Single channel recordings of reconstituted ion channel proteins: an improved technique

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Abstract
Single channel recording of reconstituted ion channels is possible by patch clamp measurements of giant liposomes formed by dehydration-rehydration of lipid films. This “hydration technique” consists of carefully controlled dehydration of a suspension of small vesicles followed by rehydration of the residue resulting in formation of large liposomes. Patch pipettes can be attached to the liposome surface, yielding stable, high resistance seals between membranes and glass pipettes. This method allows the study of the properties of reconstituted ion channels from different tissues. The hydration technique was used to characterize the reconstituted K+-channel of sarcoplasmic reticulum from rabbit skeletal muscle. In a solution of 100 mM KCl, the sarcoplasmic reticulum K+-channel studied displays a conductance γK + of 145 pS. The single channel conductance in 100 mM Rb+ and Na+ is γRb + = 98 pS and γNa + = 65 pS respectively. A concentration of 0.5 mM decamethonium causes a flickering channel block. These properties are in good agreement with the ones found in sarcoplasmic reticulum K+-channels characterized by other methods. Other ion channels have also been reconstituted and studied by this technique. This improved method is compared with previous approaches and its applicability for the characterization of reconstituted ion channel proteins is discussed.