The structure of patch-clamped membranes in high voltage Electron Microscopy

Abstract

The study of single ion channel kinetics in cell electrophysiology has been made possible by the introduction of patch-clamp techniques. Recordings can be made from a patch of membrane either attached to the cell or excised into controlled solutions. Though this technique has been widely used for more than a decade, the structure of the membrane patch and its associated cytoplasmic elements are not known except for recent work done in this laboratory . We have improved this technique to visualize membrane patches using high voltage electron microscope and also identified one class of channels in the patch by immunocytochemistry.The procedure for preserving biological structure in a glass patch pipette is basically same as described earlier as “dry mounting technique” . Briefly, after making a patch, the pipette is removed from the bath and the tip is freeze-fixed in liquid propane. A slight negative pressure is applied to the pipette while freeze-fixing in order to stretch the shape of the patch. Once fixed, the tip is not exposed to temperatures higher than-126° C. A 0.5 cm bit of the pipette tip is broken off, stored in IN2, and then freeze dried between -126° to -80° C under high vaccum (10-6Torr).