Functional characterization of cell-free expressed Kv1.3 channel using a voltage-sensitive fluorescent dye

Abstract

Using a cell-free expression system, we produced the K_v1.3 protein embedded in one step within detergent micelles. The protein was then purified and relipidated into mixed lipid bilayers. These proteoliposomes held an average of 0.8 protein per liposome. We examined channel forming activity using an oxonol VI fluorescent probe and verified its inhibition using margatoxin and ShK toxins. This assay was automatized and optimized so as to get a Z' statistical factor acceptable for venom fraction screening. We obtained a sensible amount of membrane protein using the cell-free assay, that proved to be active when embedded in liposomes. These findings emphasize the quality of the cell-free produced K_V1.3 proteoliposomes and the usefulness of a fluorescent probe. This method can benefit the field of channel characterization, as well as provide tools for the development of new inhibitors, so as to reinforce our therapeutic arsenal against autoimmune diseases.

Keywords: Cell-free protein synthesis; K(V)1.3 channel; Membrane protein; Oxonol VI; Proteoliposome.