Dynamic Study of Ceramide Channels with a Microfluidic System

Abstract

A novel microfluidic system enabling effective and robust dynamic perfusion of reagents directly to an on-chip phospholipid bilayer membrane was developed to study membrane channels. Here we report its application to the study of the dynamics of ceramide channels. The microfluidic system consists of a planar phospholipid membrane (PPM) formed across a 60-100 µm diameter aperture in a polyvinylidene chloride film formed within a thermoplastic polycarbonate substrate containing a microfabricated fluidic network. The volume of the network before the aperture is 2.3µL and the chamber beneath the aperture is 20nL. Constant pumping rates up to 5µL/min can be used without disrupting the fragile membranes and so the solution bathing the membrane can be completely replaced in less than 1 minute. Transmembrane current is monitored continuously under voltage-clamp conditions. For ceramide channel experiments, the membrane-forming solution was 5 mg diphytanoyl PC, 5 mg asolectin, 0.5 mg cholesterol, 0.13 mg C16-ceramide in 1 ml of hexanol/hexadecane (v/v 10:1). The sphingolipid, ceramide, self-assembles into large channels in phospholipid membranes and is known to play an important role in apoptosis. In our studies, channel dynamics were observed by inducing disassembly and reassembly of ceramide channels formed spontaneously in the PPMs. Perfusion with La3+ resulted in rapid disassembly. Channel reassembly occurred after washing away the La3+ using EDTA. Multiple cycles of disassembly and reassembly could be performed on the same membrane. Similar studies were performed by perfusing in Bcl-xL, a potent apoptosis inhibitor. Bcl-xL disassembles the channels rapidly but the reassembly is slow. The results are consistent with ceramide channels existing in equilibrium with ceramide monomers or aggregates in the membrane and this equilibrium can be shifted by interaction with chemical agents. Supported by grants from NIH (R21EB009485) and NSF (MCB-0641208).