Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

Summary The present studies examined some of the properties of Cl^– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP _Cl/P _K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl^– conductance of isolated medullary thick ascending limb segments. The Cl^– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca²⁺ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca²⁺ concentrations, but was inactivated by reducing KCl concentrations to 50mm.     

An anion channel of sarcoplasmic reticulum incorporated into planar lipid bilayers: Single-channel behavior and conductance properties

Summary An anion channel of sarcoplasmic reticulum vesicle has been incorporated into planar lipid bilayers by means of a fusion method and its basic properties were investigated. Analysis of fusion processes suggested that one SR vesicle contained approximately one anion channel. The conductance of this channel has several substates and shows a flickering behavior. The occupation probability of each substate was voltage dependent, which induced an inward rectification of macroscopic currents. Further, the anion channel was found to have the following properties. (1) The single-channel conductance is about 200 pS at 100mm Cl^–. (2) The channel does not select among monovalent anions but SO ₄ ^2– hardly permeates through the channel. (3) SO ₄ ^2– added to thecis side (the side to which SR vesicles were added) inhibits Cl^– current competitively in a voltage-dependent manner. (4) An analysis of this voltage dependence suggests that the binding site of SO ₄ ^2– is located at about 36% of the way across the channel from thecis entrance.     

Analysis and use of the perforated patch technique for recording ionic currents in pancreaticβ-cells

Summary To investigate the voltage dependence of the Na^–/K^– pump, current-voltage relations were determined in prophasearrested oocytes ofXenopus laevis. All solutions contained 5mm Ba^2– and 20mm tetraethylammonium (TEA) to block K^– channels. If. in addition, the Na⁺/K⁺ pump is blocked by ouabain, K⁺-sensitive currents no larger than 50 nA/cm² remain. Reductions in steady-state current (on the order of 700 nA/cm²) produced by 50 m ouabain or dihydro-ouabain or by K⁺ removal, therefore, primarily represent current generated by the Na^–/K^– pump. In Na^–-free solution containing 5mm K⁺, Na⁺/K⁺ pump current is relatively voltage independent over the potential range from –160 to +40 mV. If external [K⁺] is reduced below 0.5mm, negative slopes are observed over this entire voltage range. Similar results are seen in Na⁺- and Ca²⁺-free solutions in the presence of 2mm Ni²⁺, an experimental condition designed to prevent Na⁺/Ca²⁺ exchange. The occurrence of a negative slope can be explained by the voltage dependence of the apparent affinity for activation of the Na⁺/K⁺ pump by external K⁺, consistent with the existence of an external ion well for K^– binding. In 90mm Na⁺, 5mm K⁺ solution, Na⁺/K⁺ pump current-voltage curves at negative membrane potentials have a positive slope and can be described by a monotonically increasing sigmoidal function. At an extracellular [K⁺] of 1.3mm, a negative slope was observed at positive potentials. These findings suggest that in addition to a voltage-dependent step associated with Na⁺ translocation, a second voltage-dependent step that is dependent on external [K⁺], possibly external K⁺ binding, participates in the overall reaction mechanism of the Na⁺/K⁺ pump.     

Current-voltage relationships of a sodium-sensitive potassium channel in the tonoplast ofChara corallina

Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m^–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K⁺-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K⁺ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na⁺ and Cl^–. However, addition of Na⁺ decreases the K⁺ currents. TheI–V relationships of the open channel as measured at various K⁺ concentrations with or without Na⁺ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.     

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.     

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.     

Kinetics of voltage- and Ca2+ activation and Ba2+ blockade of a large-conductance K+ channel fromNecturus enterocytes

Summary Potassium channels in membranes of isolatedNecturus enterocytes were studied using the patch-clamp technique. The most frequent channel observed had a conductance of 170 pS and reversal potential of 0 mV in symmetrical potassium-rich solutions. Channels were highly K⁺ selective. Channel activity was modulated by membrane potential and cytosolic Ca²⁺ concentration. Channel openings occurred in characteristic bursts separated by long closures. During bursts openings were interrupted by brief closures. Two gating modes controlled channel opening. The primary gate's sensitivity to intracellular Ca²⁺ concentration and membrane potential crucially determined long duration closures and bursting. In comparison, the second gate determining brief closures was largely insensitive to voltage and intracellular Ca²⁺ concentration. The channel was reversibly blocked by cytosolic barium exposure in a voltage-sensitive manner. Blockade reduced open-state probability without altering single-channel conductance and could be described, at relatively high Ca²⁺ concentration, by a three-state model where Ba²⁺ interacted with the open channel with a dissociation constant of about 10^–4 m at 0 mV.     

Regulation of maxi-K+ channels on pancreatic duct cells by cyclic AMP-dependent phosphorylation

Summary Using the patch-clamp technique we have identified a Ca²⁺-sensitive, voltage-dependent, maxi-K⁺ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of 200 pS in excised patches bathed in symmetrical 150mm K⁺, and was blocked by 1mm Ba²⁺. Channel openstate probability (P _o ) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted theP _o /voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration,P _o remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca²⁺ concentration from 2mm to 0.1 m. However, activated channels were still blocked by 1mm Ba²⁺. ChannelP _o was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase., We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K⁺ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca²⁺. Physiologically, these K⁺ channels may contribute to the basolateral K⁺ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.     

Role of calcium ion in the excitability and electrogenic pump activity of theChara corallina membrane: II. Effects of La3+, EGTA,and calmodulin antagonists on the current-voltage relation

Summary The steady N shapeI/V curves were obtained by applying slow ramp hyper- and depolarization pulses toChara cells under the voltage-clamp condition. Application of calcium channel blocker, 20 m La³⁺, to theChara membrane caused, in about 30 min, a marked reduction of the transient inward current and later almost complete blocking of the pump current, while the steady outward current remained almost unaffected. Removal of external Ca²⁺ with 0.5mm EGTA caused similar results. Application of calmodulin antagonists, 10 m TFP or 20 m W-7, also gave very similar results, i.e., the decrease of the transient inward current and of H⁺-pump activity. These results suggest that not only the excitatory mechanisms but also the H⁺-pump activity ofChara membrane are regulated by calmodulin within a comparatively narrow range of internal Ca²⁺ level.     

Ca2+-activated K+ channels in the apical membrane ofNecturus choroid plexus

Summary The properties of Ca²⁺-activated K⁺ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K⁺ over Na⁺ and K⁺ over Cs⁺. They were blocked by high cytoplasmic Na⁺ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca²⁺ concentrations. Increasing Ca²⁺ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca²⁺ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba²⁺ reduced both channel-open probability and conductance. The sites for the action of Ba²⁺ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba²⁺ dissociation constants (K _d (0 mV) for the effects on probability and conductance, respectively. TEA⁺ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK _d (0 mV)=5.6mm. A second site, with a higher affinity,K _d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA⁺ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca²⁺-activated K⁺ channels found in other tissues.     

Characterization of the 1,25-dihydroxycholecalciferol-stimulated calcium influx pathway in CaCo-2 cells

The present studies were conducted to investigate the mechanisms underlying the 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃)-induced increase in intracellular Ca²⁺ ([Ca²⁺] _i ) in individual CaCo-2 cells. In the presence of 2mm Ca²⁺, 1,25(OH)₂D₃-induced a rapid transient rise in [Ca²⁺] _i in Fura-2-loaded cells in a concentration-dependent manner, which decreased, but did not return to baseline levels. In Ca²⁺-free buffer, this hormone still induced a transient rise in [Ca²⁺] _i , although of lower magnitude, but [Ca²⁺] _i then subsequently fell to baseline. In addition, 1,25(OH)₂D₃ also rapidly induced⁴⁵Ca uptake by these cells, indicating that the sustained rise in [Ca²⁺] _i was due to Ca²⁺ entry. In Mn²⁺-containing solutions, 1,25(OH)₂D₃ increased the rate of Mn²⁺ influx which was temporally preceded by an increase in [Ca²⁺] _i . The sustained rise in [Ca²⁺] _i was inhibited in the presence of external La³⁺ (0.5mm). 1,25(OH)₂D₃ did not increase Ba²⁺ entry into the cells. Moreover, neither high external K⁺ (75mm), nor the addition of Bay K 8644 (1 μm), an L-type, voltage-dependent Ca²⁺ channel agonist, alone or in combination, were found to increase [Ca²⁺] _i , 1,25(OH)₂D₃ did, however, increase intracellular Na⁺ in the absence, but not in the presence of 2mm Ca²⁺, as assessed by the sodium-sensitive dye, sodium-binding benzofuran isophthalate. These data, therefore, indicate that CaCo-2 cells do not express L-type, voltage-dependent Ca²⁺ channels. 1,25(OH)₂D₃ does appear to activate a La³⁺-inhibitable, cation influx pathway in CaCo-2 cells.     

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops fromChara australis

Summary Patch-clamp studies of cytoplasmic drops from the charophyteChara australis have previously revealed K⁺ channels combining high conductance (170 pS) with high selectivity for K⁺, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K⁺>Rb⁺>NH ₄ ⁺ Na⁺ and Cl^–. Divalent cytosolic ions reduce the K⁺ conductance of this channel and alter its K⁺ gating in a voltage-dependent manner. The order of blocking potency is Ba²⁺>Sr²⁺>Ca²⁺>Mg²⁺. The channel is activated by micromolar cytosolic Ca²⁺, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca²⁺ needed to open the channel. The possible role of the Ca²⁺-activated K⁺ channel in the tonoplast ofChara is discussed.     

Paramecium Na+ channels activated by Ca2+-calmodulin: Calmodulin is the Ca2+ sensor in the channel gating mechanism

Paramecium Na⁺ channels, which were Ca²⁺-calmodulin activated, were studied in the inside-out mode of patch clamp. After excision of the membrane patch, they were active in the presence of 10^–5 to 10^–3 m Ca²⁺ in the bath. They became much less active in the presence of 10^–6 m Ca²⁺, and their activity subsided completely at 10^–8 m Ca²⁺. A Hill plot showed a dissociation constant of 6 m for Ca²⁺ binding. This dissociation constant shifted to a submicromolar range in the presence of 1 mm Mg²⁺. The channels also exhibited a mild voltage dependence. When exposed to 10^–8 m Ca²⁺ for an extended period of 2–4 min, channels were further inactivated even after bath Ca²⁺ was restored to 10^–4 m. Whereas neither high voltage (+100 mV) nor high Ca²⁺ (10^–3 m) was effective in reactivation of the inactive channels, addition of Paramecium wild-type calmodulin together with high Ca²⁺ to the bath restored channel activity without a requirement of additional Mg²⁺ and metabolites such as ATP. The channels reactivated by calmodulin had the same ion conductance, ion selectivity and Ca²⁺ sensitivity as those prior to inactivation. These inactivation and reactivation of the channels could be repeated, indicating that the direct calmodulin effect on the Na⁺ channel was reversible. Thus, calmodulin is a physiological factor critically required for Na⁺ channel activation, and is the Ca²⁺ sensor of the Na⁺-channel gating machinery.We thank C. Kung for his kind support, and A. Boileau for critical reading. Supported by grants from National Institutes of Health GM 22714-20 and 36386-09.     

Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

Divalent cations as probes for structure-function relationships of cloned voltage-dependent sodium channels

1. Several cloned sodium channels were expressed in oocytes and compared with respect to their sensitivity to internal Mg²⁺ concerning the open-channel block and to external Ca²⁺ concerning open-channel block and shifts in steady-state activation. 2. A quantitative comparison between wild-type II channels and a mutant with a positive charge in the S4 segment of repeat I neutralized (K226Q) revealed no significant differences in the Mg²⁺ block. 3. The blocking effect of extracellular Ca²⁺ ions on single-channel inward currents was studied for type II, mutant K226Q and type III. A quantitative comparison showed that all three channel types differ significantly in their Ca⁺ sensitivity. 4. The influence of extracellular Ca²⁺ on the voltage dependence of steady-state activation of macroscopic currents was compared for type II and K226Q channels. Extracellular Ca⁺ increases the voltage of half-maximal activation, V_1/2, more for K226Q than for wild-type II channels; a plot of V _1/2 against [Ca] _o , is twice as steep for the mutant K226Q as for the wild-type on a logarithmic concentration scale. 5. The differential effects of extracellular Ca⁺ and intracellular Mg²⁺ on wild-type II and K226Q channels are discussed in terms of structural models of the Na⁺ channel protein.Abbreviations [Na] _i intracellular Na⁺ concentration - [Mg] intracellular Mg²⁺ concentration - [Ca] _o extracellular Ca²⁺ concentration     

Effects of D2O on permeation and gating in the Ca2+-activated potassium channel fromChara

We studied the effects of H₂O/D₂O substitution on the permeation and gating of the large conductance Ca²⁺-activated K⁺ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K⁺>Rb⁺≫Li⁺, Na⁺, Cs⁺ and Cl⁻. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D₂O as compared to H₂O. The blockade of channel conductance by cytosolic Ca²⁺ weakened in D₂O as a result of a decrease in zero voltage Ca²⁺ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D₂O primarily due to the change in Ca²⁺ binding to the channel during the activation step. The Hill coefficient for Ca²⁺ binding was 3 in D₂O and around 1 in H₂O. The values of the Ca²⁺ binding constant in the open channel conformation were 0.6 and 6 μm in H₂O and D₂O, respectively, while the binding in the closed conformation was much less affected by D₂O. The H₂O/D₂O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca²⁺.     

Veratridine induces a Ca2+ influx,cyclic GMP formation,and backward swimming inParamecium tetraurelia wildtype cells and Ca2+ current-deficient pawn mutant cells

Summary Veratridine opens voltage-dependent Na⁺ channels in many metazoans. InParamecium, which has voltage-dependent Ca²⁺ channels and a Ca/K action potential, no such Na⁺ channels are known. A Ca-inward current is correlated to an intracellular increase in cGMP. The addition of veratridine toParamecium wildtype and to pawn mutant cells, which lack the Ca-inward current, transiently increased intracellular levels of cGMP about sevenfold to 40 pmol/mg protein. A half-maximal effect was obtained with 250 m veratridine. The increase in cGMP was maximal about 15 sec after the addition of veratridine and declined rapidly afterwards. Intracellular cAMP levels were not affected. The effect of veratridine on cGMP was dependent on the presence of extracellular Ca²⁺. The time dependence and extent of stimulation closely resembled the effects observed after stimulation by Ba²⁺, which causes the repetitive firing of action potentials, Ca-dependent ciliary reversal, and cGMP formation. The effects of Ba²⁺ and veratridine were not additive. Wildtype cells and, surprisingly, also pawn mutant cells showed avoiding reactions upon addition of veratridine indicating that it induced a Ca²⁺ influx into the cilia, which causes ciliary reversal. The potency of veratridine to stimulate cGMP formation was little affected by Na⁺ in wildtype cells, three pawn mutant strains, and in the cell line fast-2, which is defective in a Ca-dependent Na-inward current. Divalent cations (Ca²⁺, Mg²⁺, and Ba²⁺) inhibited the effects the veratridine similar to metazoan cells. The results indicate that veratridine can open the voltage-operated Ca²⁺ channels inParamecium wildtype and, most interestingly, in pawn mutant cells. The pawn mutation is suggested to represent a defect in the activation of the Ca²⁺ channel. This explains the lack of differences in ciliary proteins between wildtype and pawn cells reported earlier.     

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.     

Voltage- and time dependence of apical membrane conductance during current clamp inNecturus gallbladder epithelium

Summary The effects of short (1 sec) and long (1 min) transepithelial current clamps on membrane voltages and resistances ofNecturus gallbladder were investigated. Transepithelial and cell membrane current-voltage relationships determined from 1-sec clamps revealed that: a) depolarization of the apical membrane voltage (V _mc) results in a marked decrease in apical membrane fractional resistance (fR _a), whereas hyperpolarization ofV _mc results in either no change infR _a or a small increase, and b) the voltage-dependent changes infR _a are essentially complete within 500 msec. Exposure of the tissue to 5mm TEA⁺ on the mucosal side caused no significant change in baselineV _mc (–69±2 mV) and yet virtually abolished the voltage dependence offR _a. A possible interpretation of these results is that two types of K⁺ channels exist in the apical membrane, with different voltage dependencies and TEA⁺ sensitivities. Acidification or Ba²⁺ addition to the mucosal solution also reduced the voltage-dependent changes infR _a. The time courses of the changes infR _a and in the cable properties of the epithelium were assessed during 1-min transepithelial current clamps (±200 A/cm²). No secondary change infR _a was observed with mucosa-to-serosa currents, but a slow TEA⁺-sensitive decrease infR _a (half-time of seconds) was evident with serosa-to-mucosa currents. Cable analysis experiments demonstrated that the initial (<500 msec) voltage-dependent decrease infR _a is due to a fall in apical membrane resistance. The later decrease infR _a is due to changes in both cell membrane resistances attributable to the increase in transcellular current flow resulting from a fall in paracellular conductance. The voltage dependence of the apical membrane conductance is a more significant problem in estimatingfR _a than the current-induced effects on the lateral intercellular spaces. In principle, TEA⁺ can be used to prevent the nonlinear behavior ofR _a during measurements of the voltage divider or membrane resistance ratio.     

BK Ca Channels Activating at Resting Potential without Calcium in LNCaP Prostate Cancer Cells

Large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels are activated by intracellular Ca²⁺ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK_Ca-type K⁺ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK_L. K⁺ selectivity, high conductance, activation by Mg²⁺ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK_Ca channels. However, unlike conventional BK_Ca channels, BK_L channels activated in the absence of free cytosolic Ca²⁺ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BK_Ca channels. Half-maximal Ca²⁺-dependent activation was observed at 0.4 μM for BK_L (at −20 mV) and at 4.1 μM for BK_Ca channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK_L conductance. Expression of hSlo-β1 in LNCaP cells shifted voltage-dependent activation to values between that of BK_L and BK_Ca channels and reduced the slope of the P_open (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK_Ca subunit, which is responsible for the BK_L phenotype in a dominant manner. BK_L-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK_L in LNCaP cells is regulated by serum-derived factors, however not by androgens.