Biomedical subjects

Deborah Sterling

Publications and source records attributed to Deborah Sterling.

4 recordsLinked to original sources

PubMed2004

The bicarbonate transport metabolon.

To allow cells to control their pH and bicarbonate levels, cells express bicarbonate transport proteins that rapidly and selectively move bicarbonate across the plasma membrane. Physical interactions have been identified between the carbonic anhydrase isoform, CAII, and the erythrocyte membrane Cl- /HCO3(-) anion exchanger, AE1, mediated by an acidic motif in the AE1 C-terminus. We have found that the presence of CAII attached to AE1 accelerates AE1 HCO3(-) transport activity, as AE1 moves bicarbonate either into or out of the cell. In efflux mode the presence of CAII attached to AE1 will increase the local concentration of bicarbonate at the AE1 transport site. As bicarbonate is transported into the cell by AE1, the presence of CAII on the cytosolic surface accelerates transport by consumption of bicarbonate, thereby maximizing the transmembrane bicarbonate concentration gradient experienced by the AE1 molecule. Functional and physical interactions also occur between CAII and Na+/HCO3(-) co-transporter isoforms NBC1 and NBC3. All examined bicarbonate transport proteins, except the DRA (SLC26A3) Cl-/HCO3(-) exchange protein, have a consensus CAII binding site in their cytoplasmic C-terminus. Interestingly, CAII does not bind DRA. CAIV is anchored to the extracellular surface of cells via a glycosylphosphatidyl inositol linkage. We have identified extracellular regions of AE1 and NBC1 that directly interact with CAIV, to form a physical complex between the proteins. In summary, bicarbonate transporters directly interact with the CAII and CAIV carbonic anhydrases to increase the transmembrane bicarbonate flux. The complex of a bicarbonate transporter with carbonic anhydrase forms a "Bicarbonate Transport Metabolon."

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

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↗