A laser microsurgical method of cell wall removal allows detection of large-conductance ion channels in the guard cell plasma membrane

Application of patch clamp techniques to higher-plant cells has been subjec t to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wa ll. Because the wall is an integral component of plant cells, and because c ell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell rema ins intact within its tissue environment. "Laser-assisted" patch clamping r eveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch damp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production o f individual plant protoplasts isolated from their tissue environment by en zymatic digestion of the cell wall. Given the Large conductances of the cha nnels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and elect rical signalling in guard cells.