Characterization of Cl− transport in vacuolar membrane vesicles using a Cl−-sensitive fluorescent probe: Reaction kinetic models for voltageand concentration-dependence of Cl− flux

The effects of Cl− concentration and membrane potential (Δψ) 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 ‡ψ 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 s 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 52mm.