Advanced patch-clamp techniques and single-channel analysis
| Autor: | Biskup, B, Elzenga, JTM, Homann, U, Thiel, G, Wissing, F, Maathuis, FJM |
|---|---|
| Přispěvatelé: | Elzenga lab |
| Jazyk: | angličtina |
| Rok vydání: | 1999 |
| Předmět: |
Arabidopsis thaliana
K+ CHANNELS ACTION-POTENTIAL CLAMP FABA GUARD-CELLS green fluorescent protein (GFP) macropatch HIGHER-PLANTS POTASSIUM CURRENTS action potential clamp ACCESS RESISTANCE kinetics PLASMA-MEMBRANE ION CHANNELS ARABIDOPSIS-THALIANA endocytosis permeation exocytosis VACUOLAR MEMBRANE |
| Zdroj: | Journal of Experimental Botany, 50, 1037-1054 |
| ISSN: | 0022-0957 |
| Popis: | Much of our knowledge of ion-transport mechanisms in plant cell membranes comes from experiments using voltage-clamp. This technique allows the measurement of ionic currents across the membrane, whilst the voltage is held under experimental control. The patch-clamp technique was developed to study the ionic currents flowing through single channels, but in its whole-cell configuration it is probably the most widely used voltage-clamp technique. This article provides a basic introduction to several advanced patch-clamp techniques, and evaluates methods for single-channel analysis. The initial sections 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 expression of green fluorescent protein (GFP), that allows the experimenter to identify protoplasts from specific cell types for patch-clamp studies. This is followed by descriptions of the perforated-patch technique, which allows 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 vacuoles which are too large for conventional patch-clamp techniques to be effective 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 the patch-clamp technique to investigate endocytotic and exocytotic processes through measurements of capacitance is described. The advanced patch-clamp techniques presented in this article have the potential to broaden the horizons of plant cell electrophysiology and it is expected that many will become the standard laboratory techniques of the future. |
| Databáze: | OpenAIRE |
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