pHi and serum regulate AE2-mediated Cl-/HCO3- exchange in CHOP cells of defined transient transfection status.
Anion exchanger (AE) protein-mediated anion exchange contributes to regulation of intracellular pH (pHi), Cl- concentration, and volume in vertebrate cells. We have extended the functional characterization of recombinant AE2-mediated Cl-/HCO3- exchange in single Chinese hamster ovary cells stably transfected with the polyoma large T antigen (CHOP cells) of defined transient transfection status using a novel surface marker coexpression vector. Marker expression and detection had minimal effect on the low endogenous Cl-/HCO3- exchange activity of CHOP cells, whereas coexpression of marker with AE2 elevated CHOP cell Cl-/HCO3- exchange activity 16-fold. Between pHi of 7.3 and 7.8, AE2-mediated flux of proton equivalents was activated > 11-fold by increasingly alkaline pHi without reaching saturation. This activation may be secondary to allosteric effects of pHi on AE2, in parallel with the obligatory increase in substrate intracellular HCO3- concentration. Nominal removal of CO2/HCO3- reduced AE2 activity by 90%. Addition of 10% calf serum slowly activated AE2 activity severalfold. This activation was slowly reversed after serum removal. Surface marker coexpression vectors improve both the efficiency and reliability of studies of recombinant protein function for a wide range of single cell assays in many cell types.
Jiang
L
L
Molecular Medicine Unit, Beth Israel Hospital, Boston, Massachusetts.
Stuart-Tilley
A
A
Parkash
J
J
Alper
S L
SL
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D019011:Antigens, CD7; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism; D000952:Antigens, Polyomavirus Transforming; D017920:Antiporters / Q000378:metabolism* / Q000502:physiology*; D001790:Blood Physiological Phenomena; D016466:CHO Cells / Q000378:metabolism*; D002461:Cell Line, Transformed; D027963:Chloride-Bicarbonate Antiporters; D006224:Cricetinae; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D011522:Protons; D013997:Time Factors; D014162:Transfection
D015703:Antigens, CD; D019011:Antigens, CD7; D000945:Antigens, Differentiation, T-Lymphocyte; D000952:Antigens, Polyomavirus Transforming; D017920:Antiporters; D027963:Chloride-Bicarbonate Antiporters; D005452:Fluoresceins; D011522:Protons; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester