Biochemical Journal

Research article

Transport activity of chimaeric AE2-AE3 chloride/bicarbonate anion exchange proteins

Jocelyne FUJINAGA, Frederick B. LOISELLE, Joseph R. CASEY

Abstract

Chloride/bicarbonate anion exchangers (AEs), found in the plasma membrane of most mammalian cells, are involved in pH regulation and bicarbonate metabolism. Although AE2 and AE3 are highly similar in sequence, AE2-transport activity was 10-fold higher than AE3 (41 versus 4 mM · min−1 respectively), when expressed by transient transfection of HEK-293 cells. AE2–AE3 chimaeras were constructed to define the region responsible for differences in transport activity. The level of AE2 expression was approx. 30% higher than that of AE3. Processing to the cell surface, studied by chemical labelling and confocal microscopy, showed that AE2 is processed to the cell surface approx. 8-fold more efficiently than AE3. The efficiency of cell-surface processing was dependent on the cytoplasmic domain, since the AE2 domain conferred efficient processing upon the AE3 membrane domain, with a predominant role for amino acids 322–677 of AE2. AE2 that was expressed in HEK-293 cells was glycosylated, but little of AE3 was. However, AE2 expressed in the presence of the glycosylation inhibitor, tunicamycin, was not glycosylated, yet retained 85 ± 8% of anion-transport activity. Therefore glycosylation has little, if any, role in the cell-surface processing or activity of AE2 or AE3. We conclude that the low anion-transport activity of AE3 in HEK-293 cells is due to low level processing to the plasma membrane, possibly owing to protein interactions with the AE3 cytoplasmic domain.

  • pH regulation
  • bicarbonate transport
  • cell-surface processing

Footnotes

  • Abbreviations used: AE, anion exchanger; BCECF-AM, 2′,7′-bis-2-carboxyethyl)-(5 and 6)-carboxyfluorescein acetoxymethyl ester; DMEM, Dulbecco's modified Eagle's medium; ECL(R), enhanced chemiluminescence; eGFP, enhanced green fluorescent protein; HA, haemagglutinin; HRP, horseradish peroxidase; TLCK, N-tosyl-l-lysine chloromethyl ketone; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone; sulpho-NHS-SS-biotin, sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate.