PLANT PHYSIOLOGY , Vol 110, Issue 4 1063-1068, Copyright © 1996 by American Society of Plant Biologists
Laser Microsurgery of Higher Plant Cell Walls Permits Patch-Clamp Access
G. H. Henriksen, A. R. Taylor, C. Brownlee and S. M. Assmann
Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802 (G.H.H., S.M.A)
Plasma membranes of guard cells in epidermal peels of Vicia faba and
Commelina communis can be made accessible to a patch-clamp pipet by
removing a small portion (1-3 [mu]m in diameter) of the guard cell wall
using a microbeam of ultraviolet light generated by a nitrogen laser. Using
this laser microsurgical technique, we have measured channel activity
across plasma membranes of V. faba guard cells in both cell-attached and
isolated patch configurations. Measurements made in the inside-out patch
configuration revealed two distinct K+-selective channels. Major advantages
of the laser microsurgical technique include the avoidance of enzymatic
protoplast isolation, the ability to study cell types that have been
difficult to isolate as protoplasts or for which enzymatic isolation
protocols result in protoplasts not amenable to patch-clamp studies, the
maintenance of positional information in single-channel measurements,
reduced disruption of cell-wall-mediated signaling pathways, and the
ability to investigate intercellular signaling through studies of cells
remaining situated within tissue.
