Effects of mono- and multi-valent cations on the inward-rectifying potassium channel in isolated protoplasts from maize roots (original) (raw)
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Potassium channels in plant cells
FEBS Journal, 2011
Potassium (K +) is the most abundant inorganic cation in plant cells. Unlike animals, plants lack sodium ⁄ potassium exchangers. Instead, plant cells have developed unique transport systems for K + accumulation and release. An essential role in potassium uptake and efflux is played by potassium channels. Since the first molecular characterization of K + channels from Arabidopsis thaliana in 1992, a large number of studies on plant potassium channels have been conducted. Potassium channels are considered to be one of the best characterized class of membrane proteins in plants. Nevertheless, knowledge on plant potassium channels is still incomplete. This minireview focuses on recent developments in the research of potassium transport in plants with a strong focus on voltage-gated potassium channels. Abbreviations ABA, abscisic acid; CBL, calcineurin B-like protein; CDPK, calcium-dependent protein kinases; CIPK, CBL-interacting protein kinase; ER, endoplasmic reticulum; HKT, high affinity K + uptake transporter; ROS, reactive oxygen species.
Characterization of Potassium-Dependent Currents in Protoplasts of Corn Suspension Cells
PLANT PHYSIOLOGY, 1989
Protop1est4 obtained from com (Zea mays) suspension cells were did using the whole cell patch-clamp technique. One timindependent current, as well as two time-dependent currents wer identified. All three currents were reduced by tetraethylamowium (9 millimolar), a K+ channel blocker. The time-independent current had a nearly linear current-voltage relationship and its reversal potental, defined as the voltage at which there is zerp current, was highly dependent on the extracellular potassium cnCqntralion. One of the two time-dependent currents was a;tivat9l, with rapid kinetics, by membrane hyperpolarization to potentiels mor negative than-100 millivolts. The second timedependent currnt was activated with a sigmoidal time course by membrane depolarization to potentials more positive than-60 millivots. It exhibited no inactivation and was carried primarily by potssium ions. These characteristics suggest that this latter current is caused by the voltage-dependent opening of delayedrectifier K+ channels. These three currents, which are not genera by the plasmalomma H+-ATPase, are likely to assist in the relaton of the cellular K+ fluxes and membrane potential. Ion channels play a central role in the regulation of ionic and electrical gradients that energize nutrient uptake. There is also increasing evidence of their involvement in polar growth (23), turgor regulation (22), and responses to hormones (24, 25), light (11), and environmental stress (19). Channel proteins are able to serve these many functions by virtue oftheir ability to switch, or oscillate, between open and closed states in response to changes in membrane potential or to the binding of various ligands. A variety of ion channels have been identified in plant cell membranes including Cl-channels (6, 25), Ca2+ channels (27), and K+ channels (2, 3, 9, 16, 20, 21, 24-26, 28). In particular, K+ channels are thought to modify the membrane potential under specific environmental conditions. One example, that is well documented in plants of the Characeae family, is the shift in membrane potential from the hyperpolarized, 'pump state' to the depolarized, 'K+-state' (15). This depolarization, promoted by the absence of Ca2`or concentrations of external K+ that exceed 1.0 mM, is caused by an increase in the K+ permeability (i.e. opening of K+ channels) in the plasma membrane (2-4, 16). 'Supported by Natural Sciences and Engineering Research Council of Canada and Department of Education of Quebec. K. A. K. is supported by a McGill Major Fellowship from The Friends of McGill. MATERIALS AND METHODS Cell Cultures Corn suspension cells (Zea mays, Black Mexican sweet corn University of Illinois #78-002) derived from root tissue were grown in culture medium composed of Murashige and Skoog salt mixture (Gibco Laboratories) supplemented with 1184
Journal of Membrane Biology, 1999
The activation kinetics of outward currents in protoplasts from barley root xylem parenchyma was investigated using the patch-clamp technique. The K + outward rectifying conductance (KORC), providing the main pathway for K + transport to the xylem, could be described in terms of a Hodgkin-Huxley model with four independent gates. Gating of KORC depended on voltage and the external K + concentration. An increase in the external K + concentration resulted in a shift in the voltage dependence of gating. This could be explained by a K + dependence of the rate constant  for channel closure, indicating binding of K + to a regulatory site exposed to the bath. Occasionally, KORC was observed to inactivate; this inactivation occurred and vanished spontaneously. In some of the whole cell and excised patch recordings, a stepwise increase in outward current was observed upon a depolarizing voltage pulse, indicating that several populations of 'sleepy' channels existed in the plasma membrane that activated with a certain lag time. It is discussed whether this observation can be explained by a putative subunit, which retards channel activation, or by a scheme of cooperative gating. A quantitative description of outward rectifying K + channels in xylem parenchyma cells is a major step forward towards a mathematical model of salt transport into the xylem.
Cell Type-Specific Regulation of Ion Channels Within the Maize Stomatal Complex
Plant and Cell Physiology, 2011
The stomatal complex of Zea mays is composed of two pore-forming guard cells and two adjacent subsidiary cells. For stomatal movement, potassium ions and anions are thought to shuttle between these two cell types. As potential cation transport pathways, K +-selective channels have already been identified and characterized in subsidiary cells and guard cells. However, so far the nature and regulation of anion channels in these cell types have remained unclear. In order to bridge this gap, we performed patch-clamp experiments with subsidiary cell and guard cell protoplasts. Voltage-independent anion channels were identified in both cell types which, surprisingly, exhibited different, cell-type specific dependencies on cytosolic Ca 2+ and pH. After impaling subsidiary cells of intact maize plants with microelectrodes and loading with BCECF [(2 0 ,7 0-bis-(2-carboxyethyl)-5(and6)carboxyflurescein] as a fluorescent pH indicator, the regulation of ion channels by the cytosolic pH and the membrane voltage was further examined. Stomatal closure was found to be accompanied by an initial hyperpolarization and cytosolic acidification of subsidiary cells, while opposite responses were observed during stomatal opening. Our findings suggest that specific changes in membrane potential and cytosolic pH are likely to play a role in determining the direction and capacity of ion transport in subsidiary cells.
Plant and Cell Physiology, 1996
Using the patch-clamp technique the kinetics of whole-cell and single channel inwardly rectifying K + currents were measured in enzymatically-isolated protoplasts from Avena sativa mesophyll leaf cells. The hyperpolarization-activated whole-cell current had an initial K + component (I|d) and a time-dependent K + component which reaches steady state (I Kss) within 500 ms. After an initial delay, the activation of I Kss and the deactivation of the tail K + current (I KT) followed an exponential time course. The time-constants of activation (r a), at 10 mM and 50 mM [K + ] o , and of deactivation (r d) could be described by exponential and sigmoidal dependence on membrane voltage (V m), respectively. The relative number of the activated K + channel population increased sigmoidally as a function of hyperpolarized V m. On the other hand, the relative number of the deactivated K + channel population decreased exponentially as a function of hyperpolarized V m. The kinetics of the inward rectifying K + current were dependent on V m but not on extracellular K + concentration, [K + ] o. The presence of Cs + in the bathing medium reduced r, at voltage steps less negative than-125 mV and increased r, for voltage steps between-150 mV and-200 mV. By comparison, the dependence of r d on V m was not altered significantly by changing [Cs + ] o. Cs + reversibly blocked the voltage-dependent sigmoidal rise of K + current activation and the voltage-dependent exponential decrease of current deactivation. The Cs +-induced block of K + current was both voltage and concentration dependent. Single channel current (IK), the probability of the channel being open (P o) and the mean open-time (r o) increased as a function of hyperpolarized potentials. The mean closed-time (r c) and the mean lag time before the channel opened were exponentially dependent on voltage and they became shorter as the membrane was hyperpolarized. The kinetics of single K + channels i.e. activation, deactivation and lag time, and the absence of outward single K + channel currents were consistent with whole-cell current measurements. The inward rectification of single channel currents and simulated cell currents suggest that this current rectification is an intrinsic nature of the inward rectifying K +
Plant, Cell & Environment, 2009
Potassium is a major osmolyte used by plant cells. The accumulation rates of K + in cells may limit the rate of expansion. In the present study, we investigated the involvement of ion channels in K + uptake using patch clamp technique. Ion currents were quantified in protoplasts of the elongation and emerged blade zone of the developing leaf 3 of barley (Hordeum vulgare L.). A time-dependent inwardrectifying K + -selective current was observed almost exclusively in elongation zone protoplasts. The current showed characteristics typical of Shaker-type channels. Instantaneous inward current was highest in the epidermis of the emerged blade and selective for Na + over K + . Selectivity disappeared, and currents decreased or remained the same, depending on tissue, in response to salt treatment. Net accumulation rates of K + in cells calculated from patch clamp current-voltage curves exceeded rates calculated from membrane potential and K + concentrations of cells measured in planta by factor 2.5-2.7 at physiological apoplastic K + concentrations (10-100 mM). It is concluded that under these conditions, K + accumulation in growing barley leaf cells is not limited by transport properties of cells. Under saline conditions, down-regulation of voltage-independent channels may reduce the capacity for growth-related K + accumulation.
Membrane depolarization induces K+ efflux from subapical maize root segments
New Phytologist, 2002
• The role of potassium efflux from maize (Zea mays) root segments in maintaining transmembrane electric potential difference (E m) was studied in vivo , together with the involvement of outward rectifying K + channels (ORCs). • Measurements were made of the efflux of potassium (K +) from roots when its uptake was competitively inhibited by rubidium (Rb +), of the E m of the root cells by microelectrodes and of the unidirectional fluxes of monovalent cations. • The influx of Rb + , caesium (Cs +) or ammonium (NH 4 +) into the segments induced an efflux of K +. Lithium (Li +) and sodium (Na +) were not taken up and did not induce K + efflux. The permeating cations induced membrane depolarizations, which were closely related to the values of K + efflux. Two K +-channel blockers, tetraethylammoniumchloride and quinidine, inhibited K + efflux. The inhibition was accompanied by a higher membrane depolarization induced by Rb + , whose influx was not affected. • The results suggest that a depolarizing event caused by cation uptake increased K + efflux from the cells, probably through the activation of ORCs involved in restoration and stabilization of E m .
ABA Regulation of K+-Permeable Channels in Maize Subsidiary Cells
Plant and Cell Physiology, 2006
An antiparallel-directed potassium transport between subsidiary cells and guard cells which form the graminean stomatal complex has been proposed to drive stomatal movements in maize. To gain insights into the coordinated shuttling of K þ ions between these cell types during stomatal closure, the effect of ABA on the time-dependent K þ uptake and K þ release channels as well as on the instantaneously activating non-selective cation channels (MgC) was examined in subsidiary cells. Patch-clamp studies revealed that ABA did not affect the MgC channels but differentially regulated the time-dependent K þ channels. ABA caused a pronounced rise in time-dependent outward-rectifying K þ currents (K out) at alkaline pH and decreased inward-rectifying K þ currents (K in) in a Ca 2þ-dependent manner. Our results show that the ABA-induced changes in time-dependent K in and K out currents from subsidiary cells are very similar to those previously described for guard cells. Thus, the direction of K þ transport in subsidiary cells and guard cells during ABA-induced closure does not seem to be grounded solely on the cell type-specific ABA regulation of K þ channels.