Effect of temperature on plasma membrane and tonoplast ion channels in Arabidopsis thaliana

The temperature dependence of the activity of ion channels was investigated, by means of the patch-clamp technique in the 'whole-cell' configuration, using protoplasts and vacuoles isolated form Arabidopsis thaliana L. cultured cells. The effect of temperature changes in the range 11–22°C was tested on the hyperpolarization and depolarization-activated K⁺ currents in the plasma membrane and on the hyperpolarization-activated K⁻ currents in the tonoplast (vacuolar membrane). All 3 kinds of currents were unaffected by increasing temperature up to 15°C and were activated between 15 and 20°C.     

Cation channels in the Arabidopsis plasma membrane

In vivo analyses have identified different functional types of ion channels in various plant tissues and cells. The Arabidopsis genome contains approximately 70 genes for ion channels, of which 57 might be cation-selective channels (K(+), Ca(2+) or poorly discriminating channels). Here, we describe the different families of (putative) cation channels: the Shakers, the two-P-domain and Kir K(+) channels (encoded by the KCO genes), the cyclic-nucleotide-gated channels, the glutamate receptors, and the Ca(2+) channel TPC1. We also compare molecular data with the data obtained in planta, which should lead to a better understanding of the identity of these channels and provide clues about their roles in plant nutrition and cell signalling.     

Electrogenic transport properties of growing Arabidopsis root hairs : the plasma membrane proton pump and potassium channels

Ion transport, measured using double-barreled micropipettes to obtain current-voltage relations, was examined in Arabidopsis thaliana root hairs that continued tip growth and cytoplasmic streaming after impalement with the micropipette. To do this required in situ measurements with no handling of the seedlings to avoid wounding responses, and conditions allowing good resolution microscopy in tandem with the electrophysiological measurements. Two ion transport processes were demonstrated. One was a tetraethylammonium-sensitive potassium ion current, inward at hyperpolarized potentials and outward at depolarized potentials. The addition of tetraethylammonium (a potassium channel blocker) caused the potential to hyperpolarize, indicating the presence of a net inward potassium current through the ion channels at the resting potential. The potassium influx was sufficient to “drive” cellular expansion based upon growth rates. Indeed, tetraethylammonium caused transient inhibition of tip growth. The other electrogenic process was the plasma membrane proton pump, measured by indirect inhibition with cyanide or direct inhibition by vanadate. The proton pump was the dominant contribution to the resting potential, with a very high current density of about 250 microamperes per square centimeter (seen only in young growing root hairs). The membrane potential generated by the proton pump presumably drives the potassium influx required for cellular expansion. The pump appears to be a constant current source over the voltage range −200 to 0 millivolts. With this system, it is now possible to study the physiology of a higher plant cell in dynamic living state using a broad range of cell biological and electrophysiological techniques.     

The role of proton pump and potassium channels of plasma membrane in regulation of cellulose synthesis in plants

A study was made of the influence of two activators of plasma membrane proton pump [indole-3-acetic acid (IAA) and fusicoccin (EC)] and of the blocker of potassium channels of outward direction [tetraethylammonium chloride (TEA)] on exogenous [U-14C]glucose incorporation into cellulose fraction of cell wall, and on the value of plasmalemma membrane potential. It has been shown that IAA and FC exert different influences on the intensity of [U-14C]glucose incorporation into cellulose fraction: IAA activates, while FC inhibits incorporation intensity. A conclusion is made that differences in affects of IAA and FC on the intensity of cellulose synthesis at the plasma membrane level may be due to the fact that the activating effect of IAA on plasma membrane proton pump involves activation of the inward direction potassium channels, whereas that of FC, on the contrary, is associated with their blocking. Under the action of TEA, the intensity of incorporation of radioactively labeled glucose was increased. Apparently, the role of plasma membrane in regulation of the intensity of cellulose synthesis may be associated with not only the activity of proton pump on plasma membrane, but also the functional condition of potassium channels of this membrane.     

Mitochondrial potassium and chloride channels

Channels selective for potassium or chloride ions are present in inner mitochondrial membranes. They probably play an important role in mitochondrial events such as the formation of delta pH and regulation of mitochondrial volume changes. Mitochondrial potassium and chloride channels could also be the targets for pharmacologically active compounds such as potassium channel openers and antidiabetic sulfonylureas. This review describes the properties, pharmacology, and current observations concerning the functional role of mitochondrial potassium and chloride channels.     

Red light regulates calcium-activated potassium channels in mougeotia plasma membrane

The alga Mougeotia has a large central chloroplast whose positioning is regulated by photoactivation of phytochrome, possibly via modulation of cytosolic calcium (Serlin B, Roux SJ [1984] Proc Natl Acad Sci USA 81: 6368-6372). We used the patch clamp technique to examine the effects of red and far-red light on ion channel activity in the plasma membrane of Mougeotia protoplasts to determine if ion channels play a role in chloroplast movement. Patch clamping in the cell-attached mode reveals two channels of about 2 and 4 picoamperes amplitude at 0 millivolt (inside pipette) and estimated conductances of 30 and 65 picosiemens. They are activated by red light irradiation after a lag period of about 2 to 5 minutes. Far-red light, when applied immediately after red light irradiation, reverses this activation; otherwise it has no effect. This result implicates phytochrome. The addition of the calcium ionophore, A23187, also activates ion channel activity after a lag of a few minutes. The channels are not specific for calcium since they are present when calcium is removed from the external and pipette media. They are inhibited by quaternary ammonium ions. Thus, we believe they are calcium-activated potassium channels. Their possible role in chloroplast positioning is discussed.     

The plasma membrane potential of human neutrophils. Role of ion channels and the sodium/potassium pump

Calcium-depleted human neutrophils are depolarised when suspended in calcium-free media containing sodium ions, and are repolarised by extracellular replenishment of Ca2+. The depolarisation is due to a high inward sodium current, which is blocked by calcium and by several other divalent cations, but not by barium. Addition of calcium results in a rise in the cytosolic concentration from approx. 20 nM to the resting level of approx. 130 nM. Calcium influx is strongly accelerated by a voltage-gated calcium channel. This channel might be responsible for the depolarising Na+ current in the absence of divalent cations. In the polarised state the neutrophil membrane has a high intrinsic permeability to K+, which may be low or absent in the depolarised state. Generation of membrane potential from the depolarised state is mainly due to the electrogenic sodium/potassium pump. However, the resting potential of about -75 mV is maintained primarily by the K+ conductance, and only to a small extent by the sodium/potassium pump.     

Distinct pH regulation of slow and rapid anion channels at the plasma membrane of Arabidopsis thaliana hypocotyl cells

Variations in both intracellular and extracellular pH are known to be involved in a wealth of physiological responses. Using the patch-clamp technique on Arabidopsis hypocotyl cells, it is shown that rapid-type and slow-type anion channels at the plasma membrane are both regulated by pH via distinct mechanisms. Modifications of pH modulate the voltage-dependent gating of the rapid channel. While intracellular alkalinization facilitates channel activation by shifting the voltage gate towards negative potentials, extracellular alkalinization shifts the activation threshold to more positive potentials, away from physiological resting membrane potentials. By contrast, pH modulates slow anion channel activity in a voltage-independent manner. Intracellular acidification and extracellular alkalinization increase slow anion channel currents. The possible role of these distinct modulations in physiological processes involving anion efflux and modulation of extracellular and/or intracellular pH, such as elicitor and ABA signalling, are discussed.     

Existence of memory in membrane channels: analysis of ion current through a voltage‐dependent potassium single channel

Ion current fluctuation of voltage‐dependent potassium channel in LβT2 cells has been investigated by autocorrelation function and DFA (detrended fluctuation analysis) methods. The calculation of the autocorrelation function exponent and DFA exponent of the sample was based on the digital signals or the 0–1 series corresponding to closing and opening of channels after routine evolution, rather than the sequence of sojourn times. The persistent character of the correlation of the time series was evident from the slow decay of the autocorrelation function. DFA exponent α was significantly greater than 0.5. The main outcome has been the demonstration of the existence of memory in this ion channel. Thus, the ion channel current fluctuation provided information about the kinetics of the channel protein. The result suggests the correlation character of the ion channel protein non‐linear kinetics indicates whether the channel is open or not.     

Activation of mechanosensitive ion channels in plasma membrane of K562 cells

Patch clamp method in cell-attached configuration was used to search for mechanogated ion channels in plasma membrane of human myeloid leukemia K562 cells. A reversible activation of transmembrane currents in response to negative pressure applied to membrane patch was observed. Four types of mechanosensitive channels were identified in K562 cells: two main types were characterized with conductance values of 16 and 25 pS; while two others, showing higher conductance values (about 35 and 50 pS), were rarely met. In terms of gating, all channels described here could be assigned to the stretch-activated type. No inactivation of mechanosensitive channels at the sustained stimulation was observed. The activation of mechanosensitive channels in K562 cells was not dependent upon the presence of bivalent cations in the extracellular solution.     

The demonstration of potential-dependent potassium channels in the plasma membrane of secretory cells

Outward current of the salivary gland cells membrane of chironomus larva activated by the displacement of the membrane potential to the region of positive values has been registered by the voltage-clamp method under conditions of intracellular dialysis in the presence of only the potassium transmembrane gradient. Activation threshold of the current is about +10 mV. Subsequent displacement of the membrane potential to the region of positive values causes an increase of the current. Time constant of the current activation is (652 +/- 57) ms. The current decreases with the intracellular potassium concentration, under the influence of tetraethyl-ammonium and 4-aminopyridine. Thus, high threshold potential-dependent potassium channels are presented in the secretory cells membrane.     

Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs--fundamental calcium events

Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca(2+) permeable ion channel using Ca(2+) indicators like fluo-3. These Single Channel Ca(2+) Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca(2+) sparks and Ca(2+) puffs caused by Ca(2+) release from intracellular stores (due to the opening of ryanodine receptors and IP(3) receptors, respectively). In contrast to intracellular Ca(2+) release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca(2+) handling in the vicinity of a channel with a known Ca(2+) influx, to obtain the Ca(2+) current passing through plasma membrane cation channels in near physiological solutions, to localize Ca(2+) permeable ion channels on the plasma membrane, and to estimate the Ca(2+) currents underlying those elementary events where the Ca(2+) currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca(2+) channels, and stretch-activated channels. For the L-type Ca(2+) channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca(2+) currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.     

Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana.

Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana have been studied at the single-channel level using the patch-clamp technique. The Ca2+ channel in the plasma membrane opened for extracellular Ca2+ influx. The Ca2+ channel in the vacuolar membrane opened for cytoplasmic Ca2+ influx.     

Homer regulation of native plasma membrane calcium channels in A431 cells

Homers are adapter proteins that play a significant role in the organization of calcium signaling protein complexes. Previous functional studies linked Homer proteins to calcium influx in nonexcitable cells. These studies utilized calcium imaging or whole-cell current recordings. Because of limited resolution of these methods, an identity of Homer-modulated ion channels remained unclear. There are several types of plasma membrane calcium influx channels in A431 cells. In the present study, we demonstrated that Homer dissociation resulted in specific activation of I(min) channels but not of I(max) channels in inside-out patches taken from A431 cells. In contrast, inositol 1,4,5-trisphosphate activated both I(min) and I(max) channels in inside-out patches. Short (1a) and long (1c) forms of Homer had different effects on I(min) channel activity. Homer 1a but not Homer 1c activated I(min) in the patches. This study indicates that I(min) channels are specifically regulated by Homer proteins in A431 cells.     

Electrostatic tuning of ion conductance in potassium channels

Members of the K(+) channel family display remarkable conservation of sequence and structure of the ion selectivity filter, whereas the rates of K(+) turnover vary widely within the family. Here we show that channel conductance is strongly influenced by charge at the channel's intracellular mouth. Introduction of a ring of negative charges at this position in KcsA, a bacterial K(+) channel, augments the conductance in a pH-dependent manner. These results are explained by a simple electrostatic effect based on known channel structures, where the negative charges serve to alter the electrical potential at the inner mouth and, thus, to increase the local K(+) concentration. In addition, removal of the conserved negative charges at equivalent positions in a high-conductance eukaryotic K(+) channel leads to a decrease in conductance.     

Ion channels in Arabidopsis plasma membrane : transport characteristics and involvement in light-induced voltage changes

White light (25 watts per square meter) induced an increase in plasma membrane K⁺-channel activity and a 30- to 70-millivolt transient membrane depolarization (completed in 2-3 minutes) in Arabidopsis thaliana leaf mesophyll cells. Transport characteristics of three types of ion channels in the plasma membrane were determined using inside-out patches. With 220 millimolar K⁺ on the cytoplasmic side of the patch and 50 millimolar K⁺ in the pipette, (220/50 K), the open-channel current-voltage curves of these channels were sigmoidal and consistent with an enzyme kinetic model. Two channel types were selective for K⁺ over Na⁺ and Cl⁻. One (named PKC1) had a maximum conductance (G_max) of 44 picosiemens at a membrane voltage (V_m) of −65 mV in (220/50 K) and is stimulated by light. The other (PKC2) had G_max = 66 picosiemens at V_m = 60 millivolts in (220/50 K). The third channel type (PCC1) transported K⁺ and Na⁺ about equally well but not Cl⁻. It had G_max = 109 picosiemens at V_m = 55 millivolts in (250/50 K) with 10 millimolar Ca²⁺ on the cytoplasmic side. Reducing Ca²⁺ to 0.1 millimolar increased PCC1 open-channel currents by approximately 50% in a voltage-independent manner. Averaged over time, PKC2 and PCC1 currents strongly outward rectified and PKC1 currents did so weakly. Reductants (1 millimolar dithiothreitol or 10 millimolar β-mercaptoethanol) added to the cytoplasmic side of an excised patch increased the open probability of all three channel types.