AJP - Renal Physiology, Vol 252, Issue 5 844-F855, Copyright © 1987 by American Physiological Society
Cl-/HCO-3 antiporter in red cell ghosts: a kinetic assessment with fluorescent probes
R. D. London, M. S. Lipkowitz and R. G. Abramson
The pH sensitive fluorescent probe acridine orange and membrane
potential-sensitive fluorescent probe acridine orange and membrane
potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine
iodide were used to evaluate the Cl-/HCO-3 antiporter and proton and
potassium conductances, respectively, in human red blood cell ghosts.
Acidic, chloride-loaded ghosts alkalinized rapidly in pH 8.5 chloride-free
media. Alkalinization could not be ascribed to conductive proton efflux
with either depolarizing potassium influx or chloride efflux.
Alkalinization was consequent to flux on the Cl-/HCO-3 antiporter: this
process displayed saturation kinetics, competitive inhibition by external
chloride, and inhibition by
4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. The mean Kms for
internal chloride, external bicarbonate, and external chloride were 2.19,
0.24, and 0.44 mM, respectively. These studies confirm both the asymmetry
of this carrier and the high affinity for external HCO-3; however, the
affinities for internal and external chloride are significantly greater
than prior estimates. The Km for internal chloride (2.19 mM) was
considerably lower than levels previously reported (20-65 mM) unless
external (trans) chloride was raised above 2 mM. The present studies thus
demonstrate and emphasize the critical importance of trans substrate
concentration in assessing the kinetics of a carrier whose mobility is
faster in the loaded than in the unloaded state.
