Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Mechanisms of fusicoccin action: evidence for concerted modulations of secondary K+ transport in a higher plant cell

Fusicoccin (FC) has long been known to promote K⁺ uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H⁺ pumping stimulated in FC, could help drive K⁺ uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (V _m) alone; furthermore, unidirectional flux analyses have shown that in the toxin K⁺ (⁸⁶Rb⁺) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180–192). Thus, the activities of specific pathways for K⁺ movement across the membrane could be modified in FC. We have explored a role for K⁺ channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical (⁸⁶Rb⁺) flux and in electrical current under voltage clamp was followed, using the K⁺ channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K⁺-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K⁺ channel current contributed appreciably to charge balance maintainingV _m, and adding 10 mM TEA to block the current depolarized (positive-going)V _m; TEA also reduced⁸⁶Rb⁺ efflux by 68–80%. Following treatments with 10 M FC, both K⁺ channel current and⁸⁶Rb⁺ efflux decayed, irreversbly and without apparent lag, to 10%–15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced⁸⁶Rb⁺ influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K⁺ (Rb⁺), despite membrane potentials which were 30–60 mVpositive of the K⁺ equilibrium potential. These results tentatively link (chemical) cation efflux to charge movement through the K⁺ channels. They offer evidence of an energy-coupled mechanism for K⁺ uptake in guard cells. Finally, the data reaffirm early suspicions that FC alters profoundly the K⁺ transport capacity of the cells, independent of any changes in membrane potential.Abbreviations and symbols E _K equilibrium potential for K⁺ - FC fusicoccin - Hepes 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid - G _m membrane (slope) conductance atV _m - I-V current-voltage (relationship) - apparent rate constant for exchange - K _i ⁺ , K ₀ ⁺ intracellular, extracellular K⁺ (concentration) - TEA tetraethylammonium chloride - V _m free-running membrane potential (difference)     

Ca2+-activated K+ conductance of the human red cell membrane: Voltage-dependent Na+ block of outward-going currents

Summary Human red cells were prepared with various cellular Na⁺ and K⁺ concentrations at a constant sum of 156mm. At maximal activation of the K⁺ conductance,g _K(Ca), the net efflux of K⁺ was determined as a function of the cellular Na⁺ and K⁺ concentrations and the membrane potential,V _m , at a fixed [K⁺]_ex of 3.5mm.V _m was only varied from (V _m –E _K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g _K(Ca) as a function of cellular Na⁺ and K⁺ concentrations atV _m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na⁺. Since the present Ca²⁺-activated K⁺ channels have been shown to be of the multi-ion type, the experimental data from each set of Na⁺ and K⁺ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na⁺ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na⁺ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca²⁺ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg _K(Ca) for outward currents at (V _m –E _K)>25 25 mV reflects a voltage-dependent Na⁺ block of the Ca²⁺-activated K⁺ channels.     

Potassium-proton symport inNeurospora: kinetic control by pH and membrane potential

Summary Active transport of potassium in K⁺-starvedNeurospora was previously shown to resemble closely potassium uptake in yeast,Chlorella, and higher plants, for which K⁺ pumps or K⁺/H⁺-ATPases had been proposed. ForNeurospora, however, potassium-proton cotransport was demonstrated to operate, with a coupling ratio of 1 H⁺ to 1 K⁺ taken inward so that K⁺, but not H⁺, moves against its electrochemical gradient (Rodriguez-Navarro et al.,J. Gen. Physiol. 87:649–674).In the present experiments, the current-voltage (I–V) characteristic of K⁺–H⁺ cotransport in spherical cells ofNeurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in theNeurospora plasma membrane. Addition of 5-200 M K⁺ to the bathing medium causes 10–150 mV depolarization of the unclamped membrane, and yields a sigmoidI–V curve with a steep slope (maximal conductance of 10–30 S/cm²) for voltages of –300 to –100 mV, i.e., in the normal physiologic range. Outside that range the apparentI–V curve of the K⁺-H⁺ symport saturates for both hyperpolarization and depolarization. It fails to cross the voltage axis at its predicted reversal potential, however, an effect which can be attributed to failure of theI–V difference method under reversing conditions.In the absence of voltage clamping, inhibitors—such as cyanide or vanadate—which block the primary proton pump inNeurospora also promptly inhibit K⁺ transport and K⁺-H⁺ currents. But when voltage clamping is used to offset the depolarizing effects of pump blockade, the inhibitors have no immediate effect on K⁺-H⁺ currents. Thus, the inhibition of K⁺ transport usually observed with these agents reflects the kinetic effect of membrane depolarization rather than any direct chemical action on the cotransport system itself.Detailed study of the effects of [K⁺]_o and pH_o on theI–V curve for K⁺-H⁺ symport has revealed that increasing membrane potential systematicallydecreases the apparent affinity of the transporter for K⁺, butincreases affinity for protons (K _m range: for [K⁺]_o, 15–45 M; for [H⁺]_o, 10–35 nM). This behavior is consistent with two distinct reaction-kinetic models, in which (i) a neutral carrier binds K⁺ first and H⁺ last in the forward direction of transport, or (ii) a negatively charged carrier (–2) binds H⁺ first and K⁺ last.     

Potassium-dependent,bipolar gating of K+ channels in guard cells

Summary Guard cells of higher plants control transpirational water loss and gas exchange for photosynthesis by opening and closing pores in the epidermis of the leaf. To power these turgordriven movements, guard cells accumulate (and lose) 200 to 400mm (1 to 3 pmol/cell) K⁺, fluxes thought to pass through K⁺ channels in the guard cells plasma membrane. Steady-state current-voltage (I–V) relations of intactVicia guard cells frequently show large, outward-going currents at potentials approaching 0 mV. Since this current could be carried by K⁺ channels, its pharmacology and dependence on external K⁺ (K _v ⁺ ) has been examined under voltage clamp over an extended potential range. Measurements were carried out on cells which showed little evidence of primary electrogenic transport, thus simplifying analyses. Clamping these cells away from the free-running membrane potential (V _m ) revealed an outward-rectifying current with instantaneous and time-dependent components, and sensitive to the K⁺ channel blocker tetraethylammonium chloride. The current declined also under metabolic blockade with NaCN and in the presence of diethylstilbesterol, responses which were attributed to secondary effects of these inhibitors. The putative K⁺ current rose with voltage positive toV _m but it decayed over two voltage ranges, one negative toV _m and one near +100 mV, to give steady-stateI–V relations with two regions of negative (slope) conductance. Voltage-dependent and kinetic characteristics of the current were affected by K _v ⁺ and followed the K⁺ equilibrium potential. Against a (presumably) low background of primary ion transport, the K⁺ current contributed appreciably to charge balance atV _m in 0.1mm as well as in 1 to 10mm K _v ⁺ . Thus, gating of these K⁺ channels compensates for the prevailing K⁺ conditions to ensure net K⁺ movement out of the cell.     

Intracellular K+ activities and cell membrane potentials in a K+-transporting epithelium,the midgut of tobacco hornoworm (Manduca sexta)

Summary Transbasal electrical potential (V _b) and intraepithelial potassium chemical activity ((K⁺) _i ) were measured in isolated midgut epithelium of tobacco hornworm (Manduca sexta) using double-barrelled glass microelectrodes. Values ofV _b ranging from +8 to –48 mV (relative to blood side) were recorded. For all sites, (K⁺) _i is within a few millivolts of electrochemical equilibrium with the blood side bathing solution. Sites more negative than –20 mV show relatively high sensitivity ofV _b to changes in blood side K⁺ concentration: 43% of these sites can be marked successfully with iontophoresed Lucifer yellow CH dye and shown to represent epithelial cells of all three types present in the midgut. In about half of successful marks, dye-coupling of several adjacent cells is seen. Low potential sites — those withV _b less negative than –20 mV —typically do not show high sensitivity ofVb to changes of external K⁺, but rather (K⁺) _i rapidly approaches the K⁺ activity of blood side bathing solution. These sites can seldom be marked with Lucifer yellow (4% success). The mean (K⁺) _i of the high potential sites is 95±29 (sd)mm under standard conditions, a value which is in accord with published values for the whole tissue.     

Evidence for coupling between Na+ pump activity and TEA-sensitive K+ currents in Xenopus laevis oocytes

Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na⁺-K⁺-ATPase activity from the dihydroouabain-sensitive current (I _DHO) in the presence of increasing concentrations of tetraethylammonium (TEA⁺; 0, 5, 10, 20, 40 mm), a well-known blocker of K⁺ channels. The effects of TEA⁺ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V _M) and a saturable inhibitory component affecting an outward current easily detectable at positive V _M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA⁺ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA⁺ on K⁺ channels. Interestingly, this latter component disappears when the Na⁺-K⁺-ATPase is inhibited by 10 m DHO. Conversely, TEA⁺ inhibits a component of I _DHO with a k _d of 25±4 mm at +50 mV. As the TEA⁺-sensitive current present in I _DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K⁺ channels whose activity varies in parallel with the Na⁺-K⁺-ATPase activity. Supporting this hypothesis, the inward portion of this TEA⁺-sensitive current can be completely abolished by the addition of 1 mm Ba²⁺ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA⁺-sensitive K⁺ currents and indicates that, in the absence of effective K⁺ channel inhibitors, I _DHO does not exclusively represent the Na⁺-K⁺-ATPase-generated current.     

Ion transport across leech integument

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm^-2) with an initial short-circuit current of 29.0±2.9 A·cm^-2. Removal of Na⁺ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l^-1) added to the basolateral solution, depressed the short-circuit current completely. The Na⁺ current saturated at a concentration of 90 mmol Na⁺·l^-1 in the apical solution (K _M=11.2±1.8 mmol·l^-1). Amiloride (100 mol·l^-1) on the apical side inhibited ca. 40% of the Na⁺ current and indicated the presence of Na⁺ channels. The dependence of Na⁺ current on the amiloride concentration followed Michaclis-Menten kinetics (K _i=2.9±0.4 mol·l^-1). The amiloride analogue benzamil had a higher affinity to the Na⁺ channel (K _i=0.7±0.2 mol·l^-1). Thus, Na⁺ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na⁺·l^-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na⁺ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l^-1) and isobutylmethylxanthine (1 mmol·l^-1) nearly doubled short-circuit current and increased amiloride-sensitive Na⁺ currents by 50%. By current fluctuation analysis we estimated single Na⁺ channel current (2.7±0.9 pA) and Na⁺ channel density (3.6±0.6 channels·m^-2) under control conditions. After cyclic adenosine monophosphate stimulation Na⁺ channel density increased to 5.4±1.1 channels·m^-2, whereas single Na⁺ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na⁺ channel, and show the similarities and differences to vertebrate Na⁺ channels. Whereas the channel properties are different from the classical vertebrate Na⁺ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na⁺ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na⁺ channels.Abbreviations slope of the background noise component - ADH antidiuretic hormone - cAMP cyclic adenosine monophosphate - f frequency - f _c coner frequency of the Lorentzian noise component - Hepes N-hydroxyethylpiperazine-N-ethanesulphonic acid - BMX isobutyl-methylxanthine - i _Na single Na⁺ channel current - I _Na max, maximal inhibitable Na⁺ current - I _SC short circuit current - K _i half maximal blocker concentration - K _M Michaelis constandard error of the mean - S _(f) power density of the Lorentzian noise component - S ₀ plateau value of the Lorentzian noise component - TMA tetramethylammonium - Trizma TRIS-hydroxymethyl-amino-methane - V _max maximal reaction velocity - V _T transepithelial potential - K half maximal blocker concentration     

Potassium channel currents in intact stomatal guard cells: rapid enhancement by abscisic acid

Evidence of a role for abscisic acid (ABA) in signalling conditions of water stress and promoting stomatal closure is convincing, but past studies have left few clues as to its molecular mechanism(s) of action; arguments centred on changes in H⁺-pump activity and membrane potential, especially, remain ambiguous without the fundamental support of a rigorous electrophysiological analysis. The present study explores the response to ABA of K⁺ channels at the membrane of intact guard cells ofVicia faba L. Membrane potentials were recorded before and during exposures to ABA, and whole-cell currents were measured at intervals throughout to quantitate the steady-state and time-dependent characteristics of the K⁺ channels. On adding 10 M ABA in the presence of 0.1, 3 or 10 mM extracellular K⁺, the free-running membrane potential (V _m) shifted negative-going (–)4–7 mV in the first 5 min of exposure, with no consistent effect thereafter. Voltage-clamp measurements, however, revealed that the K⁺-channel current rose to between 1.84- and 3.41-fold of the controls in the steady-state with a mean halftime of 1.1 ± 0.1 min. Comparable changes in current return via the leak were also evident and accounted for the minimal response inV _m. Calculated atV _m, the K⁺ currents translated to an average 2.65-fold rise in K⁺ efflux with ABA. Abscisic acid was not observed to alter either K⁺-current activation or deactivation.These results are consistent with an ABA-evoked mobilization of K⁺ channels or channel conductance, rather than a direct effect of the phytohormone on K⁺-channel gating. The data discount notions that large swings in membrane voltage are a prerequisite to controlling guard-cell K⁺ flux. Instead, thev highlight a rise in membranecapacity for K⁺ flux, dependent on concerted modulations of K⁺-channel and leak currents, and sufficiently rapid to account generally for the onset of K⁺ loss from guard cells and stomatal closure in ABA.     

High-conductance K+ channel in pancreatic islet cells can be activated and inactivated by internal calcium

Summary The Ca²⁺-activated K⁺ channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage (I/V) relationship showed outward rectification and the null potential was more negative than –40 mV. In symmetrical K⁺-rich solutions the single-channelI/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca²⁺-free solution containing 2mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities (P) above 0.1. Raising the free Ca²⁺ concentration in contact with the membrane inside ([Ca²⁺]_i) to 1.5×10^–7 m had little effect on the relationship between membrane potential andP. When [Ca²⁺]_i was increased to 3×10^–7 m and 6×10^–7 m smaller potential changes were required to open the channels. Increasing [Ca²⁺]_i further to 8×10^–7 m again activated the channels, but the relationship between membrane potential andP was complex. Changing the membrane potential from –50 mV to +20 mV increasedP from near 0 to 0.6 but further polarization to +50 mV decreasedP to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca²⁺]_i=1 and 2 m. In this situation a membrane potential change from –70 to +20 mV increasedP from near 0 to about 0.7 but further polarization to +80 mV reducedP to less than 0.1. The high-conductance K⁺ channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca²⁺]_i within the range 0.1 to 1 m which suggests a physiological role for this channel in regulating the membrane potential and Ca²⁺ influx through voltage-activated Ca²⁺ channels.     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

Effects of extracellular calcium and protons on osteoclast potassium currents

During resorption of mineralized tissues, osteoclasts are exposed to marked changes in the concentration of extracellular Ca²⁺ and H⁺. We examined the effects of these cations on two types of K⁺ currents previously described in these cells. Whole-cell patch clamp recordings of membrane currents were made from osteoclasts freshly isolated from neonatal rats. In control saline (1 mm Ca²⁺, pH 7.4), the voltage-gated, outwardly rectifying K⁺ current activates at approximately 45 mV and the conductance is half-maximally activated at –29 mV (V _0.5). Increasing [Ca²⁺]_out rapidly and reversibly shifted the current-voltage (I–V) relation to more positive potentials. Current at –29 mV decreased to 28 and 9% of control current at 5 and 10 mm [Ca²⁺]_out, respectively. This effect of elevating [Ca²⁺]_out was due to a positive shift of the K⁺ channel voltage activation range. Zn²⁺ or Ni²⁺ (5 to 500 m) also shifted the I–V relation to more positive potentials and had additional effects consistent with blockade of the K⁺ channel. Based on the extent to which these divalent cations affected the voltage activation range of the outwardly rectifying K⁺ current, the potency sequence was Zn²⁺ > Ni²⁺ > Ca²⁺. Lowering or raising extracellular pH also caused shifts of the voltage activation range to more positive or negative potentials, respectively. In contrast to their effects on the outwardly rectifying K⁺ current, changes in the concentration of extracellular H⁺ or Ca²⁺ did not shift the voltage activation range of the inwardly rectifying K⁺ current. These findings are consistent with Ca²⁺ and other cations affecting voltage-dependent gating of the osteoclast outwardly rectifying K⁺ channel through changes in surface charge.This work was supported by The Arthritis Society and the Medical Research Council of Canada. S.M.S. is supported by a Scientist Award and S.J.D. by a Development Grant from the Medical Research Council.     

Ca2+-activated K+ conductance of human red cell membranes exhibits two different types of voltage dependence

Summary The voltage dependence for outward-going current of the Ca-activated K⁺ conductance (g _k (Ca)) of the human red cell membrane has been examined over a wide range of membrane potentials (V _m) at constant values of [K⁺]_ex, [K⁺]_c and pH_c, the intact cells being preloaded to different concentrations of ionized calcium. Outward-current conductances were calculated from initial net effluxes of K⁺ and the corresponding (V _m-E_k) values. The basic conductance, defined as the outward-current coductance at (V _m-E_k) 20 mV and [K⁺]_ex 3mM (B. Vestergaard-Bogind, P. Stampe and P. Christophersen,J. Membrane Biol. 95:121–130, 1987) was found to be a function of cellular ionized Ca. At all degrees of Ca activationg _K(Ca) was an apparently linear function of voltage (V _m range –40 to +70 mV), the absolute level as well as the slope decreasing with decreasing activation. In a simple two-state model the constant voltage dependence can, at the different degrees of Ca activation, be accounted for by a Boltzmann-type equilibrium function with an equivalent valence of 0.4, assuming chemical equilibrium atV _m=0 mV. Alternatively, the phenomenon might be explained by a voltage-dependent block of the outward current by an intracellular ion. Superimposed upon the basic conductance is the apparently independent inward-rectifying steep voltage function with an equivalent valence of 5 and chemical equilibrium at the givenE _K value.Abbreviations CCCP carbonyl cyanidem-chlorophenylhydrazone - DIDS 4,4-diisothiocyanostilbene-2,2-disul     

Cytosolic calcium regulates a potassium current in corn (Zea mays) protoplasts

Summary The voltage- and time-dependent K⁺ current,I _K ⁺ _out , elicited by depolarization of corn protoplasts, was inhibited by the addition of calcium channel antagonists (nitrendipine, nifedipine, verapamil, methoxyverapamil, bepridil, but not La³⁺) to the extracellular medium. These results suggested that the influx of external Ca²⁺ was necessary for K⁺ current activation. The IC₅₀, concentration of inhibitor that caused 50% reduction of the current, for nitrendipine was 1 m at a test potential of +60 mV following a 20-min incubation period.In order to test whether intracellular Ca²⁺ actuated the K⁺ current, we altered either the Ca²⁺ buffering capacity or the free Ca²⁺ concentration of the intracellular medium (pipette filling solution). By these means,I _K ⁺ _out could be varied over a 10-fold range. Increasing the free Ca²⁺ concentration from 40 to 400nm also shifted the activation of the K⁺ current toward more negative potentials. Maintaining cytoplasmic Ca²⁺ at 500nm with 40nm EGTA resulted in a more rapid activation of the K⁺ current. Thus the normal rate of activation of this current may reflect changes in cytoplasmic Ca²⁺ on depolarization. Increasing intracellular Ca²⁺ to 500nm or 1 m also led to inactivation of the K⁺ current within a few minutes. It is concluded thatI _K ⁺ _out is regulated by cytosolic Ca²⁺, which is in turn controlled by Ca²⁺ influx through dihydropyridine-, and phenylalkylamine-sensitive channels.     

Factors controlling the K+ conductance inChara

Summary Previous current/voltage (I/V) investigations of theChara K⁺ state have been extended by increasing the voltage range (up to +200 mV) through blocking the action potential with La³⁺. A region of negative slope was found in theI/V characteristics at positive PD's, similar to that already observed at PD's more negative than the resting level. These decreases in membrane currents at PD's more negative than –150 mV and at PD's close to 0 or positive are thought to arise from the K⁺ channel closure. Both the negative slope regions could be reversibly abolished by 0.1mm K⁺, 20mm Na⁺, more than 10mm Ca²⁺ or 5mm tetraethylammonium (TEA). The K⁺ channels are therefore blocked by TEA, closed by low [K⁺] _o or high [Ca²⁺] _o and are highly selective to K⁺ over Na⁺. With the K⁺ channels closed, the remainingI/V profile was approximately linear over the interval of 400 mV (suggesting a leakage current), but large rectifying currents were observed at PD's more positive than +50 mV. These currents showed a substantial decrease in high [Ca²⁺] _o , sometimes displayed a slight shift to more positive PD's with increasing [K⁺] _o and were unaffected by TEA or changes in [Na⁺] _o . The slope of the linear part of theI/V profile was steeper in low [K⁺] _o than in TEA or high [Na⁺] _o (indicating participation of K⁺, but not Na⁺, in the leak current). Diethylstilbestrol (DES) was employed to inhibit the proton pump, but it was found that the leakage current and later the K⁺ channels were also strongly affected.     

Potassium channels and different states ofChara plasmalemma

Summary The current-voltage (I/V) technique was employed to investigate the different electrophysiological states of theChara plasmalemma and their interaction under a range of conditions. In K⁺ state the membrane became very permeable (conductances >20 S m 2) as [K⁺]₀ increased to 10mm. As the cells were then easily damaged by the voltage-clamp procedures, it was difficult to determine the saturation K⁺ conductance. TEA (tetraethylammonium chloride) reversibly blocked the K⁺ channels, but had no effect on theI/V curve of the pump state, indicating that the K⁺ channels were not participating in this state. Acid pH₀ (4.5) diminished the K⁺ conductance, but did not alter the response of the K⁺ channels to change in [K⁺]₀. Alkaline pH₀ (11.0) madeChara resting PD bistable: the PD either stayed near the estimatedE _K and theI/V curve showed a negative conductance region typical of the K⁺ state, or it hyperpolarized and the near-linearI/V profile of the proton-permeable state was observed.     

Selective block by α-dendrotoxin of the K+ inward rectifier at the Vicia guard cell plasma membrane

The efficacy and mechanism of -dendrotoxin (DTX) block of K⁺ channel currents in Vicia stomatal guard cells was examined. Currents carried by inward- and outward-rectifying K⁺ channels were determined under voltage clamp in intact guard cells, and block was characterized as a function of DTX and external K⁺ (K⁺) concentrations. Added to the bath, 0.1-30 nM DTX blocked the inward-rectifying K⁺ current (I_K,in), but was ineffective in blocking current through the outward-rectifying K⁺ channels (I_K,out) even at concentrations of 30 nM. DTX block was independent of clamp voltage and had no significant effect on the voltage-dependent kinetics for I_K,in, neither altering its activation at voltages negative of –120 mV nor its deactivation at more positive voltages. No evidence was found for a use dependence to DTX action. Block of I_K,in followed a simple titration function with an apparent K_1/2 for block of 2.2 nM in 3 mm K _o ⁺ . However, DTX block was dependent on the external K⁺ concentration. Raising K⁺ from 3 to 30 mm slowed block and resulted in a 60–70% reduction in its efficacy (apparent K _i = 10 mm in 10 nm DTX). The effect of K⁺ in protecting I _K,in was competitive with DTX and specific for permeant cations. A joint analysis of I_K,in block with DTX and K⁺ concentration was consistent with a single class of binding sites with a K _d for DTX of 240 pm. A K _d of 410 m for extracellular K⁺ was also indicated. These results complement previous studies implicating a binding site requiring extracellular K⁺ (K_1/2 1 mm) for I_K,in activation; they parallel features of K⁺ channel block by DTX and related peptide toxins in many animal cells, demonstrating the sensitivity of plant plasma membrane K⁺ channels to nanomolar toxin concentrations under physiological conditions; the data also highlight one main difference: in the guard cells, DTX action appears specific to the K⁺ inward rectifier.We thank J.O. Dolly (Imperial, London) and S.M. Jarvis (University of Kent, Canterbury) for several helpful discussions. This work was supported by SERC grant GR/H07696 and was aided by equipment grants from the Gatsby Foundation, the Royal Society and the University of London Central Research Fund. G.O. was supported by an Ausbildungsstipendium (OB 85/1-1) from the Deutsche Forschungsgemeinschaft. F.A. holds a Sainsbury Studentship.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.     

Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na⁺/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V _m ), the external myo-inositol concentration and the external Na⁺ concentration, yielding the kinetic parameters: K _0.5 ^MI , K _0.5 ^Na , and the Hill coefficient n. At 100 mM NaCl, K _0.5 ^MI was about 50 m and was independent of V _m . At 0.5 mm myo-inositol, K _0.5 ^Na ranged from 76 mm at V _m =–50 mV to 40 mm at V _m =–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na⁺ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K _I of 64 m at V _m =–50 mV and 130 m at V _m = –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V _m =–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na⁺/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V _m =–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na⁺-dependent uptake of ³H-d-glucose and ³H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na⁺ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V _m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model. Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812 Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554.