Techniques for studying ion channels: an introductionTester, Mark
doi: 10.1093/jxb/48.Special_Issue.353pmid: 21245214
In this Introduction to the Special Issue on Ion Channels in Plants, the techniques used to study ion channels are reviewed. In particular, the basic approaches used for the electrophysiological study of ion channels are explained, from voltage clamping membranes and observing current changes over time, to the construction of current-voltage relations and the use of tail current protocols to determine channel selectivity. This is done for whole-cell and single channel records.
Gating and permeation models of plant channelsHansen, Ulf-Peter; Keunecke, Maike; Blunck, Rikard
doi: 10.1093/jxb/48.Special_Issue.365pmid: 21245216
Models of different level are applied to plant membrane transport. For the evaluation of I/V curves, models based on enzyme kinetics, limitation by diffusion and the ion well are described. Physical models of ion-ion interaction deal with the Woodhull model, the multi-site single-file ion pore, the ion-ion ion-water interaction model and the effect of screening. The discussion of channel gating starts with pre-patch evidence of gating and the biological importance of gating. State models play a dominant role in the analysis of patch clamp records. Gating may strongly interfere with permeation models. With respect to this, the limits of temporal resolution are of great importance. The effects of Na+ and Tl+ are examples of the influence of fast gating on modelling and the problems of its detection. The highest level of modelling is achieved when the knowledge about the structure of channels is employed for the modelling of gating. Major topics in this field are blockade by ions and phosphorylation.
Anion channel activity in the Chara plasma membrane: co-operative subunit phenomena and a modelMcCulloch, Stephen R.; Laver, Derek R.; Walker, N. Alan
doi: 10.1093/jxb/48.Special_Issue.383pmid: 21245217
Anion channel activity in the plasma membrane of internodal cells of Chara australis has been studied using the patch clamp method and the records analysed with a program based on the Hidden Markov Model. The activity was variable in time, and often showed very noisy periods. At other times there were one or more of five types of channel-like activity. These are believed, like the noise, to arise from the same anion-conducting mechanism in the membrane.The channel-like activity shows many current levels, the actual levels varying on a time-scale of the order of seconds. The records show non-independent transitions between levels, and thus the activity is not the result of the patch containing many independent channels. The currents often move sequentially between adjacent levels. The current levels are at times more closely spaced as the magnitude of the current increases.Underlying this sequential transition scheme it is found that current and conductance frequently show levels that are integer multiples of a unit. Typically about 10 levels occur, a typical transition scheme being (in units) 0↔4↔7↔9. It is shown that it is possible to model such sequential transition schemes with underlying units by considering a new model based on a random walk on a semiregular fragmentary tessellation of a plane.
Oscillatory interactions between voltage gated electroenzymesGradmann, D.; Buschmann, P.
doi: 10.1093/jxb/48.Special_Issue.399pmid: 21245218
The activities of the major ion pathways in the plasma membranes of plants are sensitive to the membrane voltage, V. Therefore, these ‘electroenzymes’ interact with each other via the free running voltage under physiological conditions. A physical background is given here, of how to calculate these interactions on the basis of experimental data on these electroenzymes. Simplifying model calculations with five major electroenzymes from plant cells (H+ pump, inward and outward rectifying channels for K+, a Cl− channel, and a 2H+/Cl− symporter) show that osmotic relations are balanced in the long-term not by an appropriate steady-state, but by alternation between a state of salt uptake at V < < EK (the Nernst equilibrium voltage for K+ diffusion) and a state of salt loss at V > EK. Several specific properties of the model are discussed numerically, e.g. minimum configuration for oscillations (with two electroenzymes), temperature-compensation, the physiological impact of fast gating in plant membranes, and solution of possible paradoxes, such as flux stimulation by conductance inhibition.
Functional comparison of plant inward-rectifier channels expressed in yeastBertl, Adam; Reid, John D.; Sentenac, Hervé; Slayman, Clifford L.
doi: 10.1093/jxb/48.Special_Issue.405pmid: 21245219
Functional expression of plant ion channels in the yeast Saccharomyces cerevisiae is readily demonstrated by the successful screening of plant cDNA libraries for complementation of transport defects in especially constructed strains of yeast. The first experiments of this sort identified two potassium-channel genes from Arabidopsis thaliana, designated KAT1 and AKT1 (Anderson et al., 1992; Sentenac et al., 1992), both of which code for proteins resembling the Shaker superfamily of K+ channels in animal cells. Patch-clamp analysis, directly in yeast, of the two channel proteins (Kat1 and Akt1) reveals both functional similarities and functional differences: similarities in selectivity and in normal gating kinetics; and differences in time-dependent effects of ion replacement, in the affinities of blocking ions, and in dependence of gating kinetics on extracellular K+.Kat1, previously described in yeast (Bertl et al., 1995), is about 20-fold more permeable to K+ than to Na+ or NH+4, shows K+-independent gating kinetics, and is blocked with moderate effectiveness (30–50% at 10 mM) by barium and tetraethylammonium (TEA+) ions. Akt1, by contrast, is weakly inhibited by TEA+, more strongly inhibited by Ba2+, and very strongly inhibited by Cs+. Furthermore Na+ and NH+4, while having about the same permeance to Akt1 as to Kat1, have delayed effects on Akt1: brief replacement of extracellular K+ by Na+ enhances by nearly 100% the subsequent K+ currents after sodium removal; and brief replacement of K+ by NH+4 reduces subsequent K+ currents by nearly 75%. Furthermore, lowering of extracellular K+ concentration, by replacement with osmotically equivalent sorbitol, significantly retards the opening of Akt1 channels; that is, the gating kinetics for Akt1 are clearly influenced by the concentration of permeant ions. In this respect, Akt1 resembles the native yeast outward rectifier, Ypk1 (Duk1; Reid et al., 1996).The data suggest that all of the ions tested bind within the open channels, such that the weakly permeant species (Na+, NH+4) are easily displaced by K+, but the blocking species (Cs+, Ba2+, TEA+) are not easily displaced. With Akt1, furthermore, the permeant ions bind to a modulator site where they persist after removal from the medium, and through which they can alter the channel conductance. Extracellular K+ itself also binds to a modulator site, thereby enhancing the rate of opening of Akt1.
Function and regulation of seed aquaporinsMaurel, Christophe; Chrispeels, Maarten; Lurin, Claire; Tacnet, Frédérique; Geelen, Danny; Ripoche, Pierre; Guern, Jean
doi: 10.1093/jxb/48.Special_Issue.421pmid: 21245221
The discovery of water channel proteins named aquaporins has shed new light on the molecular mechanisms of transmembrane water transport in higher plants. As with their animal counterparts, plant aquaporins belong to the large MIP family of transmembrane channels. An increasing number of aquaporins is now being identified on both the vacuolar and plasma membranes of plant cells, but their integrated function remains unclear. Aquaporin α-TIP is specifically expressed in the membrane of protein storage vacuoles in seeds of many plant species. α-TIP was previously shown to undergo phosphorylation in bean seeds. The functional significance of this process was further investigated after heterologous expression of the protein in Xenopus oocytes. Using site-directed mutagenesis of α-TIP and in vitro and in vivo phosphorylation by animal cAMP-dependent protein kinase, it is shown that, in oocytes, direct phosphorylation of α-TIP occurs at three distinct sites and stimulates its water channel activity. In addition to aquaporin phosphorylation, other mechanisms that target aquaporin function are used by living cells to regulate their membrane water permeability. These are the fine control of aquaporin gene expression and, in animal cells only, the regulated trafficking of water channel-containing vesicles. The present work and studies by others on the phosphorylation of nodulin-26, an ion channel protein homologous to α-TIP, provide novel insights into the mechanisms of plant membrane protein regulation. These studies might help identifying and characterizing novel membrane-bound protein kinases and phosphatases. Finally, an integrated function for seed vacuolar aquaporins is discussed. During germination, the rehydration of seed cells, the drastic changes in vacuole morphology, the breakdown and the mobilization of storage products from the vacuole may create osmotic perturbations in the cytoplasm. The fine tuning of TIP aquaporin activity may help control the kinetics and amplitude of osmotic water flows across the tonoplast to achieve proper cytoplasm osmoregulation and control of vacuolar volume.
A patch clamp study of Na+ transport in maize rootsRoberts, Stephen K.; Tester, Mark
doi: 10.1093/jxb/48.Special_Issue.431pmid: 21245222
The mechanisms mediating Na+ transpdrt in higher plant roots were investigated by applying the patch clamp technique to protoplasts isolated from the cortex and stele of maize roots. In the cortex, permeation of Na+ through a time-dependent K+-selective inward rectifier was negligible. Instead, Na+ influx into maize roots probably occurs via an instantaneously-activating current. This current was partially inhibited by extracellular Ca2+, but was insensitive to extracellular TEA+, Cs+ and TTX. In outside-out patches, a plasma membrane ion channel was found which mediated an inward Na+ current which, at least in part, underlies the whole-cell instantaneously-activating current. The unitary conductance of this channel was 15 pS in 102:121 mM Na+ (outsidexytosol). Channel gating was voltage-independent and distinct from that observed for the inwardly rectifying K+-selective channel in the same cell type. Increasing extracellular Ca2+ from 0.1 to 1 mM reduced the open probability and unitary conductance of this channel. In 102 mM Na+ : 123 mM K+ (outside:cytosol) a PNa:PK of 2.1 was calculated. It is suggested that the plasma membrane Na+-permeable channel identified in the cortex of maize roots represents a pathway for low affinity Na+ uptake by intact maize roots. In the stele, permeation of Na+ through outwardly rectifying K+ channels was found to be negligible and the channels are thus unlikely to be involved in the transport of Na+ from the root symplasm.
Regulatory mechanisms of ion channels in xylem parenchyma cellsde Boer, A.H.; Wegner, L.H.
doi: 10.1093/jxb/48.Special_Issue.441pmid: 21245223
Xylem parenchyma cells surround the xylem vessels and control the composition of the transpiration stream which flows through the vessels. In the plasma membrane of the xylem parenchyma cells, one inward rectifying channel (denoted KIRC) and two outward rectifying channels (denoted KORC and NORC) have been identified. In the present study it is shown that KIRC was activated by Gpp(NH)p, in contrast to the inward rectifier in guard cells. In the inside-out patch configuration, Gpp(NH)p elicited single channel KIRC activity as well and the conclusion is, therefore, that KIRC is G-protein regulated in a membrane-delimited fashion. NORC gating is affected by the calcium buffering capacity of the pipette solution as determined by the amount of EGTA. KORC conductance is shown to be strongly dependent upon the apoplastic K+-concentration. The role of the above-mentioned transporters and their regulation mechanisms are discussed in the light of root:shoot communication and long-distance signalling.