Electrophysiological characterization of plant cation channels

Abstract

Cation channels are macromolecular protein pores in bio-membranes that catalyze passive cation influx and efflux (MacKinnon 2004). They do not use ATP energy to transport cations as opposed to active transporters such as pumps and carriers. Since cation channels are not limited by the rate of metabolic interactions, they saturate at much higher concentrations than active transporters and demonstrate low Q10 coefficients (<2.0). Cation channels consist of several transmembrane alpha helices that are also called transmembrane spans or transmembrane domains. These transmembrane domains form a pore region with a selectivity filter that selects cations over anions. Rearrangement of transmembrane domains causes pore opening (activation) or closing (deactivation). Different cation channels have different activators and inhibitors, including membrane voltage (Vm), H?, divalent cations, G-proteins, ATP, cyclic nucleotides, hormones, ROS, amino acids, stretching and gravity. Specific chemical sites in the channel macromolecule are responsible for interactions with activating and inhibiting factors. Some cation channels have fixed anion surface charges outside and/or inside of the channel entry. These charges increase a local cation concentration and modify voltage-dependence, gating and selectivity of the channel (Green and Anderson 1991; Miedema 2002). Protons and divalent cations effectively screen surface charges and cause significant changes in the channel function. Cation channels are sensitive to a range of specific and non-specific blockers. Experiments with blockers, or so-called pharmacological analysis, are necessary for the selection between several groups of channels. For example, tetraethylammonium (TEA?) is a specific blocker of K+?channels that does not affect other cation channels (reviewed by Demidchik et al. 2002a). Blockers can be of natural origin, such as Ca2+, Mg2?, Zn2 or H?, or xenobiotics, for example Ba2?, TEA?, Cs?, lanthanides, dihypropiridines, phenylalkylamines among others. Analysis of blockage provides important information about molecular determinants of the channel (Hille 1994). Cation channels play multiple physiological roles in plants. They catalyze nutritional uptake of N (taken up as NH4?), macronutrient and micronutrient cations such as K+, Ca2+, Na?, Fe2?, Cu2?, Ni2?, Co2?, Zn2??and Mn2?. Cation channels are responsible for the generation of negative resting Vm and action potentials. This is necessary for maintaining structural and functional integrity of the membrane, signaling processes and polarity. Cation channels are directly involved in osmotic balance and regulation of the turgor. This property of cation channels underlies stomata opening and closing. Calciumpermeable cation channels trigger Ca2+?signaling in plants that is involved in tissue and organ coordinated growth, development and stress responses. ROS, amino acids, purines, elicitors, hormones, gravity, different stresses and stretching act through activation of cationic channels. Having multiple physiological roles in plants, cation channels have been a subject of extensive study. Different physiological and molecular techniques have been employed for examination of physiology and structure of cation channels. Unfortunately, the crystal structure of plant cation channels remains unknown. Significant progress has been achieved in our understanding of the molecular nature of cation channels in the last decade (reviewed by Davenport 2002; Demidchik et al. 2002a; White et al. 2002; Vry and Sentenac 2003). Many genes encoding plant cation channels have been identified and characterized molecularly. Analyses of knock-out plants and plants over-expressing K+?channels showed for the first time physiological consequences of the lack or abundance of the particular channel. Nevertheless, molecular studies do not provide information regarding physiological characteristics of cation channels in intact cells. Electrophysiological techniques should be employed to establish channel properties in in vivo conditions. Here, we briefly review some of the most important electrophysiological techniques and provide examples of their use for studies of cation channels in plants.