AJP - Cell Physiology, Vol 251, Issue 5 713-C720, Copyright © 1986 by American Physiological Society
Solubilization and reconstitution of a chloride transporter from tracheal apical membrane
W. P. Dubinsky and L. B. Monti
Electrogenic Cl- transport in bovine tracheal membrane vesicles was studied
by measuring the kinetics of Cl- efflux. Efflux from KCl-loaded vesicles
was assayed as the appearance of Cl- in the medium using an anion-specific
electrode. Maximal sensitivity was obtained by passage of the vesicles
through a Sephadex column immediately before the assay to remove external
Cl-. Two components of Cl- transport could be detected. Vesicles exhibited
a characteristic spontaneous Cl- efflux that appears to be limited by the
cation permeability of the membrane. Addition of the K+-conducting
ionophore to increase selectively the permeability to K+ resulted in a
stimulation of Cl- efflux. The electrically neutral ionophore nigericin
caused only a slight transient increase in Cl- efflux. The
valinomycin-dependent flux may be assumed to represent flux through a
parallel conductive pathway. Valinomycin-dependent electrogenic Cl- flux
was 240 +/- 36 nmol X mg protein-1 X min-1 (n = 4). Soluble extracts
obtained with the nonionic detergent octyl glucoside were reconstituted by
the freeze-thaw procedure. The reconstituted vesicles exhibited a number of
similarities to the native vesicles. Efflux was maximally stimulated with
the ionophore valinomycin in the presence of an outwardly directed K+
gradient. Nigericin had no effect on efflux, suggesting the reconstituted
transporter is a conductive rather than exchange mechanism. The initial
rate of the valinomycin-dependent component was 896 +/- 63 nmol X mg
protein-1 X min-1 (n = 5), representing approximately a fourfold increase
in specific activity compared with the native membrane vesicle.
