A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters. Academic Article uri icon

Overview

abstract

  • The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl(-) channels or transporters is described. The assay is based on the measure of the efflux rate of Cl(-) from proteoliposomes following the addition of the K(+) ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H(+)/Cl(-) exchanger, as a model system. The efflux assay can be utilized to study any Cl(-) conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

publication date

  • April 20, 2015

Research

keywords

  • Chloride Channels
  • Chlorides
  • Ion Pumps
  • Proteolipids

Identity

PubMed Central ID

  • PMC4541587

Scopus Document Identifier

  • 84941254171

Digital Object Identifier (DOI)

  • 10.3791/52369

PubMed ID

  • 25938223

Additional Document Info

issue

  • 98