RESUMEN
The vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K(+) channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs). Following in planta expression TPK1 and GRF6 co-localize at the vacuolar membrane. Co-localization of wild-type TPK1, but not the TPK1-S42A mutant, indicates that phosphorylation of the 14-3-3 binding motif of TPK1 represents a prerequisite for interaction. Pull-down assays and surface plasmon resonance measurements revealed GRF6 high-affinity interaction with TPK1. Following expression of TPK1 in yeast and isolation of vacuoles, patch-clamp studies identified TPK1 as a voltage-independent and Ca(2+)-activated K(+) channel. Addition of 14-3-3 proteins strongly increased the TPK1 activity in a dose-dependent manner. However, an inverse effect of GRF6 on the activity of the slow-activating vacuolar (SV) channel was observed in mesophyll vacuoles from Arabidopsis thaliana. Thus, TPK1 seems to provide for a Ca(2+)- and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Vacuolas/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/aislamiento & purificación , Sitios de Unión , Calcio/metabolismo , Regulación hacia Abajo , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Fosforilación , Canales de Potasio de Dominio Poro en Tándem/química , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The yeast plasma-membrane potassium channel, Tok1p, is a voltage-dependent outward rectifier, the gating and steady-state conductance of which are conspicuously modulated by extracellular [K(+)] ([K(+)](o)). Activation is slow at high [K(+)](o), showing time constants (tau(a)) of approximately 90 ms when [K(+)](o) is 150 mM (depolarizing step to +100 mV), and inactivation is weak (<30%) during sustained depolarization. Lowering [K(+)](o) accelerates activation, increases peak current, and enhances inactivation, so that at 15 mM [K(+)](o) tau(a) is less than 50 ms and inactivation suppresses approximately 60% of peak current. Two negative residues, Asp292 and Asp426, near the mouth of the assembled channel, modulate both kinetics and conductance of the channel. Charge neutralization in the mutant Asp292Asn allows fast activation (tau(a) approximately 20 ms) at high [K(+)](o), peak currents diminishing with decreasing [K(+)](o), and fast, nearly complete, inactivation. The voltage dependence of tau(a) persists in the mutant, but the [K(+)](o) dependence almost disappears. Similar but smaller changes are seen in the Asp426Asn mutant, implying that pore geometry in the functional channel has twofold, not fourfold, symmetry.
Asunto(s)
Ácido Aspártico/fisiología , Activación del Canal Iónico/fisiología , Canales de Potasio/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Sustitución de Aminoácidos/genética , Ácido Aspártico/genética , Electrofisiología , Activación del Canal Iónico/efectos de los fármacos , Cinética , Potasio/metabolismo , Potasio/farmacología , Canales de Potasio/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Transformación GenéticaRESUMEN
The Arabidopsis tandem-pore K(+) (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen. AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K(+) channel after expression in Xenopus oocytes and yeast. Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K(+) currents, which were blocked by extracellular calcium and cytoplasmic protons. Functional complementation assays using a K(+) transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel. The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube. Thus, AtTPK4 represents a member of plant tandem-pore-K(+) channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Calcio/metabolismo , Membrana Celular/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Potenciales de la Membrana , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Polen/metabolismo , Canales de Potasio de Dominio Poro en Tándem/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , XenopusRESUMEN
Ionic currents related to the major potassium uptake systems in Saccharomyces cerevisiae were examined by whole cell patch-clamping, under K+ replete conditions. Those currents have the following properties. They (1) are inward under all conditions investigated, (2) arise instantaneously with appropriate voltage steps, (3) depend solely upon the moderate affinity transporter Trk2p, not upon the high affinity transporter Trk1p. They (4) appear to be independent of the extracellular K+ concentration, (5) are also independent of extracellular Ca2+, Mg2+ and Cl- but (6) are strongly dependent on extracellular pH, being large at low pH (up to several hundred pA at -200 mV and pH 4) and near zero at high pH (above 7.5). They (7) increase in proportion to log[H+]o, rather than directly in proportion to the proton concentration and (8) behave kinetically as if each transporter cycle moved one proton plus one (high pH) or two (low pH) other ions, as yet unidentified. In view of background knowledge on K+ transport related to Trk2p, the new results suggest that the K+ status of yeast cells modulates both the kinetics of Trk2p-mediated transport and the identity of ions involved. That modulation could act either on the Trk2 protein itself or on interactions of Trk2 with other proteins in a hypothetical transporter complex. Structural considerations suggest a strong analogy to the KtrAB system in Vibrio alginolyticus and/or the TrkH system in Escherichia coli.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Potasio/metabolismo , Protones , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico , Proteínas Portadoras/genética , Conductividad Eléctrica , Proteínas Fúngicas/genética , Eliminación de Gen , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/genética , Técnicas de Placa-ClampRESUMEN
Since the mid-1980s, use of the budding yeast, Saccharomyces cerevisiae, for expression of heterologous (foreign) genes and proteins has burgeoned for several major purposes, including facile genetic manipulation, large-scale production of specific proteins, and preliminary functional analysis. Expression of heterologous membrane proteins in yeast has not kept pace with expression of cytoplasmic proteins for two principal reasons: (1) although plant and fungal proteins express and function easily in yeast membranes, animal proteins do not, at least yet; and (2) the yeast plasma membrane is generally regarded as a difficult system to which to apply the standard electrophysiological techniques for detailed functional analysis of membrane proteins. Especially now, since completion of the genome-sequencing project for Saccharomyces, yeast membranes themselves can be seen as an ample source of diverse membrane proteins - including ion channels, pumps, and cotransporters - which lend themselves to electrophysiological analysis, and specifically to patch-clamping. Using some of these native proteins for assay, we report systematic methods to prepare both the yeast plasma membrane and the yeast vacuolar membrane (tonoplast) for patch-clamp experiments. We also describe optimized ambient conditions - such as electrode preparation, buffer solutions, and time regimens - which facilitate efficient patch recording from Saccharomyces membranes. There are two main keys to successful patch-clamping with Saccharomyces. The first is patience; the second is scrupulous cleanliness. Large cells, such as provided by polyploid strains, are also useful in yeast patch recording, especially while the skill required for gigaseal formation is being learned. Cleanliness is aided by (1) osmotic extrusion of protoplasts, after minimal digestion of yeast walls; (2) use of a rather spare suspension of protoplasts in the recording chamber; (3) maintenance of continuous chamber perfusion prior to formation of gigaseals; (4) preparation (pulling and filling) of patch pipettes immediately before use; (5) application of a modest pressure head to the pipette-filling solution before the tip enters the recording bath; (6) optical control for debris at the pipette tip; and (7) discarding of any pipette that does not "work" on the first try at gigaseal formation. Other useful tricks toward gigaseal formation include the making of protoplasts from cells grown aerobically, rather than anaerobically; use of sustained but gentle suction, rather than hard suction; and manipulation of bath temperature and/or osmotic strength. Yeast plasma membranes form gigaseals with difficulty, but these tend to be very stable and allow for long-term cell-attached or whole-cell recording. Yeast tonoplasts form gigaseals with ease, but these tend to be unstable and rarely allow recording for more than 15 min. The difference of stability accrues mainly because of the fact that yeast protoplasts adhere only lightly to the recording chamber and can therefore be lifted away on the patch pipette, whereas yeast vacuoles adhere firmly to the chamber bottom and are subsequently stressed by very slight relative movements of the pipette. With plasma membranes, conversion from cell-attached recording geometry to isolated ISO patch (inside-out) geometry is accomplished by blowing a fine stream of air bubbles across the pipette tip; to whole-cell recording geometry, by combining suction and one high-voltage pulse; and from whole-cell to OSO patch (outside-out) geometry, by sudden acceleration of the bath perfusion stream. With tonoplasts, conversion from the vacuole-attached recording geometry to whole-vacuole geometry is accomplished by application of a large brief voltage pulse; and further conversion to the OSO patch geometry is carried out conventionally, by slow withdrawal of the patch pipette from the vacuole, which usually remains attached to the chamber bottom.
Asunto(s)
Electrofisiología , Modelos Biológicos , Saccharomyces cerevisiae/fisiología , Membrana Celular/fisiología , Protoplastos , Control de Calidad , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The plasma membrane of the yeast Saccharomyces cerevisiae possesses a non-specific cation 'channel', tentatively dubbed NSC1, which is blocked by normal (mM) calcium and other divalent metal ions, is unblocked by reduction of extracellular free divalents below approximately 10 microM, and is independent of the identified potassium channel and porters in yeast, Duk1p, Trk1p, and Trk2p. Ion currents through NSC1, observed by means of whole-cell patch recording, have the following characteristics: Large amplitude, often exceeding 1 nA of K+/ cell at -200 mV, in tetraploid yeast, sufficient to double the normal intracellular K+ concentration within 10 s; non-saturation at large negative voltages; complicated activation kinetics, in which approximately 50% of the total current arises nearly instantaneously with a voltage-clamp step, while the remainder develops as two components, with time constants of approximately 100 ms and approximately 1.3 s; and voltage independence of both the activation time constants and the associated fractional current amplitudes.
Asunto(s)
Permeabilidad de la Membrana Celular , Canales Iónicos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Calcio/metabolismo , Cationes/metabolismo , Conductividad Eléctrica , Técnicas de Placa-Clamp , Potasio/metabolismoRESUMEN
The major voltage-dependent ion channel in the plasma membrane of Saccharomyces cerevisiae, a conspicuous outwardly rectifying K+ channel, was first dubbed YPK1 and later renamed according to its registered gene names (DUK1, TOK1). It has proven novel in both structure and function. Whole-cell patch-clamp studies of the channel directly on yeast protoplasts now extend our earlier description obtained from isolated patches of yeast membrane (Bertl & Slayman, 1992; Bertl et al., 1993), and provide new data both on the contributions of channel properties to yeast physiology and on possible contributions of molecular structure of channel properties. Three recording tactics produce completely equivalent results and thereby allow great flexibility in the design of experiments: whole-cell voltage clamp with sustained voltage steps (approximately 2.5 sec), whole-cell voltage clamp with slow voltage ramps (5 sec, -40 to +100 mV), and time-averaging of single-channel currents. Activation of Duk1 channels under steady-state conditions is dependent upon ATP in the cytoplasmic solution, and the absence of ATP results in channel "rundown"--decreasing numbers of activable channels--over periods of 10 min to 1 hr from the start of patch recording. Several putative serine- and threonine-phosphorylation sites, as well as a variant ATP-binding fold, exist in the molecule as potential mediators of the ATP effects. The channel runs down similarly following cytoplasmic acidification, but is almost completely insensitive to extracellular pH changes (8.0 to 5.5 tested). This remarkable asymmetry may depend on the protein's strongly asymmetric distribution of histidine residues, with 10 out of 12 predicted to lie close to the membrane-cytoplasm interface. Further data confirm the well-recognized observation that changes of K+ concentration, intracellular or extracellular, can shift the gating voltage of Duk1p in the direction of EK. Among the other alkali-metal cations tested, extracellular Rb+ and Cs(+)--but not Na(+)--substitute almost completely for K+. Extracellular TEA+ inhibits whole-cell K+ currents through Duk1p with a KI of 2.8 mM, and does so probably by reducing the single-channel current.
Asunto(s)
Proteínas Fúngicas/fisiología , Canales de Potasio/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Cationes Bivalentes , Cationes Monovalentes , Membrana Celular/metabolismo , Membrana Celular/fisiología , Conductividad Eléctrica , Electrofisiología , Proteínas Fúngicas/metabolismo , Canales de Potasio/metabolismo , Saccharomyces cerevisiae/metabolismoAsunto(s)
Proteínas Portadoras/fisiología , Proteínas de Transporte de Catión , Genes Fúngicos , Proteínas de la Membrana/fisiología , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/fisiología , Proteínas Portadoras/biosíntesis , Potenciales de la Membrana , Proteínas de la Membrana/biosíntesis , Técnicas de Placa-Clamp , Potasio/metabolismo , Schizosaccharomyces/genéticaRESUMEN
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 Ba(2+), 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(+), Ba(2+), 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.
RESUMEN
Potassium channel subunits have six or two transmembrane segments in addition to a conserved pore-forming (P) domain; four subunits come together to form a channel. A gene was identified in S. cerevisiae (J0911) encoding a protein with eight probable membrane-spanning segments and two such P regions. This protein (Duk1p) is a potassium channel because Xenopus oocytes injected with the corresponding RNA express potassium currents activated by depolarization that are not seen in control oocytes. Similar potassium currents were recorded from wildtype S. cerevisiae spheroplasts, but not from those in which the DUK1 locus had been disrupted. Cells carrying the duk1 delta 1::HIS disruption in addition to a chimeric gene comprising DUK1 behind the GAL1 promoter showed outward currents when grown in galactose, but not when grown in glucose. Additional sequences with the duplicate pore motif were found in C. elegans, suggesting that these proteins represent a novel structural family of potassium channel proteins.
Asunto(s)
Genes Fúngicos/genética , Canales de Potasio/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Membrana Celular , Clonación Molecular , Conductividad Eléctrica , Potenciales Evocados , Femenino , Galactosa , Expresión Génica , Datos de Secuencia Molecular , Mutación , Oocitos , Canales de Potasio/genética , ARN de Hongos , Proteínas Recombinantes de Fusión/biosíntesis , Saccharomyces cerevisiae/genética , Esferoplastos , Xenopus laevisRESUMEN
Transport-deficient strains of the yeast Saccharomyces cerevisiae have recently proven useful for cloning, by functional complementation, of cDNAs encoding heterologous membrane transporters: specifically, H(+)-amino acid symporters and K+ channels from the higher plant Arabidopsis thaliana. The present study uses whole-cell patch-clamp experiments to show that yeast strains which grow poorly on submillimolar K+ due to the deletion of two K(+)-transporter genes (TRK1 and TRK2) are in fact missing a prominent K+ inward current present in wild-type cells. Rescue of such strains for growth on low K+ by transformation with a gene (KAT1) encoding an inward-rectifying K+ channel from Arabidopsis is accompanied by the appearance of an inward current whose characteristics are in qualitative agreement with previous studies in the Xenopus oocyte system, but differ in quantitative details. The ability to make such measurements directly on Saccharomyces should facilitate structure-function studies of any electrogenic or electrophoretic ion transporters which can be expressed in the plasma membrane (or tonoplast) of that organism.
Asunto(s)
Arabidopsis/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/fisiología , Saccharomyces cerevisiae/fisiología , Animales , Proteínas de Arabidopsis , Membrana Celular/fisiología , Clonación Molecular , Cruzamientos Genéticos , ADN Complementario , Femenino , Genes Fúngicos , Genes de Plantas , Potenciales de la Membrana/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Proteínas de Plantas , Potasio/metabolismo , Potasio/farmacología , Canales de Potasio/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Sodio/farmacología , Xenopus laevisRESUMEN
A flush of technical developments and the resultant burgeoning research on channel molecules in biological membranes has, over the past two decades, led to clear operational distinctions between channel behavior and carrier behavior. These have evinced the tacit assumption that the two types of behavior are associated with distinctly different structural motifs (though certainly having some elements in common). Although a full survey of the physiological situation is hampered by low activation probabilities for most channels and by the small currents for most carrier systems (necessitating study of large ensembles of molecules), it is nevertheless clear that many intermediate types of behavior do exist, either in comparison of different channel and carrier molecules or in the responses of a given (class of) molecule at different times and in different membranes. Then in order really to understand the molecular mechanisms of transport, we must explicitly design experiments to isolate and quantify these unconventional modes of behavior.
Asunto(s)
Membrana Celular/metabolismo , Canales Iónicos/metabolismo , Plantas/metabolismo , Animales , Transporte Biológico Activo , Proteínas Portadoras/metabolismoRESUMEN
Disruption of genes encoding endogenous transport proteins in Saccharomyces cerevisiae has facilitated the recent cloning, by functional expression, of cDNAs encoding K+ channels and amino acid transporters from the plant Arabidopsis thaliana [1-4]. In the present study, we demonstrate in whole-cell patch clamp experiments that the inability of trk1deltatrk2delta mutants of S. cerevisiae to grow on submillimolar K+ correlates with the lack of K+ inward currents, which are present in wild-type cells, and that transformation of the trk1deltatrk2delta double-deletion mutant with KAT1 from Arabidopsis thaliana restores this phenotype by encoding a plasma membrane protein that allows large K+ inward currents. Similar K+ inward currents are induced by transformation of a trk1 mutant with AKT1 from A. thaliana.
Asunto(s)
Proteínas de Arabidopsis , Proteínas de Transporte de Catión , Cinesinas , Proteínas de Plantas/fisiología , Canales de Potasio de Rectificación Interna , Canales de Potasio/fisiología , Potasio/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Arabidopsis , Proteínas Portadoras/genética , Proteínas Fúngicas/genética , Genes de Plantas , Prueba de Complementación Genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Transporte Iónico , Proteínas de la Membrana/genética , Técnicas de Placa-Clamp , Proteínas de Plantas/genética , Canales de Potasio/genética , Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Transactivadores/genética , Transactivadores/fisiologíaRESUMEN
The plasma membrane of the yeast Saccharomyces cerevisiae has been investigated by patch-clamp techniques, focusing upon the most conspicuous ion channel in that membrane, a K(+)-selective channel. In simple observations on inside-out patches, the channel is predominantly closed at negative membrane voltages, but opens upon polarization towards positive voltages, typically displaying long flickery openings of several hundred milliseconds, separated by long gaps (G). Elevating cytoplasmic calcium shortens the gaps but also introduces brief blocks (B, closures of 2-3 msec duration). On the assumption that the flickery open intervals constitute bursts of very brief openings and closings, below the time resolution of the recording system, analysis via the beta distribution revealed typical closed durations (interrupts, I) near 0.3 msec, and similar open durations. Overall behavior of the channel is most simply described by a kinetic model with a single open state (O), and three parallel closed states with significantly different lifetimes: long (G), short (B) and very short (I). Detailed kinetic analysis of the three open/closed transitions, particularly with varied membrane voltage and cytoplasmic calcium concentration, yielded the following stability constants for channel closure: K1 = 3.3 x e-zu in which u = eVm/kT is the reduced membrane voltage, and z is the charge number; KG = 1.9 x 10(-4) ([Ca2+].ezu)-1; and KB = 2.7 x 10(3)([Ca2+].ezu)2. Because of the antagonistic effects of both membrane voltage (Vm) and cytoplasmic calcium concentration ([Ca2+]cyt) on channel opening from the B state, compared with openings from the G state, plots of net open probability (Po) vs. either Vm or [Ca2+] are bell-shaped, approaching unity at low calcium (microM) and high voltage (+150 mV), and approaching 0.25 at high calcium (10 mM) and zero voltage. Current-voltage curves of the open channel are sigmoid vs. membrane voltage, saturating at large positive or large negative voltages; but time-averaged currents, along the rising limb of Po (in the range 0 to +150 mV, for 10 microM [Ca2+]) make this channel a strong outward rectifier. The overall properties of the channel suggest that it functions in balancing charge movements during secondary active transport in Saccharomyces.
Asunto(s)
Membrana Celular/fisiología , Activación del Canal Iónico/fisiología , Canales de Potasio/fisiología , Saccharomyces cerevisiae/fisiología , Calcio/fisiología , Membrana Celular/ultraestructura , Potenciales de la Membrana/fisiología , Canales de Potasio/ultraestructura , Saccharomyces cerevisiae/ultraestructura , Factores de TiempoRESUMEN
Detailed patch-clamp studies have been made of ion channels in the plasma membrane and tonoplast of the yeast Saccharomyces cerevisiae. The predominant tonoplast channel is a high-conductance cation-selective inward rectifier (passing ions easily into the cytoplasm from the vacuole), with its open probability (Po) peaking at about -80 mV (cytoplasm negative) and falling to near zero at +80 mV. It has a maximal slope conductance of approximately 150 pS in 100 mmol l-1 KCl, and conducts Na+, K+ and Ca2+. Elevated cytoplasmic Ca2+ concentration, alkaline pH and reducing agents can activate the channel, its likely physiological function being to adjust cytoplasmic Ca2+ concentration from the vacuolar reservoir. The predominant plasma-membrane channel is a strongly outward rectifying K+ channel (passing K+ easily out of the cytoplasm to the extracellular medium), which is activated by positive-going membrane voltages as well as by elevated cytoplasmic Ca2+ concentration and alkaline pH. Interaction between membrane voltage and [Ca2+]cyt is complex and defines three parallel closed states for the channel: a Ca(2+)-independent brief closure (I), a calcium-inhibited long closure (G) and, at large positive voltages, a calcium-induced brief blockade (B). This channel is likely to function in steady-state turgor regulation and in charge balancing during proton-coupled substrate uptake.
Asunto(s)
Canales Iónicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Cationes/metabolismo , Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Vacuolas/metabolismoRESUMEN
Ion channels in both the tonoplast and the plasma membrane of Saccharomyces cerevisiae have been characterized at the single channel level by patch-clamp techniques. The predominant tonoplast channel is cation selective, has an open-channel conductance of 120 pS in 100 mM KCl, and conducts Na+ or K+ equally well, and Ca2+ to a lesser extent. Its open probability (Po) is voltage-dependent, peaking at about -80 mV (cytoplasm negative), and falling to near zero at +80 mV. Elevated cytoplasmic Ca2+, alkaline cytoplasmic pH, and reducing agents activate the channel. The predominant plasma membrane channel is highly selective for K+ over anions and other cations, and shows strong outward rectification of the time-averaged current-voltage curves in cell-attached experiments. In isolated inside-out patches with micromolar cytoplasmic Ca2+, this channel is activated by positive going membrane voltages: mean Po is zero at negative membrane voltages and near unity at 100 mV. At moderate positive membrane voltages (20-40 mV), elevating cytoplasmic Ca2+ activates the channel to open in bursts of several hundred milliseconds duration. At higher positive membrane voltages, however, elevating cytoplasmic Ca2+ blocks the channel in a voltage-dependent fashion for periods of 2-3 ms. The frequency of these blocking events depends on cytoplasmic Ca2+ and membrane voltage according to second-order kinetics. Alternative cations, such as Mg2+ or Na+, block the yeast plasma-membrane K+ channel in a similar but less pronounced manner.
Asunto(s)
Canales Iónicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Concentración de Iones de Hidrógeno , Potenciales de la Membrana , Vacuolas/metabolismoRESUMEN
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 Ca(2+) on the cytoplasmic side. Reducing Ca(2+) 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 beta-mercaptoethanol) added to the cytoplasmic side of an excised patch increased the open probability of all three channel types.
RESUMEN
The vacuolar membrane of the yeast Saccharomyces cerevisiae, which is proposed as a system for functional expression of membrane proteins, was examined by patch-clamp techniques. Its most conspicuous feature, in the absence of energizing substrates, is a cation channel with a characteristic conductance of approximately 120 pS for symmetric 100 mM KCl solutions and with little selectivity between K+ and Na+ (PNa+/PK+ approximately 1) but strong selectivity for cations over anions (PCl-/PK+ less than 0.1). Channel gating is voltage-dependent; open probability, Po, reaches maximum (approximately 0.7) at a transmembrane voltage of -80 mV (cytoplasmic surface negative) and declines at both more negative and more positive voltages (i.e., to 0 around +80 mV). The time-averaged current-voltage curve shows strong rectification, with negative currents (positive charges flowing from vacuolar side to cytoplasmic side) much larger than positive currents. The open probability also depends strongly on cytoplasmic Ca2+ concentration but, for ordinary recording conditions, is high only at unphysiologically high (greater than or equal to 1 mM) Ca2+. However, reducing agents such as dithiothreitol and 2-mercaptoethanol poise the channels so that they can be activated by micromolar cytoplasmic Ca2+. The channels are blocked irreversibly by chloramine T, which is known to oxidize exposed methionine and cysteine residues specifically.