Advanced patch-clamp techniques and single-channel analysis

Much of our knowledge of ion-transport mechanisms in plant cell membranes c omes from experiments using voltage-clamp. This technique allows the measur ement of ionic currents across the membrane, whilst the voltage is held und er experimental control. The patch-clamp technique was developed to study t he ionic currents flowing through single channels, but in its whole-cell co nfiguration it is probably the most widely used voltage-clamp technique. Th is article provides a basic introduction to several advanced patch-clamp te chniques, and evaluates methods for single-channel analysis. The initial se ctions describe the fabrication and use of perfusion pipettes, which enable the experimenter to manipulate the composition of solutions on both sides of the membrane, and a molecular-genetic approach, based on cell-specific e xpression of green fluorescent protein (GFP), that allows the experimenter to identify protoplasts from specific cell types for patch-clamp studies. T his is followed by descriptions of the perforated-patch technique, which al lows ionic currents to be determined in cells whose cytoplasm is relatively undisturbed and whose endogenous signal transduction cascades are intact, and the use of 'macropatches' for studying ion-transport processes in vacuo les which are too large for conventional patch-clamp techniques to be effec tive and are unsuitable for impaling electrodes. Methods for modelling the kinetics and permeation of ion channels using single-channel recordings are presented. How the ionic currents underlying an action potential (AP) can be identified using the AP-clamp method is discussed. Finally, the use of t he patch-clamp technique to investigate endocytotic and exocytotic processe s through measurements of capacitance is described. The advanced patch-clam p techniques presented in this article have the potential to broaden the ho rizons of plant cell electrophysiology and it is expected that many will be come the standard laboratory techniques of the future.