We determined EC50 and relative Vmax for Cl- in an Cl-/H+ exchange assay for both transport directions and found a slight asymmetry in transporter kinetics.
Background and Assay Description:
Assays for Chloride influx and efflux have been performed using different chloride concentrations ranging from 1 mM to 300 mM on the same sensor.
The vesicles have been equilibrated in Cl- free solution during sensor preparation. For the Cl- influx assay, Clc is activated by a chloride concentration jump. When the Cl- efflux assay is performed, the sensor is first flushed with Cl- containing solution to pre-load the vesicles with chloride. Then a chloride is removed by rinsing the sensor with a chloride free solution (negative concentration jump).
By normalizing each dataset to the peak current obtained for 300 mM chloride, datasets obtained from multiple sensors can be averaged.
Impact of the data:
We have determined EC50 and relative Vmax for Cl- influx and efflux, showing that Influx has a transport rate three times faster than efflux, but a lower apparent affinity.
Due to the experimental design, the slight asymmetry of transporter kinetics may be a result of small differences in the experimental conditions: In the efflux assay, loading of the vesicles with chloride during the experiment generates a membrane potential which might affect the kinetics of Cl- efflux.
Author: Nanion Technologies, in collaboration with Merritt Maduke, Stanford University School of Medicine
Target: EriC, ClcA, ClC-ecl
Family / Type: Chloride Carrier/Channel Family (ClCF, TCDB: 2.A.49)
Mode of transport: H+/2Cl- exchange
Organism: Escherichia coli
Sample: Proteoliposomes from purified protein, LPR 20
Expression: Recombinant overexpression in E.coli
Platform: SURFE²R N1