Characterization of Cl- transport in vacuolar membrane vesicles using a Cl(-)-sensitive fluorescent probe: reaction kinetic models for voltage- and concentration-dependence of Cl- flux.
The effects of Cl- concentration and membrane potential (delta psi) on Cl- influx in isolated vesicles of vacuolar membrane (tonoplast) from red beet (Beta vulgaris L.) storage tissue have been characterized using the Cl(-)-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). The initial rate of Cl- transport into the vesicles was enhanced both by the imposition of a positive delta psi and by increases in extravesicular Cl- concentration. The kinetic mechanism underlying these responses was investigated by examining the accuracy with which the data could be described by several transport models. A model based on constant field theory yielded a poor description of the data, but satisfactory fits were generated by pseudo-two-state reaction kinetic models based on classical carrier schemes. Fits were equally good when it was assumed that charge translocation accompanied Cl- entry, or when charge was carried by the unloaded transport system, as long as only a single charge is translocated in each carrier cycle. Expansion of the models to three states enabled description of the Cl- concentration dependence of transport by changes in a single, voltage insensitive rate constant which is tentatively identified with Cl- binding at the external surface of the membrane. The derived value of the dissociation constant between Cl- and the transport system is estimated at between 30 and 52 mM.
Pope
A J
AJ
AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Hertfordshire, United Kingdom.
Jennings
I R
IR
Sanders
D
D
Leigh
R A
RA
D001692:Biological Transport; D002712:Chlorides / Q000378:metabolism*; D004058:Diffusion; D005456:Fluorescent Dyes; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008564:Membrane Potentials; D008954:Models, Biological; D010944:Plants / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*
D002712:Chlorides; D005456:Fluorescent Dyes; D011188:Potassium