Efficient methods for the analysis of ion channel proteins

Abstract

Methods combining the creation of small bilayer membranes with the fusion of native cell membrane vesicles into those bilayer membranes. By placing a voltage gradient across the lipid bilayer and recording the transmembrane current, the activity of some membrane proteins, such as ion channels, can be analyzed. The methods permit access to a large variety of membrane proteins. The native vesicles may be derived from a variety of cells and intracellular organelles, and as the native cell membrane vesicles may be stored at low temperatures over long periods, the methods reduce the need to maintain cell cultures or obtain primary cells. Fluorescence measurements also may be performed on the bilayer membranes.

I claim:  
     
         1 . A method of analyzing membrane proteins, comprising: 
 positioning a vesicle on a substantially insulating carrier;    forming a lipid membrane on the carrier;    forming one or more smaller vesicles that contain at least one membrane protein, where the smaller vesicles are derived from a cell membrane or organelle membrane;    incorporating the membrane protein in the lipid membrane by fusing the small vesicles with the lipid membrane; and    analyzing the membrane protein.    
     
     
         2 . The method of  claim 1 , where the step of analyzing comprises electrical analysis of the membrane protein.  
     
     
         3 . The method of  claim 1 , where the step of analyzing comprises optical analysis of the membrane protein.  
     
     
         4 . The method of  claim 1 , where the carrier separates two fluid compartments, and includes a small aperture between the two fluid compartments.  
     
     
         5 . The method of  claim 4 , where the vesicle is positioned on the carrier across the aperture by generating a voltage difference between the fluid compartments.  
     
     
         6 . The method of  claim 1 , where the carrier is in contact on one side with at least one fluid compartment that is confined to such a small area that the lipid membrane will form at a particular position.  
     
     
         7 . The method of  claim 1 , where one carrier comprises a plurality of distinct sites for analyzing membrane proteins.  
     
     
         8 . The method of  claim 1 , where a surface of the carrier is chemically or physically modified so as to enhance the formation of a high electrical seal with the lipid bilayer.  
     
     
         9 . The method of  claim 1 , where the membrane protein is analyzed using voltage-clamp techniques.  
     
     
         10 . The method of  claim 1 , where the carrier further comprises a conductive material, and where the step of analyzing comprises analyzing the membrane protein using impedance spectroscopy.  
     
     
         11 . The method of  claim 1 , farther comprising orienting the lipid bilayer in the focal plane of an objective lens.  
     
     
         12 . The method of  claim 3 , where the optical analysis comprises confocal observation.  
     
     
         13 . The method of  claim 1 , where the smaller vesicles are derived by endocytosis, exocytosis, or synaptic vesicle release.  
     
     
         14 . The method of  claim 1 , where the smaller vesicles are derived by mechanical destruction of a cell or chemical destruction of a cytoskeleton.  
     
     
         15 . The method of  claim 1 , further comprising sorting the smaller vesicles according to origin and membrane protein content prior to incorporating them into the lipid bilayer.  
     
     
         16 . The method of  claim 1 , where the step of incorporating is promoted by the presence of polyvalent cations, fusogenic proteins, or both.  
     
     
         17 . The method of  claim 1 , where the step of incorporating is promoted by the application of osmotic stress on the lipid bilayer or the smaller vesicles or both.  
     
     
         18 . A method of analyzing membrane proteins, comprising: 
 positioning a vesicle on a substantially insulating carrier;    forming a lipid membrane on the carrier;    forming one or more membrane fractions that contain at least one membrane protein, where the membrane fractions are derived by solubilization of a cell membrane or organelle membrane;    incorporating the membrane protein in the lipid membrane by integrating the membrane fractions into the lipid membrane; and    analyzing the membrane protein.