transport metabolon, which maximizes the bicarbonate transport rate. To determine whether cell surface-anchored carbonic anhydrase IV (CAIV) interacts with AE proteins to accelerate the bicarbonate transport rate, AE1-mediated bicarbonate transport was monitored in transfected HEK293 cells. Expression of the inactive CAII V143Y mutant blocked the interaction between endogenous cytosolic CAII and AE1, AE2, and AE3 and inhibited their transport activity (53 +/- 3, 49 +/- 10, and 35 +/- 1% inhibition, respectively). However, in the presence of V143Y CAII, expression of CAIV restored full functional activity to AE1, AE2, and AE3 (AE1, 101 +/- 3; AE2, 85 +/- 5; AE3, 108 +/- 1%). In Triton X-100 extracts of transfected HEK293 cells, resolved by sucrose gradient ultracentrifugation, CAIV recruitment to the position of AE1 suggested a physical interaction between CAIV and AE1. Gel overlay assays showed a specific interaction between CAIV and AE1, AE2, and AE3. Glutathione S-transferase pull-down assays revealed that the interaction between CAIV and AE1 occurs on the large fourth extracellular loop of AE1. We conclude that AE1 and CAIV interact on extracellular loop 4 of AE1, forming the extracellular component of a bicarbonate transport metabolon, which accelerates the rate of AE-mediated bicarbonate transport.

Amino Acid Sequence↗

PubMed2002

Bicarbonate transport proteins.

Bicarbonate is not freely permeable to membranes. Yet, bicarbonate must be moved across membranes, as part of CO2 metabolism and to regulate cell pH. Mammalian cells ubiquitously express bicarbonate transport proteins to facilitate the transmembrane bicarbonate flux. These bicarbonate transporters, which function by different transport mechanisms, together catalyse transmembrane bicarbonate movement. Recent advances have allowed the identification of several new bicarbonate transporter genes. Bicarbonate transporters cluster into two separate families: (i) the anion exachanger (AE) family of Cl-/HCO3- exchangers is related in sequence to the NBC family of Na+/HCO3- cotransporters and the Na(+)-dependent Cl/HCO3- exchangers and (ii) some members of the SLC26a family of sulfate transporters will also transport bicarbonate but are not related in sequence to the AE/NBC family of transporters. This review summarizes our understanding of the mammalian bicarbonate transporter superfamily.

Amino Acid Sequence↗

PubMed2002

Meeting report: membrane proteins in health and disease.

This article summarizes the scientific presentations made at a Canadian Society of Biochemistry and Molecular & Cellular Biology Symposium on "Membrane Proteins in Health and Diseases" and two satellite meetings on "Bicarbonate Transporters" and "Nucleoside Transporters" held in Banff, Alberta, 20-24 March 2002. Membrane proteins are encoded by about 1/3 of genes and are involved in a wide range of essential functions, including the transport of nutrients, ions, and waste products across biological membranes. Mutations or changes in the expression of these genes cause an equally wide range of diseases. Membrane proteins are also common drug targets or provide drug entry mechanisms. The importance of membrane proteins in biology and medicine was highlighted by the presentations made at this exciting meeting by an international group of experts.

Animals↗

PubMed2002

The functional and physical relationship between the DRA bicarbonate transporter and carbonic anhydrase II.

COOH-terminal cytoplasmic tails of chloride/bicarbonate anion exchangers (AE) bind cytosolic carbonic anhydrase II (CAII) to form a bicarbonate transport metabolon, a membrane protein complex that accelerates transmembrane bicarbonate flux. To determine whether interaction with CAII affects the downregulated in adenoma (DRA) chloride/bicarbonate exchanger, anion exchange activity of DRA-transfected HEK-293 cells was monitored by following changes in intracellular pH associated with bicarbonate transport. DRA-mediated bicarbonate transport activity of 18 +/- 1 mM H+ equivalents/min was inhibited 53 +/- 2% by 100 mM of the CAII inhibitor, acetazolamide, but was unaffected by the membrane-impermeant carbonic anhydrase inhibitor, 1-[5-sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-dimethyl-4-phenyl-pyridinium perchlorate. Compared with AE1, the COOH-terminal tail of DRA interacted weakly with CAII. Overexpression of a functionally inactive CAII mutant, V143Y, reduced AE1 transport activity by 61 +/- 4% without effect on DRA transport activity (105 +/- 7% transport activity relative to DRA alone). We conclude that cytosolic CAII is required for full DRA-mediated bicarbonate transport. However, DRA differs from other bicarbonate transport proteins because its transport activity is not stimulated by direct interaction with CAII.

Acetazolamide↗

PubMedDate not supplied

Structural and functional characterization of the human NBC3 sodium/bicarbonate co-transporter carboxyl-terminal cytoplasmic domain.

The sodium bicarbonate co-transporter, NBC3, is expressed in a range of tissues including heart, skeletal muscle and kidney, where it modulates intracellular pH and bicarbonate levels. NBC3 has a three-domain structure: 67 kDa N-terminal cytoplasmic domain, 57 kDa membrane domain and an 11 kDa C-terminal cytoplasmic domain (NBC3Ct). The role of C-terminal domains as important regulatory regions is an emerging theme in bicarbonate transporter physiology. This study determined the functional role of human NBC3Ct and characterized its structure using biochemical techniques. The NBC3 C-terminal domain deletion mutant (NBC3DeltaCt) had only 12 +/- 5% of wild-type transport activity. This low activity is attributable to low steady-state levels of NBC3DeltaCt and almost complete retention inside the cell, as assessed by immunoblots and confocal microscopy, suggesting a role of NBC3Ct in cell surface processing. To characterize the structure of NBC3Ct, amino acids 1127-1214 of NBC3 were expressed as a GST fusion protein (GST.NBC3Ct). GST.NBC3Ct was cleaved with PreScission Protease and native NBC3Ct could be purified to 94% homogeneity. Gel permeation chromatography and sedimentation velocity ultracentrifugation of NBC3Ct indicated a Stokes radius of 26 and 30 angstroms, respectively. Shape modelling revealed NBC3Ct as a prolate shape with long and short axes of 19 and 2 nm, respectively. The circular dichroism spectra of NBC3Ct did not change over the pH 6.2-7.8 range, which rules out a large change of secondary structure as a component of pH sensor function. Proteolysis with trypsin and chymotrypsin identified two proteolytically sensitive regions, R1129 and K1183-K1186, which could form protein interaction sites.

Amino Acid Sequence↗

PubMedDate not supplied

Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl-/HCO3- exchanger, AE1.

Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO2 to HCO3- + H+. Bicarbonate transport proteins, which catalyze the transmembrane movement of membrane-impermeant bicarbonate, function in cooperation with CA. Since CA and bicarbonate transporters share the substrate, bicarbonate, we examined whether novel competitive inhibitors of CA also have direct inhibitory effects on bicarbonate transporters. We expressed the human erythrocyte membrane Cl-/HCO3- exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl-/NO3- exchange assays, which were independent of CA activity, and in Cl-/HCO3- exchange assays. Transport was measured by following changes of intracellular [Cl-] and pH, using the intracellular fluorescent reporter dyes 6-methoxy-N-(3-sulfopropyl)quinolinium and 2',7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, respectively. We examined the effect of 16 different carbonic anhydrase inhibitors on AE1 transport activity. Among these 12 were newly-reported compounds; two were clinically used non-steroidal anti-inflammatory drugs (celecoxib and valdecoxib) and two were anti-convulsant drugs (topiramate and zonisamide). Celecoxib and four of the novel compounds significantly inhibited AE1 Cl-/NO3- exchange activity with EC50 values in the range 0.22-2.8 microM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 microM of each compound was only 22-53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl-/HCO3- exchange assays, which depend on functional CA to produce transport substrate, 40 microM celecoxib inhibited AE1 by 62+/-4%. We conclude that some carbonic anhydrase inhibitors, including clinically-used celecoxib, will inhibit bicarbonate transport at clinically-significant concentrations.

Anion Exchange Protein 1, Erythrocyte↗