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

Abstract

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.

Ion channel gating in plants: Physiological implications and integration for stomatal function

“…This permeability sequence is characteristic of several anion channels derived from vacuolar membranes (cf. Hedrich, Flugge & Fernandez, 1986;Pope & Leigh, 1988;Pope et al, 1990;. Most striking, however, is the voltage dependence of the channel.…”

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

“…These experiments demonstrated that CI-could be electrically driven into the vesicle interior, and also suggested that C1-may enter via a saturable uniport whether driven by A 0 or by a C1-concentration gradient alone ( Fig. 4; see also Pope et al 1990). By imposing a range of K+-diffusion potentials, at a fixed external C1-concentration, flux voltage (F-V) curves could be generated (e.g.…”

“…These are directly analgous to current-voltage (I-V) curves which are a major tool in electrophysiological studies. In a separate paper, we have used such data to investigate the kinetic mechanisms underlying the responses of C1-flux to changes in both A 0 and C1-concentration (Pope et al 1990). At a C1-concentration of 10 mol.m -3, influx was most sensitive to AO at values between +25 mV and + 55 mV (Fig.…”