A simple method to isolate vacuoles and protoplasts for patch-clamp experiments

Protoplasma 213 (1-2): 39-45 (2000)

Trebacz, K. and Schönknecht, G.

It was not possible to obtain protoplasts or vacuoles from the thallus of the liverwort Conocephalum conicum by applying cell wall degrading enzymes. Therefore, a surgical method was developed to isolate protoplasts and vacuoles. A thallus was plasmolysed and cut. The few protoplasts along the cutting edge that were not destroyed emerged from the edge under deplasmolysis and became thus accessible for a patch-pipette. Whereas under slightly hypoosmolar conditions the emerging protoplast remained largely intact, more hypoosmolar conditions gave rise to isolated vacuoles. This method to isolate protoplasts and vacuoles could also be applied to other plant tissues like leaves of Arabidopsis thaliana. Patch-clamp measurements were performed with isolated vacuoles and excised tonoplast patches. A slow activating vacuolar (SV) channel in C. conicum displayed the characteristic features of higher plant SV channels.