RCDM/Res/Audit/1036/12/0305.

36 Cl Ϫ efflux at pHo 7.4 and acid shifted the pH o vs. activity profile of wild-type AE2, suggesting that His residues might be involved in pH sensing. Single His mutants of AE2 were generated and expressed in oocytes. Although mutation of H1029 to Ala severely reduced transport and surface expression, other individual His mutants exhibited wild-type or near-wild-type levels of Cl Ϫ transport activity with retention of pHo sensitivity. In contrast to the effects of DEPC on wild-type AE2, pH o sensitivity was significantly alkaline shifted for mutants H1144Y and H1145A and the triple mutants H846/H849/H1145A and H846/H849/H1160A. Although all functional mutants retained sensitivity to pH i, pHi sensitivity was enhanced for AE2 H1145A. The simultaneous mutation of five or more His residues, however, greatly decreased basal AE2 activity, consistent with the inhibitory effects of DEPC modification. The results show that multiple TMD His residues contribute to basal AE2 activity and its sensitivity to pH i and pHo. pH regulation; histidine residues; Cl Ϫ /HCO 3 Ϫ exchange THE SLC4 BICARBONATE TRANSPORTER gene superfamily includes the anion exchanger gene family of Na ϩ -independent Cl Ϫ / HCO 3 Ϫ exchangers, AE1, AE2, and AE3. In mammalian cells, these anion exchangers are involved in the control of intracellular Cl Ϫ , pH, and cell volume (2). The anion exchanger polypeptides share a highly conserved COOH-terminal transmembrane domain (TMD) of ϳ500 amino acids (aa) with a short COOH-terminal cytoplasmic tail. The TMD is preceded by a less extensively conserved ϳ400-aa (AE1) to ϳ700-aa (AE2 and AE3) NH 2 -terminal cytoplasmic tail (1). The COOH-terminal TMD can mediate anion exchange in the absence of the NH 2 -terminal cytoplasmic domain (12, 21, 37), but the physiological regulation of AE2 transport activity by pH requires the NH 2 -terminal cytoplasmic domain.The anion exchanger polypeptides are expressed in tissueand cell-specific patterns and differ in their acute response to a change of pH. Structural elements contributing to this pH sensitivity have been localized to the COOH TMD and NH 2 -terminal cytoplasmic domain (37, 46). AE1-mediated Cl Ϫ / HCO 3 Ϫ and Cl Ϫ /Cl Ϫ exchange in erythrocytes (10) and Xenopus oocytes exhibits a broad pH vs. activity profile (17,46). In contrast, Na ϩ -independent Cl Ϫ /HCO 3 Ϫ exchange in nonerythroid cells in culture is often characterized by strong pH sensitivity (24,35) and is most likely mediated by AE2 and AE3 gene products and/or by polypeptides of the SLC26 gene family. A high sensitivity to intracellular pH (pH i ) and/or extracellular pH (pH o ) has been observed for recombinant AE2 and AE3 expressed in tissue culture cells (19,23) and Xenopus oocytes (37, 46). AE2 Ϫ/Ϫ mice are achlorhydric and fail to survive weaning (11). AE3 Ϫ/Ϫ mice display enhanced susceptibility to pharmacologically induced seizures (14).Structure-function studies have localized to the AE2 TMD a putative "pH sensor" that confers sensitivity of the anion transporter to changes in pH o and pH i...

HighlightsEVD is associated with life-threatening electrolyte imbalance and organ dysfunction.Clinical staging/early warning scores can be useful EVD prognostic indicators.Enhanced protocolized care is a blueprint for future treatment in low-resource settings.

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