Contribution of a H+ pump in determining the resting potential of neuroblastoma cells

The aim of this work was to examine the effects of changes in external K⁺ concentration (K _o ) around its physiological value, of various K⁺ channels blockers, including internal Cs⁺, of vacuolar H⁺-ATPase inhibitors and of the protonophore CCCP on the resting potential and the voltage-dependent K⁺ current of differentiated neuroblastoma x glioma hybrid NG108-15 cells using the whole-cell patch-clamp technique. The results are as follows: (i) under standard conditions (K _o =5 mm) the membrane potential was –60±1 mV. It was unchanged when K _o was decreased to 1 mm and was depolarized by 4±1 mV when K_o was increased to 10 mm. (ii) Internal Cs⁺ depolarized the membrane by 21±3 mV. (iii) The internal application of the vacuolar H⁺-ATPase inhibitors N-ethylmaleimide (NEM), NO ₃ ^– and bafilomycin A1 (BFA) depolarized the membrane by 15±2, 18±2 and 16±2 mV, respectively, (iv) When NEM or BFA were added to the internal medium containing Cs⁺, the membrane was depolarized by 45±1 and 42±2 mV, respectively. (v) The external application of CCCP induced a transient depolarization followed by a prolonged hyperpolarization. This hyperpolarization was absent in BFA-treated cells. The voltage-dependent K⁺ current was increased at negative voltages and decreased at positive voltages by NEM, BFA and CCCP. Taken together, these results suggest that under physiological conditions, the resting potential of NG108-15 neuroblastoma cells is maintained at negative values by both voltage-dependent K⁺ channels and an electrogenic vacuolar type H⁺-ATPase.This work was supported by a grant from INSERM (CRE 91 0906).     

An Amiloride-sensitive Na+ conductance in the basolateral membrane of toad urinary bladder

Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 cm². The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm Na⁺, and serosal Na⁺ is substituted by K⁺, Cs⁺, or N-methyl-d-glucamine, the basolateral membrane expresses what appears to be a large Na⁺ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca²⁺. In Cl-free SO ₄ ^2– Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K⁺. This pathway can be blocked by serosal amiloride (K _i=13.1 m) or serosal Na⁺ ions (K _i 10 to 20mm). It also conducts Li⁺ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec^–1 at 0 mV.     

K+-Sensitive Gating of the K+ Outward Rectifier in Vicia Guard Cells

The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K⁺ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K⁺ current was monitored under voltage clamp in 0.1–30 mm K⁺ and in equivalent concentrations of Rb⁺, Cs⁺ and Na⁺. From a conditioning voltage of −200 mV, clamp steps to voltages between −150 and +50 mV in 0.1 mm K⁺ activated current through outward-rectifying K⁺ channels (I _{K, out}) at the plasma membrane in a voltage-dependent fashion. Increasing [K⁺] _o shifted the voltage-sensitivity of I _{K, out} in parallel with the equilibrium potential for K⁺ across the membrane. A similar effect of [K⁺] _o was evident in the kinetics of I _{K, out} activation and deactivation, as well as the steady-state conductance- (g _K ⁻) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near −130 mV in 0.1 mm K⁺, −80 mV in 1.0 mm K⁺, and −20 mV in 10 mm K⁺. Similar data were obtained with Rb⁺ and Cs⁺, but not with Na⁺, consistent with the relative efficacy of cation binding under equilibrium conditions (K⁺≥ Rb⁺ > Cs⁺ > > Na⁺). Changing Ca²⁺ or Mg²⁺ concentrations outside between 0.1 and 10 mm was without effect on the voltage-dependence of g _K or on I _{K, out} activation kinetics, although 10 mm [Ca²⁺] _o accelerated current deactivation at voltages negative of −75 mV. At any one voltage, increasing [K⁺] _o suppressed g _K completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K⁺ was sensitive to voltage, varying from 0.5 to 20 mm with clamp voltages near −100 to 0 mV, respectively. These, and additional data indicate that extracellular K⁺ acts as a ligand and alters the voltage-dependence of I _{K, out} gating; the results implicate K⁺-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of two K⁺ ions outside affects the distribution between closed states of the channel. Received: 27 November 1996/Revised: 4 March 1997     

Dissociation of cellular K+ accumulation from net Na+ transport by toad urinary bladder

Summary A number of published data suggest a variable stoichiometry between the rates of cellular potassium uptake and net sodium transport (J _Na) across the urinary bladder of the toad. This problem was examined by simultaneously studying the intracellular chemical activity of potassium (a _K) with open-tip K⁺-selective microelectrodes and micropipets, and monitoringJ _Na by measuring the short-circuit current (SCC). When bathed in the short-circuited state with solutions containing ana _K of 2.7mm, the mean ±sem values for intracellulara _K were 43±0.6mm.Ouabain, at a concentration of 10^–2 m, reduced intracellulara _K by 56–67% and SCC by 96–100%. At 5×10^–4 m, ouabain reversibly reduced intracellulara _K by 40–55%, and SCC by 63–68%; the inhibition of SCC was only partly reversible during the period of observation.Removal of external potassium reduced intracellulara _K by 69–80% and SCC by 51–76%. Restoration of external potassium entirely returned intracellulara _K to its control value, but only partially reversed the inhibition of SCC during the period of study. Furthermore, recovery ofa _K began 19–43 min before that of SCC; recovery ofa _K was 90–97% complete before any increase in SCC could be measured. Although other interpretations are possible, the simplest interpretation of the data is that the processes responsible for potassium accumulation and transepithelial sodium transport are not identical. We propose the existence of a separate transfer mechanism at the basolateral cell membrane, responsible for accumulating intracellular potassium, and not directly coupled to active sodium transport.     

Ba2+-Induced conductance fluctuations of spontaneously fluctuating K+ channels in the apical membrane of frog skin (Rana temporaria)

Summary We studied the influence of mucosal Ba²⁺ ions on the recently described (Zeiske & Van Driessche, 1979a, J. Membrane Biol. 47:77) transepithelial, mucosa towards serosa directed K⁺ transport in the skin ofRana temporaria. The transport parametersG (conductance), PD (potential difference),I _sc (short-circuit current, K⁺ current), as well as the noise ofI _sc were recorded. Addition of millimolar concentrations of Ba²⁺ to the mucosal K⁺-containing solution resulted in a sudden but quickly reversible drop inI _sc.G andI _sc decreased continuously with increasing Ba²⁺ concentration, (Ba²⁺) _o . The apparent Michaelis constant of the inhibition by Ba²⁺ lies within the range 40–80 m. The apical membrane seems to remain permselective for K⁺ up to 500 m (Ba²⁺) _o . Higher (Ba²⁺) _o , however, appears to induce a shunt (PD falls,G increases). This finding made an accurate determination of the nature of the inhibition difficult but our results tend to suggest a K⁺-channel block by K⁺–Ba²⁺ competition. In the presence of Ba²⁺, the power spectrum of the K⁺ current shows a second Lorentzian component in the low-frequency range, in addition to the high-frequency Lorentzian caused by spontaneous K⁺-channel fluctuations (Van Driessche & Zeiske, 1980). Both Lorentzian components are only present with mucosal K⁺ and can be depressed by addition of Cs⁺ ions, thus indicating that Ba²⁺ ions induce K⁺-channel fluctuations. The dependence of the parameters of the induced Lorentzian on (Ba²⁺) _o , shows a rise in the plateau values to a maximum around 60 m (Ba²⁺) _o , followed by a sharp and progressive decrease to very low values. The corner frequency which reflects the rate of the Ba²⁺-induced fluctuations, however, increases quasi-linearly up to 1mm (Ba²⁺) _o with a tendency to saturate at higher (Ba²⁺) _o . Based on a three-state model for the K⁺ channel (having one open state, one closed by the spontaneous fluctuation and one blocked by Ba²⁺) computer calculations compared favorably with our results. The effect of Ba²⁺ could be explained by assuming reversible binding at the outer side of the apical K⁺ channel, thereby blocking the open channel in competition with K⁺. The association-dissociation of Ba²⁺ at its receptor site is thought to cause a chopping of the K⁺ current, resulting in modulated current fluctuations.     

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.     

Enzyme kinetics of the prime K+ channel in the tonoplast of Chara: Selectivity and inhibition

Summary The prime potassium channel from the tonoplast of Chara corallina has been analyzed in terms of an enzyme kinetic model (Gradmann, Klieber & Hansen 1987, Biophys. J. 53:287) with respect to its selectivity for K⁺ over Rb⁺ and to its blockage by Cs⁺ and by Ca²⁺. The channel was investigated by patchclamp techniques over a range of membrane voltages (V _m , referred to an extracytoplasmic electrical potential of zero) from –200 mV to + 200 mV under various ionic conditions (0 to 300 mM K⁺, Rb⁺, Cs⁺, Ca²⁺, and Cl^–) on the two sides of isolated patches. The experimental data are apparent steady-state currentvoltage relationships under all experimental conditions used and amplitude histograms of the seemingly noisy open-channel currents in the presence of Cs⁺. The used model for K⁺ uniport comprises a reaction cycle of one binding site through four states, i.e., (1) K⁺-loaded and charged, facing the cytoplasm, (2) K⁺-loaded and charged facing the vacuole, (3) empty, facing the vacuole, and (4) empty, facing the cytoplasm. V _m enters the system in the form of a symmetric Eyring barrier between state 1 and 2. The numerical results for the individual rate constants are (in 10⁶s^–1 for zero voltage and 1 m substrate concentration): k ₁₂: 1,410, k ₂₁: 3,370, k ₂₃: 105,000, k ₃₂: 10,600, k ₃₄: 194, k ₄₃: 270, k ₄₁: 5,290, k ₁₄: 15,800. For the additional presence of an alternate transportee (here Rb⁺), the model can be extended in an analog way by another two states ((5) Rb⁺-loaded and charged, facing cytoplasm, and (6) Rb⁺-loaded and charged, facing vacuole) and six more rate constants (k ₄₅: 300, k ₅₄: 240, k ₅₆: 498, k ₆₅: 4,510, k ₆₃: 4,070, k ₃₆: 403). This six-state model with its unique set of fourteen parameters satisfies the complete set of experimental data. If the competing substrate can be bound but not translocated (here Cs⁺ and Ca²⁺), k ₅₆ and k ₆₅ of the model are zero, and the stability constants K _cyt (= k ₃₆/k ₆₃) and K _vac (= k ₄₅/k ₅₄) turn out to be K _cyt(Ca²⁺): 250 m ^–1 · exp(V _m /(64 mV)), k _vac(Ca²⁺): 10 m ^–1 · exp(–V _m /(66 mV)), K _cyt(Cs⁺): 0, and K _vac(Cs⁺): 46 m ^–2 · exp(–V _m /(12.25 mV)). With the assumption that the current fluctuations in the presence of Cs⁺ consist of incompletely resolved, short periods of complete openings and complete closures, the amplitude histograms of the noisy open channel currents can be described by a beta distribution, yielding the rate constants for binding (92 · 10⁶ sec^–1 · m ^–2 · exp(–V _m /(22.5 mV)) and debinding (2, 10⁶ sec^–1 · m ^–2 · exp(V _m /(22.5 mV)) of Cs⁺ to the vacuolar side of the channel as functions of the [Cs⁺] and of V _m . Considering these data and those from the literature, an asymmetry of the channel can be assessed, with a high charge density at the cytoplasmic side (Eisenman-series Nr. XI) and a low charge density at the vacuolar side (Eisenman-series Nr. I). Furthermore, the results provide an example for intimate linkage between conduction and switching of a channel.This work has been supported by the Deutsche Forschungsgemeinschaft.     

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: Modes of operation,dependence on extracellular and intracellular Na+, kinetics,pH dependency and the effect of cell volume and N-ethylmaleimide

Summary The effect of extracellular and intracellular Na⁺ (Na _o ⁺ , Na _i ⁺ ) on ouabain-resistant, furosemide-sensitive (FS) Rb⁺ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na⁺+1 K⁺+2 Cl^–) cotransport system operates in two different modes: modei) promoting bidirectional 11 (Na⁺–K⁺) cotransport, and modeii) a Na _o ⁺ -independent 11 K _o ⁺ /K _i ⁺ exchange requiring Na _i ⁺ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb⁺ uptake through Rb _o ⁺ /K _i ⁺ exchange contributes about 25% to total Rb⁺ uptake in 145mm NaCl media containing 5mm RbCl at normal Na _i ⁺ (pH 7.4). Na⁺–K⁺ cotransport into the cells occurs largely additive to K⁺/K⁺ exchange. Inward Na⁺–Rb⁺ cotransport exhibits a substrate inhibition at high Rb _o ⁺ . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K⁺/K⁺ exchange (apparent pK close to pH 7.4). The apparentK _m ^Rb _o ⁺ of Na⁺–K⁺ cotransport is low (2mm) and almost independent of pH, and high for K⁺/K⁺ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na⁺+1 K⁺+2 Cl^–) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl^–-dependent K⁺ transport pathway that is independent of both Na _o ⁺ and Na _i ⁺ . This pathway differs in many properties from the basal, Na _o ⁺ -independent K⁺/K⁺ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na _o ⁺ -independent FS K⁺ transport pathway which most probably is not identical to basal K⁺/K⁺ exchange. K _o ⁺ o ⁺

o ⁺ o ²⁺ reduce furosemide-resistant Rb⁺ inward leakage relative to choline _o ⁺ .     

Regulatory volume increase in Ehrlich ascites tumor cells is mediated by the 1Na ∶ 1K ∶ 2Cl cotransport system

Summary After swelling in hyposmotic solution, Ehrlich ascites tumor cells shrink towards their original volume. Upon restoration of isosmolality (300 mOsm) the cells initially shrink but subsequently recover volume. This regulatory volume increase (RVI) is completely blocked when [Na⁺] _o or [Cl^–] _o is reduced by 50% in the presence of normal [K⁺] _o . With normal [NaCl] _o but less than 2 mm [K⁺] _o , not only is volume recovery blocked but the cells lose KCl and shrink. When [K⁺] _o is increased to 5 mm there is a rapid net uptake of K⁺ and Cl^– which results in volume recovery. This suggests that the reswelling phase requires the simultaneous presence of Na⁺, K⁺, and Cl^–. Although ouabain has no effect on volume recovery, bumetanide completely blocks RVI by inhibiting a cotransport pathway that mediates the net uptake of Na⁺, K⁺ and Cl^– in the ratio of 1Na1K2Cl. Na⁺ that accumulates is then replaced by K⁺ via the Na/K pump.I wish to thank my colleague, Dr. Thomas C. Smith for advice and helpful comments during the course of these studies. The excellent technical assistance provided by Rebecca Corcoran-Merrill is gratefully acknowledged.This investigation was supported by Grant CA 32927 from the National Cancer Institute, U.S. Public Health Service.     

Voltage dependence of current through the Na,K-exchange pump ofRana oocytes

Summary We have studied current (I _Str) through the Na, K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically,I _Str was measured as the strophanthidin-sensitive current in the presence of Ba^2–, Cd²⁺ and gluconate (in place of external Cl^–). In addition,I _Str was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a meanK _0.5 of 0.4 m, with 2–5 m producing 84–93% pump' block. From baseline data with 12 Na⁺-preloaded oocytes, voltage clamped in the range [–170, +50 mV] with and without 2–5 m Str, the averageI _Str depended directly onV _m up to a plateau at 0 mV with interpolated zero current at –165 mV. In three oocytes, lowering the external [Na⁺] markedly decreased the voltage sensitivity ofI _p , while producing only a small change in the maximal outwardI _Str. In contrast, decreasing the external [K⁺] from 25 to 2.5mm reducedI _Str at 0 mV without substantially affecting its voltage dependence. At K⁺ concentrations of 1mm, both the absolute value ofI _Str at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averagedI _Str by [K⁺] _o over the voltage interval [–30, +30 mV] was well fit by the Hill equation, with K=1.7±0.4mm andnH (the minimum number of K⁺ binding sites) =1.7±0.4. The results unequivocally establish that the cardiotonic-sensitive current ofRana oocytes displays only a positive slope conductance for [K⁺] _o >1mm. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na⁺ and K⁺ are consistent with results obtained in other tissues and may reflect an ion-well effect.     

Single-file diffusion multi-ion mechanism of permeation in paracellular epithelial channels

Summary The transepithelial fluxes, conductances and permeabilities of Li⁺, Na⁺, K⁺, Cs⁺, NH ₄ ⁺ and H₃CNH ₃ ⁺ were studied under ionic concentrations ranging from 12 to 250mm inBufo arenarum gallbladders. When these measurements are carefully corrected in order to get only the component due to the paracellular cation channels, the following results are obtained: (1) The permeability ratios (cationic/anionic) are a decreasing function of salt concentration. (2) The partial conductances through paracellular cationic channels show nonlinear saturable concentration kinetics. (3) Moreover, partial conductance kinetics of K⁺, Cs⁺ and NH ₄ ⁺ present a maximum followed, at higher concentratons, by a negative-slope region. (4) The selectivity sequences obtained from biionic potentials do not agree with those obtained from partial conductance measurements. (5) The unidirectional²²Na tracer flux (serosal to mucosal) is inhibited by 63% when the K⁺ symmetrical concentration in the bathing solutions is raised from 25 to 200mm. (6) When the unidirectional⁴²K fluxes (serosal to mucosal) at 200mm KCl Na-free solutions are compared with K⁺ partial conductance by means of the Hodgkin and Keynes (Hodgkin, A.L., Keynes, R.D. 1955.J. Physiol London 128:61–88) expression, then factor is 2.0. These results indicate that cations do not follow the independence principle and behave as in single-file diffusion multi-ion pores when crossing the paracellular cation channels ofBufo arenarum gallbladder epithelium.     

Cesium,Na+-K+ pump and pacemaker potential in cardiac purkinje fibers

The mechanisms of the hyperpolarizing and depolarizing actions of cesium were studied in cardiac Purkinje fibers perfused in vitro by means of a microelectrode technique under conditions that modify either the Na⁺-K⁺ pump activity or I_f. Cs⁺ (2 mM) inconsistently increased and then decreased the maximum diastolic potential (MDP); and markedly decreased diastolic depolarization (DD). Increase and decrease in MDP persisted in fibers driven at fast rate (no diastolic interval and no activation of I_f). In quiescent fibers, Cs⁺ caused a transient hyperpolarization during which elicited action potentials were followed by a markedly decreased undershoot and a much reduced DD. In fibers depolarized at the plateau in zero [K⁺]_o (no I_f), Cs⁺ induced a persistent hyperpolarization. In 2 mM [K⁺]_o, Cs⁺ reduced the undershoot and suppressed spontaneous activity by hyperpolarizing and thus preventing the attainment of the threshold. In 7 mM [K⁺]_o, DD and undershoot were smaller and Cs⁺ reduced them. In 7 and 10 mM [K⁺]_o, Cs⁺ caused a small inconsistent hyperpolarization and a net depolarization in quiescent fibers; and decreased MDP in driven fibers. In the presence of strophanthidin, Cs⁺ hyperpolarized less. Increasing [Cs⁺]_o to 4, 8 and 16 mM gradually hyperpolarized less, depolarized more and abolished the undershoot. We conclude that in Purkinje fibers Cs⁺ hyperpolarizes the membrane by stimulating the activity of the electrogenic Na⁺-K⁺ pump (and not by suppressing I_f); and blocks the pacemaker potential by blocking the undershoot, consistent with a Cs⁺ block of a potassium pacemaker current.     

K+-conductance and electrogenic Na+/K+ transport of cultured bovine pigmented ciliary epithelium

Summary Using intracellular microelectrode technique, we investigated the changes in membrane voltage (V) of cultured bovine pigmented ciliary epithelial cells induced by different extracellular solutions. (1)V in 213 cells under steady-state conditions averaged –46.1±0.6 mV (sem). (2) Increasing extracellular K⁺ concentration ([K⁺] _o ) depolarizedV. Addition of Ba²⁺ could diminish this response. (3) Depolarization on doubling [K⁺] _o was increased at higher [K⁺] _o (or low voltage). (4) Removing extracellular Ca²⁺ decreasedV and reduced theV amplitude on increasing [K⁺] _o . (5)V was pH sensitive. Extra-and intracellular acidification depolarizedV; alkalinization induced a hyperpolarization.V responses to high [K⁺] _o were reduced at acidic extracellular pH. (6) Removing K _o ⁺ depolarized, K _o ⁺ readdition after K⁺ depletion transiently hyperpolarizedV. These responses were insensitive to Ba²⁺ but were abolished in the presence of ouabain or in Na⁺-free medium. (7) Na⁺ readdition after Na⁺ depletion transiently hyperpolarizedV. This reaction was markedly reduced in the presence of ouabain or in K⁺-free solution but unchanged by Ba²⁺. It is concluded that in cultured bovine pigmented ciliary epithelial cells K⁺ conductance depends on Ca²⁺, pH and [K⁺] _o (or voltage). An electrogenic Na⁺/K⁺-transport is present, which is stimulated during recovery from K⁺ or Na⁺ depletion. This transport is inhibited by ouabain and in K⁺-or Na⁺-free medium.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

Basolateral potassium membrane permeability of A6 cells and cell volume regulation

The K⁺ permeabilities (⁸⁶Rb(K) transport) of the basolateral membranes (J_bK) of a renal cell line (A6) were compared under isosmotic and hypo-osmotic conditions (serosal side) to identify the various components involved in cell volume regulation.Changing the serosal solution to a hypo-osmotic one (165 mOsm) induced a fast transient increase in Ca _i (max <1 min) and cell swelling (max at 3–5 min) followed by a regulatory volume decrease (5–30 min) and rise in the SCC (stabilization at 30 min). In isosmotic conditions (247 mOsm), the ⁸⁶Rb(K) transport and the SCC were partially blocked by Ba²⁺, quinidine, TEA and glibenclamide, the latter being the least effective. Changing the osmolarity from isosmotic to hypo-osmotic resulted in an immediate (within the first 3–6 min) stimulation of the ⁸⁶Rb(K) transport followed by a progressive decline to a stable value higher than that found in isosmotic conditions. A serosal Ca²⁺-free media or quinidine addition did not affect the initial osmotic stimulation of J_bK but prevented its secondary regulation, whereas TEA, glibenclamide and DIDS completely blocked the initial J_bK increase. Under hypo-osmotic conditions, the initial J_bK increase was enhanced by the presence of 1 mm of barium and delayed with higher concentrations (5 mm). In addition, cell volume regulation was fully blocked by quinidine, DIDS, NPPB and glibenclamide, while partly inhibited by TEA and calcium-free media.We propose that a TEA- and glibenclamide-sensitive but quinidine-insensitive increase in K⁺ permeability is involved in the very first phase of volume regulation of A6 cells submitted to hypo-osmotic media. In achieving cell volume regulation, it would play a complementary role to the quinidine-sensitive K⁺ permeability mediated by the observed calcium rise.This work was supported by grants from the Commissariat à l'Energie Atomique and the Centre National de Recherche Scientifique URA 638.     

Noise analysis reveals K+ channel conductance fluctuations in the apical membrane of rabbit colon

Summary In this paper we describe current fluctuations in the mammalian epithelium, rabbit descending colon. Pieces of isolated colon epithelium bathed in Na⁺ or K⁺ Ringer's solutions were studied under short-circuit conditions with the current noise spectra recorded over the range of 1–200 Hz. When the epithelium was bathed on both sides with Na⁺ Ringer's solution (the mucosal solution contained 50 m amiloride), no Lorentzian components were found in the power spectrum. After imposition of a potassium gradient across the epithelium by replacement of the mucosal solution by K⁺ Ringer's (containing 50 m amiloride), a Lorentzian component appeared with an average corner frequency,f _c=15.6±0.91 Hz and a mean plateau valueS _o=(7.04±2.94)×10^–20 A² sec/cm². The Lorentzian component was enhanced by voltage clamping the colon in a direction favorable for K⁺ entry across the apical membrane. Elimination of the K⁺ gradient by bathing the colon on both sides with K⁺ Ringer's solutions abolished the noise signal. The Lorentzian component was also depressed by mucosal addition of Cs⁺ or tetraethylammonium (TEA) and by serosal addition of Ba²⁺. The one-sided action of these K⁺ channel blockers suggests a cellular location for the fluctuating channels. Addition of nystatin to the mucosal solution abolished the Lorentzian component. Serosal nystatin did not affect the Lorentzian noise. This finding indicates an apical membrane location for the fluctuating channels. The data were similar in some respects to K⁺ channel fluctuations recorded from the apical membranes of amphibian epithelia such as the frog skin and toad gallbladder. The results are relevant to recent reports concerning transcellular potassium secretion in the colon and indicate that the colon possesses spontaneously fluctuating potassium channels in its apical membranes in parallel to the Na⁺ transport pathway.     

Voltage-gated K+ channels in the mouse interleukin 3-dependent cell line,FDC-P2

Summary The electrical properties of a mouse interleukin (IL)-3-dependent cell line, FDC-P2, were examined using the tightseal whole-cell clamp technique. Under current clamp conditions with 140mM K⁺ in the pipette, the cells had a resting potential of –30 mV. Under voltage-clamp conditions, a transient outward current was elicited upon depolarization from a holding potential of –80 mV. The current was activated at potentials more positive than –10 mV and had a delayed-rectifying property. It showed rapid activation and slow inactivation during command steps. The current was abolished by Cs⁺ in the pipette, indicating that K⁺ is the charge carrier. The K⁺ current was suppressed by tetraethylammonium withK _i of <0.1mM and was not affected by scorpion toxin. Recovery from inactivation was steeply voltage dependent: As the holding potential was more hyperpolarized, the recovery became faster. Thus, with a holding potential of –80 mV, the current showed slight use-dependent inactivation, while the current decreased prominently by repetitive depolarization at a holding potential of –40 mV. These properties of the K⁺ current are similar to those of thel-type K⁺ channel current in mature T lymphocytes. The K⁺ current in FDC-P2 cells was dramatically reduced after culture in the IL-3-free medium for 1–2 days. When IL-3 was re-added to the medium, the current was re-expressed. These observations suggest that expression of the K⁺ current depends on extracellular IL-3, and that the current may play some roles in proliferation of these cells.     

Permeation of Ca2+ through K+ channels in the plasma membrane of Vicia faba guard cells

Summary The whole-cell patch-clamp method has been used to measure Ca²⁺ influx through otherwise K⁺-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K⁺ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K⁺ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K⁺ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca²⁺ is more negative than or equal to the K⁺ equilibrium potential (–47 mV), indicating that the current is due to Ca²⁺ influx through the K⁺ channels prior to their closure. Decreasing internal [Ca²⁺] (Ca _i ) from 200 to 2 nm or increasing the external [Ca²⁺] (Ca _o ) from 1 to 10 mm increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca²⁺ influx also decrease the amplitude of time-activated current, indicating that Ca²⁺ permeation is slower than K⁺ permeation, and so causes a partial block. Increasing Ca _o also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. –160 mV, indicating that the Ca^2– block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K⁺ channels in Zea mays guard cells also appear to have a Ca _i -, and Ca _o -dependent ability to mediate Ca²⁺ influx. We suggest that the inwardly rectiying K⁺ channels are part of a regulatory mechanism for Ca _i . Changes in Ca _o and (associated) changes in Ca _i regulate a variety of intracellular processes and ion fluxes, including the K⁺ and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments.     

A Calcium-activated and nucleotide-sensitive nonselective cation channel in M-1 mouse cortical collecting duct cells

We recently reported that M-1 mouse cortical collecting duct cells show nonselective cation (NSC) channel activity (Proc. Natl. Acad. Sci. USA 89:10262–10266, 1992). In this study, we further characterize the M-1 NSC channel using single-channel current recordings in excised inside-out patches. The M-1 NSC channel does not discriminate between Na⁺, K⁺, Rb⁺, Cs⁺, and Li⁺. It has a linear I-V relation with a conductance of 22.7±0.5 pS (n=78) at room temperature. The P_cation/ P_anion ratio is about 60 and there is no measurable conductance for NMDG, Ca²⁺, Ba²⁺, and Mn²⁺. Cytoplasmic calcium activates the M-1 NSC channel at a threshold of 10^–6 m and depolarization increases channel activity (NP _o ). Cytoplasmic application of adenine nucleotides inhibits the M-1 NSC channel. At doses of 10^–4 m and 10^–3 m, ATP reduces NP _o by 23% and 69%, respectively.Furthermore, since ADP (10^–3 m) reduces NP _o by 93%, the inhibitory effect of adenine nucleotides is not dependent on the presence of a -phosphoryl group and therefore does not involve protein phosphorylation. The channel is not significantly affected by 8-Br-cGMP (10^–4 m) or by cGMP-dependent protein kinase (10^–7 m) in the presence of 8-Br-cGMP (10^–5 m) and ATP (10^–4 m). The NSC channel is not sensitive to amiloride (10^–4 m cytoplasmic and/or extracellular) but flufenamic acid (10^–4 m) produces a voltage-dependent block, reducing NP _o by 35% at depolarizing voltages and by 80% at hyperpolarizing voltages.We conclude that the NSC channel of M-1 mouse cortical collecting duct cells belongs to an emerging family of calcium-activated and nucleotide-sensitive nonselective cation channels. It does not contribute to amiloride-sensitive sodium absorption and is unlikely to be a major route for calcium entry. The channel is normally quiescent but may be activated under special physiological conditions, e.g., during volume regulation.The expert technical assistance of U. Fink and I. Doering-Hirsch is gratefully acknowledged. We thank A. Rabe and Dr. J. Disser for programming the computer software.This work was supported by a grant from the Deutsche Forschungsge-meinschaft (DFG grant Fr 233/9-1) and a grant from the National Institutes of Health (NIH grant DK-17433).     

Effect of amiloride,ouabain and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia

Summary Effect of amiloride, ouabain, and Ba⁺⁺ on the nonsteady-state Na–K pump flux and short-circuit current in isolated frog skin epithelia.The active Na⁺ transport across isolated frog skin occurs in two steps: passive diffusion across the apical membrane of the cells followed by an active extrusion from the cells via the Na⁺–K⁺ pump at the basolateral membrane. In isolated epithelia with a very small Na⁺ efflux, the appearing Na⁺-flux in the basolateral solution is equal to the rate of the pump, whereas the short-circuit current (SCC) is equal to the active transepithelial Na⁺ transport. It was found that blocking the passive diffusion of Na⁺ across the apical membrane (addition of amiloride) resulted in an instantaneous inhibition of the SCC (the transepithelial Na⁺ transport, whereas the appearing flux (the rate of the Na⁺–K⁺ pump) decreased with a halftime of 1.9 min. Addition of the Na⁺–K⁺ pump inhibitor ouabain (0.1mm) resulted in a faster and bigger inhibition of the appearing flux than of the SCC. Thus, by simultaneous measurement of the SCC and the appearing Na⁺ flux one can elucidate whether an inhibitor exerts its effect by inhibiting the pump or by decreasing the passive permeability. Addition of the K⁺ channel inhibitor Ba⁺⁺, in a concentration which gave maximum inhibition of the SCC, had no effect on the appearing flux (the rate of the Na–K pump) in the first 2 min, although the inhibition of the SCC was already at its maximum.It is argued that in the short period, where the Ba⁺⁺-induced inhibition of SCC is at its maximum and the appearing flux in unchanged, the decrease in the SCC (SCC) is equal to the net K⁺ flux via the Na⁺–K⁺ pump, and the coupling ratio () of the Na⁺–K⁺ pump can be calculated from the following equation =SCC _t=0/SCC where SCC _t=0 is the steady-state SCC before the addition of Ba⁺⁺.