Patch-Clamping and Other Molecular Approaches for the Study of Plasma Membrane Transporters Demystified

Abstract

As the boundary between the inside and the outside of the cell, the plasma membrane is one of the most important structures that cells use to regulate their interna1 composition and activities. Proteins embedded in the plasma membrane control which solutes are accumulated and which are excluded; their activity creates gradients of solutes across the membrane. This process is important for many aspects of plant growth and development, including cell expansion, mineral nutrition, long-distance transport of assimilated carbon and nitrogen, and cellular and whole-plant responses to environmental signals.Electrophysiology is the study of the transport of charged solutes across membranes and the proteins that carry out this transport. Electrophysiological studies have been performed on plants for at least 50 years. In the last 15 years the highest-resolution electrophysiological method, the patch-clamp technique, has been used extensively in plants. As a result, many plasma membrane transport activities, especially ion channel activities, have been characterized in detail. These activities can now be linked to structural information of proteins and, as a result, we can begin to understand the mechanisms of transport at the molecular level.Neither traditional genetic approaches nor Iibrary screening using heterologous probes has been useful for obtaining transport protein genes. A unique set of molecular tools, including expression cloning in yeast, heterologous expression in Xenopus laevis oocytes, reverse genetics to obtain knockout mutants, and high-resolution electrophysiological assays, is being used to begin to create a full description of transport protein structure, function, expression pattern, targeting, and regulation.This Update provides a practical guide for nonelectrophysiologists to understand the results from patch-clamp experiments. This is important because the patch-clamp technique will have additional applications in the future as knockout mutants and heterologous expression become more common. Furthermore, patch-clamp results are difficult to understand for many scientists who have not had personal experience with the technique. The current molecular approaches mentioned above, which are aiding in the rapid progression of the cloning of cDNAs of transport proteins, are also presented here to provide a glimpse into the cutting edge of electrophysiology, where mutant trans-* E-rnail wardauni-tuebingen.de; fax 049-07071-29-3287. porters are selected for desired characteristics on a functional basis and where knockout/ replacement studies will finally be possible in plants.

A vacuolar phosphate transporter essential for phosphate homeostasis inArabidopsis

“…6 G and H). Among the three classes of transporter proteins (pumps, carriers, and ion channels), only ion channels can be measured in an excised patch of membrane (30). The VPT1 single-channel characteristics are comparable to those of vacuolar anion channels described previously (31,32), and, notably, the VPT1 also has a flickering behavior and a large conductance (Fig.…”

Processes Modulating Calcium Distribution in Citrus Leaves. An Investigation Using X-Ray Microanalysis with Strontium as a Tracer

“…Further, pulse-chase approaches could be used to study secondary redistribution processes that might occur after initial uptake into cells. The technique is relatively noninvasive and so has the potential to identify sites of high rates of uptake or redistribution that can then be subsequently investigated using cellspecific techniques such as ion-selective microelectrodes (Miller et al, 2001), single-cell sampling (Tomos and Leigh, 1999), patch clamp (Ward, 1997), uptake into isolated protoplasts (Dietz et al, 1992), or microarray gene expression analysis (Leonhardt et al, 2004). Such cell-based approaches have already been used to demonstrate differences in gene expression and components of signaling pathways between stomatal guard cells and mesophyll cells (Sutton et al, 2000;Leonhardt et al, 2004) and may be useful in resolving mechanisms underlying the intercellular compartmentation of calcium.…”

Functional Properties of the Arabidopsis Peptide Transporters AtPTR1 and AtPTR5

“…Membrane potentials in plants are usually between Ϫ100 and Ϫ150 mV, although potentials as low as Ϫ250 mV were also measured (29,30). Consistently, the dipeptide-induced currents never saturated at the potentials applied (Յ Ϫ140 mV), which is a common feature among many transporters from plants.…”