Expressing and characterizing mechanosensitive channels in Xenopus oocytes

Abstract

The oocytes of the African clawed frog (Xenopus laevis) comprise one of the most widely used membrane protein expression systems. While frequently used for studies of transporters and ion channels, the application of this system to the study of mechanosensitive ion channels has been overlooked, perhaps due to a relative abundance of native expression systems. Recent advances, however, have illustrated the advantages of the oocyte system for studying plant and bacterial mechanosensitive channels. Here we describe in detail the methods used for heterologous expression and characterization of bacterial and plant mechanosensitive channels in Xenopus oocytes.

Fig. 1

Image of a custom-made gridded petri dish. Plastic mesh is glued to the bottom of a 35-mm dish. Each cell of the grid accommodates a single oocyte

Fig. 2

Setup for oocyte injection. A manually driven micropipettor (on the right) should be capable of reproducibly and accurately injecting 50 nl of solution into a single oocyte. Inset: RNAse-free water is sucked into the pipette from a drop in a petri dish

Fig. 3

Diagram of the experimental setup and typical results. (a) Schematic representation of the patch-clamp setup. (b) Illustration of a membrane patch with applied suction and a single MSC opening in response to increased membrane tension. (c) Representative trace showing five opening and closing MSL10 channels. A 50 mV command potential and a −120 mmHg symmetric pressure ramp were used. The trace was sampled at 20 kHz with a 5 kHz low-pass Bessel filter (Axopatch 200B), then filtered at 1 kHz and exported as graphics via Clampfit software (Molecular Devices)

Fig. 4

Experimental setup, shown from two angles. The glass patch pipette (1) is located above the ground electrode (2) immersed into a petri dish filled with bath buffer (3). The microscope objective is located underneath. Also shown are the pressure-clamp headstage (4), the pipette holder (5), the common ground point (6), and the inverted microscope condenser (7)

Fig. 5

A laser-scanning confocal micrograph of the fluorescent signal from the periphery of an oocyte RNA 7 days after injection with MscS-GFP cRNA. An oocyte in ND96 buffer was placed onto a glass slide with concaved bottom and covered with a coverslip. Under bright field the lens was focused on the edge of the oocyte, then the confocal scan with GFP filters (488/510 nm) setup was performed. Inset: oocyte image in bright field