Avoiding the formation of vesicles by patch excision from Xenopus oocytes

Abstract

The patch-clamp technique is well-established to investigate the function of ion channels. Several patch configurations have been described, including the inside-out patch configuration providing the unique advantage of having free access to the patch from the cytosolic side. An inside-out patch is predominantly built from a cell-attached patch by pulling the patch pipette back. However, when using pipettes with high resistance (>8 MΩ), often a vesicle is formed instead of the desired inside-out patch, preventing proper recording of ion currents. Using quartz pipettes with high resistance we studied the benefit of a simple alternative excision manoeuvre that significantly enhances the efficiency to obtain an inside-out patch from Xenopus oocytes. We show that the formation of vesicles depends on the direction of patch excision: after a cell-attached patch has formed, pushing the patch pipette first into the depth of the oocyte and exposing the patch only then to the bath solution generated a success rate of 89% (16 out of 18) for a proper inside-out patch, as evaluated by the current flowing through HCN2 channels which were heterologously expressed in the oocytes. In contrast, with the same type of pipettes and oocytes only 22% (4 out of 18) of the patches developed HCN2 currents when pulling the pipette in the backward direction as usual. The difference in the success rate favours the idea to use "pushed inside-out patches" instead of "pulled inside-out patches" when studying ion channels expressed in Xenopus oocytes.