LASER MICROSURGERY OF HIGHER-PLANT CELL-WALLS PERMITS PATCH-CLAMP ACCESS

Plasma membranes of guard cells in epidermal peels of Vicia faba and C ommelina communis can be made accessible to a patch-clamp pipet by rem oving a small portion (1-3 mu m in diameter) of the guard cell wall us ing a microbeam of ultraviolet light generated by a nitrogen laser. Us ing this laser microsurgical technique, we have measured channel activ ity across plasma membranes of V. faba guard cells in both cell-attach ed and isolated patch configurations. Measurements made in the inside- out patch configuration revealed two distinct K+-selective channels. M ajor advantages of the laser microsurgical technique include the avoid ance of enzymatic protoplast isolation, the ability to study cell type s that have been difficult to isolate as protoplasts or for which enzy matic isolation protocols result in protoplasts not amenable to patch- clamp studies, the maintenance of positional information in single-cha nnel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling throu gh studies of cells remaining situated within tissue.