Studying Calcium Channels from the Plasma Membrane of Plant Root Cells in Planar Lipid Bilayers

Abstract

Abstract The influx of Ca 2+ to plant root cells through Ca 2+ -permeable ion channels increases their cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) and, thereby, initiates cellular responses to numerous developmental cues and environmental challenges. Three classes of Ca 2+ -permeable channels have been identified in the plasma membrane of plant root cells. These are the hyperpolarisation-activated Ca 2+ channels (HACCs), the depolarisation-activated Ca 2+ channels (DACCs) and the voltage-independent cation channels (VICCs). This chapter describes how the electrophysiological characterisation of these channels in planar lipid bilayers (PLBs) has contributed to our understanding of their transport properties and physiological roles. The electrophysiological characteristics of DACCs and VICCs have been determined following the incorporation of plasma membrane vesicles from cereal roots into PLBs. Two distinct DACCs have been observed. These have been termed the maxi cation channel (or VDCC1) and the root calcium channel ( rca /VDCC2). In a root cell, both these channels would be activated by plasma membrane depolarisation and facilitate a transient Ca 2+ influx. Several distinct VICCs have also been reported. These are generally insensitive to cytosolic regulators. The dominant VICC would be open 60–80% of the time in a typical root cell. Under most physiological conditions this VICC would catalyse a perpetual Ca 2+ influx to root cells. By contrast, HACCs have rarely been recorded when plasma membrane vesicles from plant roots have been incorporated into PLBs. However, HACCs may be formed by annexins, and the electrical properties of the Ca 2+ -permeable ion channels formed by purified plant annexins have been characterised in both artificial and natural membranes. Using data from PLB studies, cation fluxes through both DACCs and VICCs have been modelled. Predictions from these models have been tested using a variety of electrophysiological, radio-tracer and [Ca 2+ ] cyt imaging techniques on intact root cells or their protoplasts. The roles of each class of Ca 2+ channel in generating [Ca 2+ ] cyt signals is under investigation. Recent evidence suggests that HACCs provide the sustained increase in [Ca 2+ ] cyt required for the elongation of root cells and may also raise [Ca 2+ ] cyt in response to pathogens. The DACCs are thought to transduce general stress-related signals that are initiated by plasma membrane depolarisation, and have been implicated in both plant–microbe interactions and in acclimatory responses to low temperatures. The VICCs are thought to maintain the basal Ca 2+ influx required for [Ca 2+ ] cyt homeostasis in unstimulated root cells. Unfortunately, although HACCs and DACCs are relatively selective for Ca 2+ under physiological conditions, VICCs are not. Indeed, VICCs appear to mediate most of the (toxic) Na + and Cs + influx to root cells. Thus, the same system that allows root cells to maintain [Ca 2+ ] cyt homeostasis may also poison them.