A study of Functional ion Transport Using Tethered Membranes

Abstract

Tethered membranes (t-BLMs) are lipid bilayers that are chemically attached to a solid surface. In the present studies the surface is gold on polycarbonate and the attachment chemistry, sulphur to gold. t-BLMs are more stable making possible longer and more complex experiments of model and biomembranes. t-BLMs have been used here to study the ion conduction properties of both native and mutant forms of four ion channels families including: the Na+/H+ co-transport protein; the Voltage Dependent Anion Channel transporter (VDAC); the Chloride Intracellular Channel Family (CLIC); the Mechano-Sensitive Channels (MscL) and (MscS). Of the four channel types CLIC spontaneously inserts into the t-BLM, the MscL and MscS were transferred via a detergent rinse, and the VDAC and co-transporter families were transferred from proteoliposomes. A novel mixed tethered/mobile t-BLM system was used to overcome previous limitations impeding the insertion of ion channels into tethered membranes and overcoming the poor electrical seals which previously prevented the observation of the ion conductance of the inserted ion channels. The successful approach employs a mix of tethered and mobile lipid species. Tether densities in the range 10%-40% were used, permitting the incorporation of intrinsic membrane proteins with molecular weights in the range 15-50kDa into the untethered free lipid patches. The results of these studies will be compared with results for the same proteins studied using classical patch clamp techniques.