Institut für Physiologie II, Jena University Hospital, Jena 07743, Germany.
Institut für Physiologie II, Jena University Hospital, Jena 07743, Germany.
J Neurosci Methods. 2014 Mar 30;225:29-31. doi: 10.1016/j.jneumeth.2014.01.005. Epub 2014 Jan 20.
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.
膜片钳技术是研究离子通道功能的成熟方法。已经描述了几种膜片钳构型，包括内面向外膜片钳构型，该构型具有从细胞质侧自由进入膜片的独特优势。内面向外膜片主要由细胞贴附膜片通过向后拉动膜片管来构建。然而，当使用高电阻 (>8 MΩ) 的微电极时，通常会形成囊泡而不是所需的内面向外膜片，从而阻止离子电流的正确记录。
使用高电阻的石英微电极，我们研究了一种简单替代切除操作的益处，该操作可显著提高从非洲爪蟾卵母细胞中获得内面向外膜片的效率。
我们表明，囊泡的形成取决于膜片切除的方向：在形成细胞贴附膜片后，首先将膜片管推向卵母细胞的深处，然后仅将膜片暴露于浴液中，这会生成 89%（16/18）的成功率，适用于通过在卵母细胞中异源表达的 HCN2 通道流过的电流来评估的正确内面向外膜片。
相比之下，使用相同类型的微电极和卵母细胞，当按照常规向后拉动微电极时，只有 22%（4/18）的膜片会产生 HCN2 电流。
成功率的差异表明，在研究在非洲爪蟾卵母细胞中表达的离子通道时，使用“推进式内面向外膜片”而不是“拉动式内面向外膜片”更有利。
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