Ion channels in the membrane ofChara inflata

Summary Voltage-clamped steps in the electric potential difference (PD) across the membrane in cells of the green alga,Chara inflata, cause voltage- and time-dependent current flows, interpreted to arise from opening and closing of various types of ion channel in the membrane. With cells in the light, these channels are normally closed, and the resting PD is probably determined by the operation of an H⁺ efflux pump. Positive steps in PD from the resting level often caused the opening of K⁺ channels with sigmoid kinetics. The channels began to show opening when the PD–120 mV for an external concentration of K⁺ of 1.0mm. Return of the PD to the resting level caused closing of the channels with complex kinetics. Various treatments of the cell could cause these K⁺ channels to open, and remain open continuously, with the PD then lying closer to the Nernst PD for K⁺. The K⁺ channels have been identified by the blocking effects of TEA⁺. Another group of channels, probably Cl^– and Ca²⁺ associated with the action potential open when the PD is stepped to values less negative than –50 mV. Negative steps from the resting PD cause the slow opening, with a time course of seconds, of yet another type of channel, probably Cl^–.     

Increased Resistance to Extracellular Cation Block by Mutation of the Pore Domain of the Arabidopsis Inward-rectifying K+ Channel KAT1

Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K⁺ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root hair cells. K⁺ channel blockers are potent tools for studying the physiological functions and structural properties of K⁺ channels. In the present study the structural and biophysical mechanisms of Cs⁺ and TEA⁺ block of a cloned Arabidopsis inward-rectifying K⁺ channel (KAT1) were analyzed. Effects of the channel blockers Cs⁺ and TEA⁺ were characterized both extracellularly and intracellularly. Both external Cs⁺ and TEA⁺ block KAT1 currents. A mutant of KAT1 (``m2KAT1'; H267T, E269V) was produced by site-directed mutagenesis of two amino acid residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs⁺ and TEA⁺ than the wild-type K⁺ channel. Internal TEA⁺ and Cs⁺ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K⁺ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested for outward-rectifying K⁺ channels. Received: 31 July 1995/Revised: 5 January 1996     

Modulation of HERG channel inactivation by external cations

We investigated the effect of external cations on the permeability characteristics and gating kinetics of the human ether-à-go-go-related gene (HERG) current using the whole-cell patch-clamp technique. Inward HERG currents were recorded on hyperpolarization in 140 mM external Cs⁺ and Rb⁺, as well as K⁺. The permeability ratios of Rb⁺ and Cs⁺ relative to K⁺ were 1.25 and 0.56, respectively. Biphasic outward currents were recorded on depolarization in 140 mM Cs⁺ and in Rb⁺ with much smaller amplitude. The voltage dependence of inactivation was affected by external cations, such that the half-inactivation voltage shifted from –69.4±3.7 mV in K⁺ to –30.7±1.6 mV in Cs⁺ and to –35.8±1.9 mV in Rb⁺ (n=5). The time constants of inactivation were also changed significantly by external cations; of inactivation at +40 mV was 16.4±2.2 ms in 140 mM K⁺, 181±20.3 ms in Cs⁺, and 94.1±7.6 ms in Rb⁺ (n=5). Voltage dependence of activation was not altered significantly. The inhibition of the rapid inactivation mechanism by large cations may suggest that the foot-in-the-door model of gating is involved in HERG channel inactivation.     

Ca2+-activated K+ currents inNecturus choroid plexus

Summary The tight-seal whole-cell recording method has been used to studyNecturus choroid plexus epithelium. A cell potential of –59±2 mV and a whole cell resistance of 56±6 M were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110mm NaCl Ringer solution and the interior of the cell contained a solution of 110mm KCl and 5nm Ca²⁺, stepping the membrane potential from a holding value of –50 to –10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca²⁺-activated K^_ channels. Based on the voltage dependence of the activation of Ca²⁺-activated K⁺ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K⁺ as their reversal potential at different K⁺ concentration gradients followed the Nernst potential for K⁺. These currents were reduced by the addition of TEA⁺ to the bath solution and were eliminated when Cs⁺ or Na⁺ replaced intracellular K⁺. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t _1/2) to the steady value. Increasing the pipette Ca²⁺ also decreased the delay and decreasedt _1/2. For instance, when pipette Ca²⁺ was increased from 5 to 500nm, the delay andt _1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K⁺ currents through Ca²⁺-activated K⁺ channels.At the resting membrane potential of –60 mV, Ca²⁺-activated K⁺ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20–30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca²⁺. Thus the Ca²⁺-activated K⁺ channels may play a specific role in maintaining intracellular K⁺ concentrations during CSF secretion.     

Novel actions of ryanodine and analogues—Perturbers of potassium channels

The effects of ryanodine, 9,21-didehydroryanodine and 9,21-didehydroryanodol on two types of K⁺ channel (a maxi, Ca²⁺-activated, 170 pS channel (BK channel) and an inward rectifier, stretch-sensitive channel of 35 pS conductance (IK channel) found in the plasma membrane of locust skeletal muscle have been investigated. 10^–9M-10^–5M ryanodine irreversibly induced a dose-dependent reduction of the reversal potential (V_rev) of the currents of both channels, i.e. from 60 mV in the absence of the alkaloid to 15 mV for 10^–5M ryanodine, measured under physiologically normal K⁺ and Na⁺ gradients. In both cases the change in the ionic selectivity was Ca²⁺-independent. 9,21-didehydroryanodine and 9,21-didehyroryanodol also reduced V_rev, but only to 35 mV during application of 10^–5M of these compounds. Additionally, 9,21-didehydroryanodine reversibly diminished the conductances of the two K⁺ channels. To test the hypothesis that ryanoids increase Na⁺ permeability by enlarging the K⁺ channels, the channels were probed with quaternary ammonium ions during ryanoid application. When applied to the cytoplasmic face of inside-out patches exised from locust muscle membrane, TEA blocked the K⁺ channels in a voltage-dependent fashion. The dissociation constant (K_d(0)) for TEA block of the IK channel was reduced from 44 mM to 1 mM by 10^–7 M ryanodine, but the voltage-dependence of the block was unaffected. Qualitatively similar data were obtained for the BK channel. Ryanodine had no effect on the K_d for cytoplasmically-applied TMA. However, the voltage-dependence for TMA block was increased for both K⁺ channels, from 0.47 to 0.8 with 10^–6M ryanodine. The effects of ryanodine on TEA and TMA block support the hypothesis that ryanodine enlarges the K⁺ channels so as to facilitate permeation of partially hydrated Na⁺ ions.     

Comparative study of the effects of cromakalim (BRL 34915) and diazoxide on membrane potential, [Ca2+] i and ATP-sensitive potassium currents in insulin-secreting cells

Summary Patch-clamp and single cell [Ca²⁺] _i measurements have been used to investigate the effects of the potassium channel modulators cromakalim, diazoxide and tolbutamide on the insulin-secreting cell line RINm5F. In intact cells, with an average cellular transmembrane potential of –62±2 mV (n=42) and an average basal [Ca²⁺] _i of 102±6nm (n=37), glucose (2.5–10mm): (i) depolarized the membrane, through a decrease in the outward K_ATP current, (ii) evoked Ca²⁺ spike potentials, and (iii) caused a sharp rise in [Ca²⁺] _i . In the continued presence of glucose both cromakalim (100–200 m) and diazoxide (100 m) repolarized the membrane, terminated Ca²⁺ spike potentials and attenuated the secretagogue-induced rise in [Ca²⁺] _i . In whole cells (voltage-clamp records) and excised outside-out membrane patches, both cromakalim and diazoxide enhanced the current by opening ATP-sensitive K⁺ channels. Diazoxide was consistently found to be more potent than cromakalim. Tolbutamide, a specific inhibitor of ATP-sensitive K⁺ channels, reversed the effects of cromakalim on membrane potential and K_ATP currents.     

Blockade of cardiac outwardly rectifying K+ channels by TEA and class III antiarrhythmics — evidence against a single drug-sensitive channel site

Elementary K⁺ currents through cardiac outwardly rectifying K⁺ channels were recorded in insideout patches excised from cultured neonatal rat cardiocytes at 19 °C and at 9 °C. By studying the inhibitory effects of tetraethylammonium (TEA), quinidine and verapamil, the properties of this novel type of K⁺ channel were further characterized. Internal TEA (50 mmol/1) evoked a reversible decline of i_unit to 62.7 + 2.7% of control (at –7 mV), without significant changes of open state kinetics, indicating a blockade of the open K⁺ pore with kinetics too fast to be resolvable at 1 kHz. This TEA blockade was e-fold voltage-dependent, with a decrease of the apparent K_{D( TEA)} from 102 mmol/1 at –37 mV to 65 mmol/1 at +33 mV and, furthermore, became accentuated on lowering the internal K⁺ concentration. Thus, TEA competes with the permeant K⁺ for a site located in some distance from the cytoplasmic margin, within the K⁺ pore. Quinidine (100 mol/l), like verapamil (40 mol/1) reversibly depressed i_unit to about 80% of the control value (at –7 mV), but drug-induced fast flicker blockade proved voltage-insensitive between –27 mV and +23 mV These drugs gain access to a portion of the pore distinct from the TEA binding site whose occupancy by drugs likewise blocks K⁺ permeation. Both drugs showed a greater potency to depress P_o which, with quinidine,decreased reversibly to38.6 ± 11.1% (at –7 mV) and, with verapamil to 24.9 ± 9.1%(at –7 mV), mainly by an increase of the prolonged closed state (C,). This alteration of the gating process also includes a sometimes dramatic shortening of the open state. Most probably, cardiac K_{(outw.-rect.)} ⁺K⁺ _outw.-rect. channels possess a second drug-sensitive site whose occupancy by quinidine or verapamil may directly or allosterically stabilize their non-conducting configuration. Correspondence to: M. Kohlhardt     

Potassium accumulation in growing Methanobacterium thermoautotrophicum and its relation to the electrochemical proton gradient

Cultures of Methanobacterium thermoautotrophicum (Marburg) growing on media low in potassium accumulated the cation up to a maximal concentration gradient ([K⁺]_{intracellular}/[K⁺]_{extracellular}) of approximately 50,000-fold. Under these conditions, the membrane potential was determined by measuring the equilibrium distribution of the lipophilic cation (¹⁴C) tetraphenylphosphonium (TPP⁺). This cation was accumulated by the cells 350-to 1,000-fold corresponding to a membrane potential (inside negative) of 170–200 mV. The pH gradient, as measured by equilibrium distribution of the weak acid, benzoic acid, was found to be lower than 0.1 pH units (extracellular pH=6.8). The addition of valinomycin (0.5–1 nmol/mg cells) to the culture reduced the maximal concentration gradient of potassium from 50,000-to approximately 500-fold, without changing the membrane potential. After dissipation of the membrane potential by the addition of ¹²C-TTP⁺ (2 mol/mg cells) or tetrachlorosalicylanilide (3 nmol/mg cells), a rapid and complete efflux of potassium was observed.These data indicate that potassium accumulation in the absence of valinomycin is not in equilibrium with the membrane potential. It is concluded that at low extracellular K⁺ concentrations potassium is not accumulated by M. thermoautotrophicum via an electrogenic uniport mechanism.Non-common abbreviations TPP⁺ Tetra phenylphosphonium bromide - DTE Dithioerythritol - TCS 3,5,3,4-Tetrachlorosalycylanilide     

Channel formation in phospholipid bilayer membranes by the toxin ofHeminthosporium maydis,race T

Summary Southern Corn Leaf Blight is caused by a toxin produced by a virulent form ofHelminthosporium maydis (Race T). The toxin has been shown to uncouple oxidative phosphorylation and dissipate Ca²⁺ gradients in mitochondria isolated from susceptible, but not resistant, corn. The possibility that the toxin acted by increasing the permeability of membranes to ions was tested using a planar bilayer membrane system. Addition of the toxin to the bilayer system, under voltage-clamp conditions, resulted in stepwise increases in current across the phospholipid bilayer, a response characteristic for channel formers. Single-channel conductance in 1m KCl is 27 pS which corresponds to 1.7×10⁷ ions sec^–1 channel^–1 at 100 mV applied potential. The toxin channels are: (i) fairly uniform in conductance, (ii) ideally selective for K⁺ over Cl^–, and (iii) most conductive to H⁺. The channel showed the following selectivity for alkali metal cations: Rb⁺>K⁺>Cs⁺>Na⁺>Li⁺ (169731) based on the most frequently observed conductance in 1m chloride salts. The toxin showed no voltage dependence over the range of –100 to +100 mV. Channel formation was also a property of a synthetic analog (Cmpd IV) of the toxin. The ability of the native toxin to form channels may be a mode of toxin action on mitochondrial membranes from susceptible corn.     

Action potentials inAcetabularia: Measurement and simulation of voltage-gated fluxes

Summary Amounts and temporal changes of the release of the tracer ions K⁺ (⁸⁶Rb⁺),²²Na⁺, and³⁶Cl^– as well as of H⁺ in the course of action potentials inAcetabularia have been recorded. New results and model calculations confirm in quantitative terms the involvement of three major ion transport systemsX in the plasmalemma: Cl^– pumps, K⁺ channels, and Cl^– channels (which are marked in the following by the prefixes,P, K andC) with their equilibrium voltages _X V _e and voltage/time-dependent conductances, which can be described by the following, first approximation. Let the maximum (ohmic) conductance of each of the three populations of transporter species be about the same (_P L, _KL,_C L=1) but voltage gating be different: the pump ( _{p V e} about –200 mV) being inactivated (open,oclosed,c) at positive going transmembrane voltages,V _m; the K⁺ channels (_K V _e about –100 mV) are inactivated at negative goingV _m; and the Cl^– channels (_C V _e: around 0 mV), which are normally closed (c) at a restingV _m (near_PV_e) go through an intermediate open (o) state at more positiveV _m before they enter a third shut state (s) in series. Model calculations, in which voltage sensitivities are expressed by the factorf=exp(V _mF/(2RT)), simulate, the action potential fairly well with the following parameters (_PK_co10/f ks^–1,_PK_oc1000·f ks^–1,_KK_co200·f ks^–1,_Kk_oc2/f ks^–1,_cK_co500·f ks^–1,_CK_oc5/f ks^–1,_CK_so0.1/f ks^–1,_Ck_os20·f ks^–1). It is also shown that the charge balance for the huge transient Cl^– efflux, which frequently occurs during an action potential, can be accounted for by the observation of a corresponding release of Na⁺.     

Transport of potassium inChara australis: II. Kinetics of a symport with sodium

Summary An electrogenic K⁺–Na⁺ symport with a high affinity for K⁺ has been found inChara (Smith & Walker, 1989). Under voltage-clamp conditions, the symport shows up as a change in membrane current upon adding either K⁺ or Na⁺ to the bathing medium in the presence of the other. Estimation of kinetic parameters for this transport has been difficult when using intact cells, since K⁺–Na⁺ current changes show a rapid falling off with time at K⁺ concentrations above 50 m. Cytoplasm-enriched cell fragments are used to overcome this difficulty since they do not show the rapid falling off of current change seen with intact cells. Current-voltage curves for the membrane in the absence or presence of either K⁺ or Na⁺ are obtained, yielding difference current-voltage curves which isolate the symport currents from other transport processes. The kinetic parameters describing this transport are found to be voltage dependent, withK _m for K⁺ ranging from 30 down to 2 m as membrane potential varies from –140 to –400 mV, andK _m for Na⁺ ranging between 470 and 700 m over a membrane potential range of –140 to –310 mV.Two different models for this transport system have been investigated. One of these involves the simultaneous transport of both the driver and substrate ions across the membrane, while the other allows for the possibility of the two ions being transported consecutively in two distinct reaction steps. The experimental results are shown to be consistent with either of these cotransport models, but they do suggest that binding of K⁺ occurs before that of Na⁺, and that movement of charge across the membrane (the voltage-dependent step) occurs when the transport protein has neither K⁺ nor Na⁺ bound to it.     

Satellite glial cells In Situ within mammalian prevertebral ganglia express K+ channels active at rest potential

Patch-clamp experiments were performed on satellite glial cells wrapped around sympathetic neurons in the rabbit coeliac ganglion. With the cleaning method used, the glial cells could be kept in place and were directly accessible to the patch-clamp pipettes. Whole-cell recordings showed that glial cells had almost ohmic properties. Their resting potential (–79.1±1.2 mV) was found to be very nearly the same as the K⁺ reversal potential and 20 mV more negative than that of the neurons they encapsulated. Unitary currents from ionic channels present in the glial membrane were recorded in the cell-attached configuration with pipettes filled with various amounts of K⁺, Na⁺ and gluconate. Only K⁺-selective channels with slight inwardly rectifying properties (in the presence of 150 mM [K⁺]₀) were detected. These channels were active (P ₀=0.7–0.8) at the cell resting potential. The channel conductance, but not its opening probability, was dependent on the [K⁺] in the pipette. Cl^–-selective channels (outwardly rectifying and large conductance channels) were detected in excised patches.The properties of the K⁺ channels (increased inward current with [K⁺] and detectable outward current at low [K⁺]) are well suited for siphoning the K⁺ released by active neurons.     

The voltage-dependent potassium-uptake channel of corn coleoptiles has permeation properties different from other K+ channels

The initial response of coleoptile cells to growth hormones and light is a rapid change in plasma-membrane polarization. We have isolated protoplasts from the cortex of maize (Zea mays L.) coleoptiles to study the electrical properties of their plasma membrane by the patch-clamp techniqueUsing the whole-cell configuration and cell-free membrane patches we could identify an H⁺-ATPase, hyperpolarizing the membrane potential often more negative than -150 mV, and a voltage-dependent, inward-rectifying K⁺ channel (unit conductance 5–7 pS) as the major membrane conductan-ces Potassium currents through this channel named CKC1_in (for Coleoptile K ⁺ Channel inward rectifier) were elicited upon voltage steps negative to -80 mV, characterized by a half-activation potential of -112 mV. The kinetics of activation, well described by a double-exponential process, were strongly dependent on the degree of hyperpolarization and the cytoplasmic Ca²⁺ level. Whereas at nanomolar Ca²⁺ concentrations K⁺ currents increased with a t_1/2=16 ms (at -180 mV), higher calcium levels slowed the activation process about fourto fivefoldUpon changes in the extracellular K⁺ concentration the reversal potential of the K⁺ channel followed the Nernst potential for potassium with a 56-mV shift for a tenfold increaseThe absence of a measurable conductance for Na⁺, Rb⁺, Cs⁺ and a permeability ratio P_{NH 4} ⁺ /P_K+ around 0.25 underlines the high selectivity of CKC1_in for K⁺In contrast to Cs⁺, which at submillimolar concentration blocks the channel in a voltage-dependent manner, Rb⁺, often used as a tracer for K+, does not permeate this type of K⁺ channelThe lack of Rb⁺ permeability is unique with respect to other K⁺ transporters. Therefore, future molecular analysis of CKC1_in, considered as a unique variation of plant inward rectifiers, might help to understand the permeation properties of K⁺ channels in general.Abbreviations CKC1_in Coleoptile K ⁺ Channel inward rectifier - U membrane voltage - I_ss steady-state currents - I_tail tail currents Experiments were conducted in the laboratory of F.G. during the stay of RHas a guest professor sponsored by Special Project RAISA, subproject N2.1, paper N2155.     

The influence of pH and external H+ concentration on caesium toxicity and accumulation inEscherichia coli andBacillus subtilis

Summary Toxicity screening ofEscherichia coli NCIB 9484 andBacillus subtilis 007, NCIB 168 and NCIB 1650 has shown Cs⁺ to be the most toxic Group 1 metal cation. However, toxicity and accumulation of Cs⁺ by the bacteria was affected by two main external factors; pH and the presence of other monovalent cations, particularly K⁺. Over the pH range 6–9 bothE. coli andB. subtilis showed increasing sensitivity towards caesium as the pH was raised. The presence of K⁺ and Na⁺ in the laboratory media used lowered caesium toxicity and lowered acumulation of the metal. In order to assess accurately Cs⁺ toxicity towards the bacterial strains it was therefore necessary to define the K⁺:Cs⁺ ratio in the external medium. The minimum inhibitory K⁺:Cs⁺ concentration ratio for theBacillus strains tested was in the range 12–13 whileE. coli had a minimum inhibitory K⁺:Cs⁺ concentration ratio of 16.     

Mechanism of blue-light-induced plasma-membrane depolarization in etiolated cucumber hypocotyls

A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H⁺-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca²⁺-channel blockers (verapamil, La³⁺) or by reducing extracellular free Ca²⁺ by treatment with ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca²⁺ is involved. No effects of the K⁺-channel blocker tetraethylammonium (TEA⁺) were detected. Vanadate and KCN, used to inhibit the H⁺-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11–26% after 1–2 min of BL. Input resistance of trichome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H⁺-ATPase with subsequent transient activation of one or more types of ion channels.Abbreviations and Symbols BL blue light - CI current injection - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - TEA⁺ tetraethylammonium - V_m membrane voltage We wish to thank Drs. Adam Bertl and Clifford L. Slayman, Yale School of Medicine, New Haven, Conn., USA, for helpful discussions. This work was supported by a Natural Sciences and Engineering Research Council of Canada Scholarship (E.P.S.) and National Science Foundation Grant DMB-8351030 (D.J.C.).     

Functional identification of ATP-sensitive K<Superscript>+</Superscript> uniporter in mitochondria from sugar beet taproot

Mitochondria isolated from sugar beet (Beta vulgaris L.) taproot were shown to swell spontaneously after the transfer from a sucrose-containing isolation medium to isoosmotic potassium chloride solutions. The kinetics of this process was strongly retarded after the replacement of potassium with sodium in the incubation medium and was substantially stimulated by the electron-transport chain activity and valinomycin. At neutral pH of the incubation medium, the rate of K⁺-dependent swelling of mitochondria decreased by 30–50% after adding 1 mM ATP but was insensitive to other nucleotides (GTP, UTP, and CTP). In the medium acidified to pH 6.0, the addition of ATP caused shrinkage of mitochondria that had been swollen in the KCl medium. In the absence of this nucleotide, the kinetics of K⁺-dependent swelling of mitochondria was considerably decelerated upon the acidification of the incubation medium. The effects of ATP were independent of the presence or absence of oligomycin and atractyloside. However, the ATP-dependent shrinkage of mitochondria was inhibited in the presence of quinine, and this agent also inhibited K⁺-dependent swelling of organelles in potassium acetate solutions. The presence of K⁺ ions in the incubation medium caused a rapid dissipation of the mitochondrial membrane potential () that was generated during succinate oxidation. The addition of ATP to the reaction medium resulted in the oligomycin-insensitive restoration of . The results are regarded as evidence that the membrane of taproot mitochondria is endowed with functionally active ATP-sensitive K⁺ uniporter. This system is likely to represent a K⁺ channel that catalyzes the electrogenic transfer of potassium ions to the mitochondrial matrix. It is supposed that the membrane of taproot mitochondria also contains a quinine-sensitive K⁺/H⁺ antiporter that catalyzes the efflux of potassium from the matrix or, on the contrary, the accumulation of K⁺ in the presence of potassium acetate.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 209–215.Original Russian Text Copyright © 2005 by Shugaev, Andreev, Vyskrebentseva.This revised version was published online in April 2005 with a corrected cover date.     

Evidence for a K+ channel in bovine chromaffin granule membranes: single-channel properties and possible bioenergetic significance

A K⁺ channel was incorporated into voltage-clamped planar lipid bilayers from bovine chromaffin granules and resealed granule membranes (ghosts). It was not incorporated from plasma membrane-rich fractions from the adrenal medulla. The channel had a conductance of 400 pS in symmetric 450 mM KCI, with the permeability sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, and was insensitive to both Ca²⁺ and charybdotoxin. It exhibited complex gating kinetics, consistent with the presence of multiple open and closed states, and its gating was voltage-dependent. The channels appeared to incorporate into bilayers with the same orientation, and were blocked from one side (the side of vesicle addition) by 0.2-1 mM TEA'. The block was slightly voltage-dependent. Acidification of resealed granule membranes in response to external ATP (which activated the vacuolartype ATPase) was significantly reduced in the presence of 1 mM intralumenal TEACI (with 9 mM KCl), and parallel measurements with the potential-sensitive dye Oxonol V showed that such vesicles tended to develop higher internal-positive membrane potentials than control vesicles containing only 10 mM KCI. 1 mM TEA⁺ had no effect on proton-pumping activity when applied externally, and did not directly affect either the proton-pumping or ATP hydrolytic activity of the partially-purified ATPase. These results suggest that chromaffin granule membranes contain a TEA⁺-sensitive K⁺ channel which may have a role in regulating the vesicle membrane potential. Correspondence to: R. H. Ashley     

Characterization of single K+ channels inHelix pomatia neurons

The measurements of unitary outward ion currents in unidentified neurons of the snailHelix pomatia with the patch-clamp technique in a cell-attached configuration showed the presence of several types of K⁺ channels. We investigated three types of K⁺ channels: with big (75 pS, BKC), medium (22 pS, MKC), and small (6.2 pS, SKC) unitary conductance. BKC and MKC were activated at a membrane potential of about –30 mV, whereas SKC were activated at more negative potentials, with opening probability of the latter channels significantly decreasing at potentials more positive than –30 mV. Pharmacological investigation showed that BKC and MKC channel activity disappeared after 8–10 min of cell patching with a pipette solution containing 60 mM Cs⁺, whereas MKC channels remained unaffected. BKC and MKC were proved to be more sensitive to TEA (20 mM), whereas SKC were selectively sensitive to 4-AP (10 mM). Cd²⁺ (100 µM) in the pipette solution decreased the unitary conductance of BKC channels by 55 % and that of MKC channels by about 31 %. In contrast, the unitary conductance of SKC channels was not changed by the above blocker. Bath application of 10 µM 5-HT showed that MKC were suppressed by 5-HT, whereas SKC and BKC were insensitive to this transmitter. It is supposed that BKC can be classified as big-conductance Ca²⁺-dependent K⁺ channels (KCa) or to 5-HT-sensitive K⁺ channels (S-type channels), while MKC correspond to intermediate-conductance KCa, and SKC channels comply well with the characteristics of A-type K⁺ current.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 250–259, November–December, 1996.     

Two steady states in membrane potential deflection in relation to chemoreception and chemotaxis byPhysarum polycephalum

Summary In the plasmodium ofPhysarum polycephalum application of various monovalent cation salts elicited either a depolarization or a hyperpolarization of the membrane potential. The hyperpolarization was restricted to phosphates and bicarbonates of large cations (Na⁺, Li⁺, NMe₄ ⁺, NEt₄ ⁺). More than 50 other combinations of cations (K⁺, Rb⁺, Cs⁺, NH₄ ⁺) and anions (Cl^–, NO₃ ^–, SO₄ ^2–, acetate, lactate, citrate, etc.) induced the depolarization. In both cases the magnitude of the deflection in membrane potential () varied linearly with logarithm of concentration above the threshold C_th (10^–4 M for all monovalent cation salts examined) according to the following equation: =± R log (C/C_th).The value of R was 10–15 mV, and plus and minus signs correspond to depolarization and hyperpolarization, respectively. Depolarizing and hyperpolarizing agents competed with each other and exhibited a sharp transition between the two states of the membrane which were characterized by — R and R in the above equation or displayed a strong hysteresis, depending on which agents had first been applied to the plasmodial membrane. This transition in the membrane potential corresponded to the transition between positive and negative taxis at the behavioral level.     

Evidence for two K+ currents activated upon hyperpolarization ofParamecium tetraurelia

Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K⁺ solutions elicits a complex of Ca²⁺ and K⁺ currents. The tail current that accompanies a return to holding potential (–40 mV) contains two K⁺ components. The tail current elicited by a step to –110 mV of 50-msec duration contains fast-decaying (3.5 msec) and slow-decaying (20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K⁺], suggesting that they represent pure K⁺ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K⁺ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca²⁺ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K⁺ currents are inhibited by extracellular TEA⁺ in a concentration-dependent, noncooperative manner, whereas the fast K⁺ current alone is inhibited by 0.7mm quinidine.