Using Giant Uni-Lamellar Vesicles to Study Ion Channel Activity and Interactions

Abstract

Unraveling the complicated dynamics of ion channel activity and interactions requires experimental systems in which ion channel activity and distribution can be measured while controlling ion channel concentration and membrane composition, tension and voltage. Many of the constraints imposed by existing techniques, such as planar Black Lipid Membranes (BLM), could potentially be circumvented by using Giant Uni-Lamellar Vesicles (GUVs). To explore this possibility, a method was developed to produce GUVs containing KvAP, a bacterial voltage-gated potassium channel [1]. Protein was first purified, fluorescently labeled and reconstituted into Small Unilamellar Vesicles (SUVs) which were then used to grow GUVs via an electro-formation procedure [2]. Incorporation of the fluorescently-labeled channels was confirmed via confocal microscopy while channel activity was studied with the patch-clamp technique. In parallel, attempts were made to adapt the “whole-cell” patch-clamp geometry for GUVs. While the absence of a cytoskeleton made the “whole-GUV” configuration quite difficult, this geometry is possible and its further development is important since it permits simultaneous control of the voltage inside the GUV and measurement of the entire membrane current. These results confirm the potential of GUVs for ion channel studies, and experiments measuring the effect of membrane tension, composition and curvature on KvAP activity and distribution are currently underway.[1] Ruta et. al., Nature, Volume 422, p180-185 (2003)[2] Montes et. al., Biophysical Journal, Volume 93, p3548-3554 (2007).