BK channels in human glioma cells

Ion channels in inexcitable cells are involved in proliferation and volume regulation. Glioma cells robustly proliferate and undergo shape and volume changes during invasive migration. We investigated ion channel expression in two human glioma cell lines (D54MG and STTG-1). With low [Ca2+]i, both cell types displayed voltage-dependent currents that activated at positive voltages (more than +50 mV). Current density was sensitive to intracellular cation replacement with the following rank order; K+ > Cs+ approximately = Li+ > Na+. Currents were >80% inhibited by iberiotoxin (33 nM), charybdotoxin (50 nM), quinine (1 mM), tetrandrine (30 microM), and tetraethylammonium ion (TEA; 1 mM). Extracellular phloretin (100 microM), an activator of BK(Ca2+) channels, and elevated intracellular Ca2+ negatively shifted the I-V curve of whole cell currents. With 0, 0.1, and 1 microM [Ca2+]i, the half-maximal voltages, V(0.5), for whole cell current activation were +150, +65, and +12 mV, respectively. Elevating [K+]o potentiated whole cell currents in a fashion proportional to the square-root of [K+]o. Recording from cell-attached patches revealed large conductance channels (150-200 pS) with similar voltage dependence and activation kinetics as whole cell currents. These data indicate that human glioma cells express large-conductance, Ca2+ activated K+ (BK) channels. In amphotericin-perforated patches bradykinin (1 microM) activated TEA-sensitive currents that were abolished by preincubation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). The BK channels described here may influence the responses of glioma cells to stimuli that increase [Ca2+]i.     

Met-enkephalin-induced mobilization of intracellular Ca2+ in rat intracardiac ganglion neurones.

The effects of Met-enkephalin on Ca2+-dependent K+ channel activity were investigated using the cell-attached patch recording technique on isolated parasympathetic neurones of rat intracardiac ganglia. Large-conductance, Ca2+-dependent K+ channels (BK(Ca)) were examined as an assay of agonist-induced changes in the intracellular free calcium ion concentration ([Ca2+]i). These BK(Ca) channels had a conductance of approximately 200 pS and were charybdotoxin- and voltage-sensitive. Caffeine (5 mM), used as a control, evoked a large increase in BK(Ca) channel activity, which was inhibited by 10 microM ryanodine. Met-enkephalin (10 microM) evoked a similar increase in BK(Ca) channel activity, which was dependent on the presence of extracellular Ca2+ and inhibited by either ryanodine (10 microM) or naloxone (1 microM). In Fura-2-loaded intracardiac neurones, Met-enkephalin evoked a transient increase in [Ca2+]i. Met-enkephalin-induced mobilization of intracellular Ca+ may play a role in neuronal excitability and firing behaviour in mammalian intracardiac ganglia.     

Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons

The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-clamp recording technique. In symmetrical 140 mM K+, the single-channel slope conductance was linear in the voltage range -60/+60 mV, and was 207+/-19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+]i) with a Hill plot giving a half-saturating [Ca2+] (K0.5) of 1.35 microM and slope of approximately equals 3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 microM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+]i <100 nM and the increased channel activity evoked by ionomycin was consistent with a rise in [Ca2+]i to > or =0.3 microM. TEA (0.2-1 mM) increased the action potential duration approximately equals 1.5-fold and reduced the amplitude and duration of the afterhyperpolarization (AHP) by 26%. Charybdotoxin (100 nM) did not significantly alter the action potential duration or AHP amplitude but reduced the AHP duration by approximately equals 40%. Taken together, these data indicate that BK channel activation contributes to the action potential and AHP duration in rat intracardiac neurons.     

Ion channel regulation of the dynamical instability of the resting membrane potential in saccular hair cells of the green frog (Rana esculenta)

AIMS: We investigated the ion channel regulation of the resting membrane potential of hair cells with the aim to determine if the resting membrane potential is poised close to instability and thereby a potential cause of the spontaneous afferent spike activity. METHODS: The ionic mechanism and the dynamic properties of the resting membrane potential were examined with the whole-cell patch clamp technique in dissociated saccular hair cells and in a mathematical model including all identified ion channels. RESULTS: In hair cells showing I/V curves with a low membrane conductance flanked by large inward and outward rectifying potassium conductances, the inward rectifier (K(IR)), the delayed outward rectifier (K(V)) and the large conductance, calcium-sensitive, voltage-gated potassium channel (BK(Ca)) were all activated at rest. Under current clamp conditions, the outward current through these channels balanced the inward current through mechano-electrical transduction (MET) and Ca2+ channels. In 45% (22/49) of the cells, the membrane potential fluctuated spontaneously between two voltage levels determined by the voltage extent of the low membrane conductance range. These fluctuations were not influenced by blocking the MET channels but could be reversibly stopped by increasing [K+]o or by blocking of K(IR) channels. Blocking the BK(Ca) channels induced regular voltage oscillations. CONCLUSIONS: Two intrinsic dynamical instabilities of V(m) are present in hair cells. One of these is observed as spontaneous voltage fluctuations by currents through K(IR), K(V) and h-channels in combination with a steady current through MET channels. The other instability shows as regenerative voltage changes involving Ca2+ and K(V) channels. The BK(Ca) channels prevent the spontaneous voltage fluctuations from activating the regenerative system.     

Properties of large conductance calcium-activated potassium channels in pyramidal neurons from the hippocampal CA1 region of adult rats

The properties of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels were studied in rat hippocampal CA1 pyramidal neurons by using the patch-clamp technique in the excised-inside-out-patch configuration. The lowest [Ca(2+)](i) in which BK(Ca) channel activities were observed was 0.01 microM with the membrane potential of +20 mV and the [Ca(2+)](i) at which P(O) of the channel is equal to 0.5 was 2 microM. The unitary conductance of the single BK(Ca) channel was 245.4 pS with symmetrical 140 mM K(+) on both sides of the excised membrane. With a fixed [Ca(2+)](i) of 2 microM, P(O) increased e-fold with a 17.0 mV positive change in the membrane potential. Two exponentials, with time constants of 2.8 ms and 19.2 ms at the membrane potential of +120 mV with 2 microM [Ca(2+)](i), were required to describe the observed open time distribution of BK(Ca) channel, suggesting the existence of two distinct open channel states with apparently normal conductance. A BK(Ca) channel occasionally entered an apparent third open channel state with the single channel current amplitude about 45% of the normal amplitude. The properties of BK(Ca) channel, which were found in this study to be more steeply dependent on voltage and more sensitive to [Ca(2+)](i) in adult hippocampal neurons than in cultured or immature hippocampal neurons, may be responsible for the shortened duration of action potential in hippocampal CA1 pyramidal neurons of adult rat.     

Ca2+ and voltage dependence of cardiac ryanodine receptor channel block by sphingosylphosphorylcholine

The effect of sphingosylphosphorylcholine (SPC) on the cytoplasmic Ca(2+) and voltage dependence of channel gating by cardiac ryanodine receptors (RyR) was examined in lipid bilayer experiments. Micromolar concentrations of the lysosphingolipid SPC added to cis solutions rapidly and reversibly decreased the single-channel open probability (P(o)) of reconstituted RyR channels. The SPC-induced decrease in P(o) was marked by an increase in mean closed time and burst-like channel gating. Gating kinetics during intraburst periods were unchanged from those observed in the absence of the sphingolipid, although SPC induced a long-lived closed state that appeared to explain the observed decrease in channel P(o). SPC effects were observed over a broad range of cis [Ca(2+)] but were not competitive with Ca(2+). Interestingly, the sphingolipid-induced, long-lived closed state displayed voltage-dependent kinetics, even though other channel gating kinetics were not sensitive to voltage. Assuming SPC effects represent channel blockade, these results suggest that the blocking rate is independent of voltage whereas the unblocking rate is voltage dependent. Together, these results suggest that SPC binds directly to the cytoplasmic side of the RyR protein in a location in or near the membrane dielectric, but distinct from cytoplasmic Ca(2+) binding sites on the protein.     

Properties of the inwardly rectifying K+ conductance in the toad retinal pigment epithelium.

An inwardly rectifying K+ current was analysed in isolated toad retinal pigment epithelial (RPE) cells using the perforated-patch clamp technique. The zero-current potential (Vo) of RPE cells averaged -71 mV when the extracellular K+ concentration ([K+]o) was 2 mM. Increasing [K+]o from 0.5 to 5 mM shifted V0 by +43 mV, indicating a relative K+ conductance (TK) of 0.74. At [K+]o greater than 5 mM, TK decreased to 0.53. Currents were larger in response to hyperpolarizing voltage pulses than depolarizing pulses, indicating an inwardly rectifying conductance. Currents were time independent except in response to voltage pulses to potentials positive to 0 mV, where the outward current decayed with an exponential time course. Both the inwardly rectifying current and the transient outward current were eliminated by the addition of 0.5 mM Ba2+, 5 mM Cs+ or 2 mM Rb+ to the extracellular solution. The current blocked by these ions reversed near the K+ equilibrium potential (EK) over a wide range of [K+]o, indicating a highly selective K+ channel. The current-voltage relationship of the isolated K+ current exhibited mild inward rectification at voltages negative to -20 mV and a negative slope conductance at voltages positive to -20 mV. The Cs(+)- and Ba(2+)-induced blocks of the K+ current were concentration dependent but voltage independent. The apparent dissociation constants were 0.8 mM for Cs+ and 40 microM for Ba2+. The K+ conductance decreased when extracellular Na+ was removed. Increasing [K+]o decreased the K+ chord conductance (gK) at negative membrane potentials. In the physiological voltage range, increasing [K+]o from 2 to 5 mM caused gK to decrease by approximately 25%. We conclude that the inwardly rectifying K+ conductance represents the resting K+ conductance of the toad RPE apical membrane. The unusual properties of this conductance may enhance the ability of the RPE to buffer [K+]o changes that take place in the subretinal space at the transition between dark and light.     

Purinergic activation of BK channels in clonal kidney cells (Vero cells)

We have studied the activation of a high-conductance channel in clonal kidney cells from African green monkey (Vero cells) using patch-clamp recordings and microfluorometric (fura-2) measurements of cytosolic Ca2+. The single-channel conductance in excised patches is 170 pS in symmetrical 140 mM KCl. The channel is highly selective for K+ and activated by membrane depolarization and application of Ca2+ to the cytoplasmatic side of the patch. The channel is, thus, a large-conductance Ca2+-activated K+ channel (BK channel). Cell-attached recordings revealed that the channel is inactive in unstimulated cells. Extracellular application of less than 0.1 microM ATP transiently increased the cytosolic Ca2+ concentration ([Ca2+]i) to about 550 nM, and induced membrane hyperpolarization caused by Ca2+-activated K+ currents. ATP stimulation also activated BK channels in cell-attached patches at both the normal-resting potential and during membrane hyperpolarization. The increase in [Ca2+]i was owing to Ca2+ release from internal stores, suggesting that Vero cells express G-protein-coupled purinergic receptors (P2Y) mediating IP3-induced release of Ca2+. The P2Y receptors were sensitive to both uracil triphosphate (UTP) and adenosine diphosphate (ADP), and the rank of agonist potency was ATP > UTP >/= ADP. This result indicates the presence of both P2Y1 and P2Y2 receptors or a receptor subtype with untypical agonist sensitivity. It has previously been shown that hypotonic challenge activates BK channels in both normal and clonal kidney cells. The subsequent loss of KCl may be an important factor in cellular volume regulation. Our results support the idea of an autocrine role of ATP in this process. A minute release of ATP induced by hypotonically evoked membrane stretch may activate the P2Y receptors, subsequently increasing [Ca2+]i and thus causing K+ efflux through BK channels.     

Kinetics of the Ca(2+)-activated K+ channel in rat hippocampal neurons

The kinetics of the large-conductance Ca(2+)-activated K+ channel (235 pS in symmetrical 150 mM K+) were examined in the inside-out mode of the patch clamp technique. The open probability of the channel increased when [Ca2+]i, [Sr2+]i, or [Ba2+]i was increased. The [Ca2+]i-response relation was fitted with a Hill coefficient of 2 and half-maximum concentrations of 185, 80, 14.5, and 5.5 microM at -40, -20, +20, and +40 mV, respectively. The channel was blocked by TEA or Ba2+. The open-time histogram showed a single exponential component and the closed-time histogram showed at least two exponential components at various [Ca2+]i. Increasing [Ca2+]i decreased the time constant of the slow component of the closed-time histogram. Cell-attached patch recording revealed activation of the large-conductance Ca(2+)-activated K+ channel (BK channel) during the action potential. The deactivation time course was consistent with the fast after-hyperpolarization. A minimum model of the channel, close(2)-close(1)-open, where the transition from close(2) to close(1) requires the binding of 2 Ca2+, reconstructed quick activation of the channel if [Ca2+]i of 40 microM was assumed.     

Inhibition of BK channels contributes to the second phase of the response to TRH in clonal rat anterior pituitary cells

AIM: Thyrotropin-releasing hormone (TRH) induces biphasic changes in the electrical activity, the cytosolic free Ca2+ concentration ([Ca2+]i), and prolactin secretion from both GH cells and native lactotrophs. It is well established that inhibition of erg channels contributes to the second phase of the TRH response. We have investigated if BK channels are also involved. RESULTS: The BK channels may be active at the resting membrane potential (open probability, Po=0.01) in clonal rat anterior pituitary cells (GH4), which makes it possible that inhibition of these channels may contribute to the reduced K+ conductance during the TRH response. The specific BK channel blocker iberiotoxin (IbTx, 100 nm) had no effect on the resting conductance at holding potentials negative to -40 mV, but significantly reduced the conductance at shallower membrane potentials. This corresponds to the voltage dependency of the sustained [Ca2+]i. Furthermore, IbTx increased the action potential frequency by 36% in spontaneously firing cells. During the second phase of the TRH response, the action potential frequency increased by 34%, concomitantly with 61% reduction of the Po of single BK channels. The protein kinase C (PKC)-activating phorbol ester TPA had no significant effect on BK channel Po within the normal range of the resting potential. CONCLUSION: The BK channels may contribute to the resting membrane conductance, and they are partially inhibited by TRH during the second phase. This modulation seems not to depend on PKC. We propose that inhibition of erg and BK channels acts in concert to enhance the cell excitability during the second phase of the response to TRH.     

Properties of a Ca(2+)-activated large conductance K(+) channel with ATP sensitivity in human renal proximal tubule cells

The properties of a native Ca(2+)-activated large conductance K(+) channel (BK channel) present in the surface membrane of cultured human renal proximal tubule epithelial cells (RPTECs) were investigated by using the patch-clamp technique. The slope conductance of the BK channel was about 295 pS, and the channel was selective to K(+) over Na(+), with a selectivity ratio of about 12.2. The activity of the channel was almost maximally enhanced by 10(-4 )M or more Ca(2+) in the cytoplasmic surface of the patch membrane and was markedly diminished by reducing the cytoplasmic Ca(2+) to 10(-6) M at the membrane potential of about 0 mV. The depolarization of the patch membrane also enhanced the channel activity, and hyperpolarization lowered it. K(+) channel blockers, Ba(2+) (0.1-1 mM), tetraethylammonium (1 mM), and charybdotoxin (100 nM), were effective for the suppression of channel activity. A significant feature of the K(+) channel was that channel activity maintained by 10(-5)-10(-4 )M Ca(2+) in inside-out patches was inhibited by the addition of ATP (1-10 mM) to the bath solution. ATPgammaS, and a nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), also had inhibitory effects on channel activity. However, an inhibitor of ATP-sensitive K(+) channels, glibenclamide (0.1 mM), induced no appreciable change in channel activity from both intra- and extracellular sides. These results suggest that besides the common natures of the BK channel family such as regulation by cytoplasmic Ca(2+) and membrane potential, the BK channel in RPTECs is directly inhibited by intracellular ATP independent of phosphorylation processes and sulfonylurea receptor.     

Cytoplasmic Ca2+ at low submicromolar concentration stimulates mitochondrial metabolism in rat luteal cells

The cytoplasmic Ca2+ signal is transferred to the mitochondrial matrix and activates mitochondrial dehydrogenases. The requirement for supramicromolar cytoplasmic [Ca2+] ([Ca2+]i) in perimitochondrial microdomains in this response has been suggested. We studied the correlation between [Ca2+]i, mitochondrial [Ca2+] ([Ca2+]m) and mitochondrial formation of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] in the presence of submicromolar [Ca2+]i in cultured rat "large" luteal cells. [Ca2+]i was monitored fluorimetrically with fura-PE3, [Ca2+]m with rhod-2 and NAD(P)H with autofluorescence. In intact cells, prostaglandin F2alpha, which induces both intracellular Ca2+ release and Ca2+ entry, stimulated mitochondrial NAD(P)H formation. Thapsigargin-induced Ca2+ release and subsequent capacitative Ca2+ entry, both resulting in Ca2+ responses not exceeding 150-200 nM, also enhanced the reduction of pyridine nucleotides. As shown in inhibitor studies, the increased steady-state NAD(P)H level was due to activation of Ca2+-dependent dehydrogenases. [Ca2+]m, measured in permeabilized cells, increased moderately, but significantly, following elevation of [Ca2+]i from 50 to 180 nM, showed a further gradual increase at higher submicromolar [Ca2+]i values and rose steeply at supramicromolar [Ca2+]i. In summary, our results demonstrate that, in a steroid-producing cell type, net mitochondrial Ca2+ uptake and mitochondrial dehydrogenation can be activated even by low submicromolar increases of [Ca2+]i.     

Surface potential reflected in both gating and permeation mechanisms of sodium and calcium channels of the tunicate egg cell membrane

1. Threshold changes of Na and Ca currents due to various polyvalent cations (stabilizing cations) or H(+) ions were studied in the egg cell membrane of a tunicate, Halocynthia roretzi, by using the voltage-clamp technique.2. With an increase in [Ca](o) or a decrease in pH in the external solution, the current-voltage (I-V) relations for the peak of the Na and Ca currents shifted along the voltage axis in the positive direction. These voltage shifts in the I-V relations, measured at a potential of V((1/2)) where inward current attains its half-maximum, were shown to be identical to shifts in voltage-dependence of the time courses of Na and Ca currents, and also identical to shifts in the inactivation curves of Na current along the voltage axis.3. The shifts in V((1/2)) produced by various polyvalent cations or H(+) ions were analysed by the Gouy-Chapman equation for the diffuse double layer, by assuming that a change in V((1/2)) directly corresponds to a change in the surface double layer potential.4. The V((1/2))-divalent cation concentration relations of Na current were exactly described by the predictions of the theory with a constant value of the surface charge density of 1e(-)/(9 A)(2). The weak stabilizing effects of Mg(2+), Sr(2+) and Ba(2+) were quite similar to each other and were explained in terms of a ;screening' effect. Other divalent cations, such as Ca(2+), Mn(2+) and Ni(2+), showed various different stabilizing effects which were explained in terms of a ;binding' effect. The binding constants (K(1)'s) for Ca(2+), Mn(2+) and Ni(2+) were 0.21, 0.45 and 0.94 M(-1), respectively.5. H(+) ions showed a powerful stabilizing effect upon the Na current with a K(H) of 6 x 10(4)M(-1). This value indicates that the acidic sites around Na channels have a pK(a) of 4.78. La(3+) ions also acted as a strong stabilizer upon the Na current with a K(La) of 15 M(-1). For both H(+) and La(3+), the V((1/2))-concentration relations were also exactly described by the Gouy-Chapman equation with the same charge density of 1e(-)/(9 A)(2) as estimated by varying divalent cations.6. The stabilizing effect of permeant cations such as Ca(2+), Sr(2+) and Ba(2+) on Ca channel currents was analysed. The effect of lowering pH was also studied. It was found that the surface charge density of 1e(-)/(9 A)(2) estimated by Na current is also applicable to the explanation for the V((1/2))-divalent cation concentration or - pH relationships. The estimated binding constants for H(+), Ca(2+) and Sr(2+) were 1.2x10(5), 0.58 and 0.035 M(-1), respectively. Ba(2+) does not bind to charged sites near to the Ca channels.7. It was noticed that a considerable reduction in the conductances of Na and Ca currents occurred in parallel with a stabilizing effect. This reduction was ascribed to a decrease in the concentration of permeant cations at the external surface of the cell membrane, as predicted by the theory of the diffuse double layer. The Goldman, Hodgkin-Katz equation for ionic currents was applied to explain the conductance suppression.8. The conductance suppressions of Na and Ca channel currents due to Ca(2+), Sr(2+) and Ba(2+) were found to be apparent ones, only reflecting decreases in the surface concentration of permeant cations without any changes in the permeability. After correction for the apparent suppression, the real permeability ratio among Ca(2+), Sr(2+) and Ba(2+) for Ca channels was determined as 1.00, 0.56 and 0.21 respectively.9. The conductance suppression of Na current by lowering pH was explained in terms of a real suppression or blocking which is superimposed on the apparent suppression. Considering the surface [Na](o), the plot of P(Na) against the surface pH yielded a blocking curve of Na channel by H(+) ions, which implies that two H(+) ions are necessary to block each Na channel. For Ca channels no real blockage was observed in acidic pH.10. It was concluded from the present experiment that there exists a surface potential capable of affecting both gating and permeation mechanisms of ionic channels in this tunicate egg cell membrane.     

L-type Ca2+ channels are not involved in coronary endothelial Ca2+ influx mechanism responsible for endothelium-dependent relaxation

Effects of L-type Ca2+ channel blockers on intracellular Ca2+ concentration ([Ca2+]i) changes evoked by the stimulations which cause endothelium-dependent relaxation were examined in freshly isolated pig coronary endothelial cells using fura-2 fluorescent analysis. Substance P and bradykinin produced endothelium-dependent relaxations of pig coronary arteries. The relaxations were inhibited significantly but not completely by N(omega)-nitro-L-arginine (L-NNA) or aspirin, suggesting that nitric oxide (NO), prostacyclin (PGI2) and endothelium-derived hyperpolarizing factor (EDHF) were involved in the responses. Both substance P and bradykinin elevated coronary endothelial [Ca2+]i in a biphasic manner: An initial transient increase was observed within a minute, which was followed by the subsequent sustained increase declining with time. In the medium without Ca2+, substance P-induced elevation of [Ca2+]i was markedly reduced. L-type Ca2+ channel blockers (nicardipine, diltiazem and verapamil) did not affect substance P-induced increase in endothelial [Ca2+]i. In consistent with this finding, Bay k 8644 failed to increase [Ca2+]i in partially depolarized endothelial cells. In contrast, substance P-induced elevation of endothelial [Ca2+]i was suppressed in high K+ solutions. These findings indicate that: (1) Substance P and bradykinin relax pig coronary artery via production/release of NO, PGI2 and EDHF from the endothelium; (2) The synthesis and release of these endothelium-derived factors are accompanied by an increase in endothelial [Ca2+]i; (3) Activation of L-type Ca2+ channels is not involved in coronary endothelial elevation of [Ca2+]i responsible for the production/release of these endothelium-derived factors. L-type Ca2+ channel blockers seem to be advantageous in the application for the disorders of coronary circulation with respect to that they do not prevent endothelial function to produce/release of endogenous vasorelaxants.     

Calcium-activated potassium channels in goldfish hair cells.

1. Characteristics of Ca(2+)-activated K+ channels in the basolateral membrane of hair cells isolated from the caudal part of the goldfish saccular macula were studied mainly with the inside-out mode of the patch clamp method. 2. Several types of Ca(2+)-activated K+ channels differing in unitary conductance were identified. The conductances (n = 156) ranged from 130 to 320 pS (when measured in symmetrical 125 mM KCl) and could be roughly separated into four groups, centred on values of 150, 200, 250 and 300 pS. The pharmacological profile, assessed by, for example, tetraethylammonium blockade, and the relatively large conductance indicated that these channels can be classified as large-conductance Ca(2+)-activated K+ channels (BK channels). The relative permeability of these channels to different ion species was in the order K+ (1.0) > Rb+ (0.8) > NH4+ (0.14) > Na+, Cs+ (< 0.05). 3. Curves relating open state probability to [Ca2+]i, for membrane potentials between -50 and +50 mV, were similar to those observed for BK channels of rat muscle. However, the maximum open state probability (100-1000 microM [Ca2+]i and 50 mV membrane potential) was 0.4-0.9, and always less than 1. 4. These channels had a short arithmetic mean open time ranging from 0.08 to 1.2 ms (0.08-0.5 ms in 88% of cases) and an arithmetic mean shut time ranging from 0.24 to 1.2 ms (10 microM [Ca2+]i and 50 mV membrane potential). The shut intervals were more sensitive to changes in [Ca2+]i and membrane potential than were the open intervals. 5. The distribution of individual open and shut intervals was fitted with the sum of exponential functions. Except for the slowest shut component, which only accounted for less than 1% of shut events, all other components had time constants shorter than 1 ms. As a result of these short open and shut intervals, the current trace had a flickery pattern rather than a burst-interburst pattern. 6. There was a rough correlation between unitary conductance and mean open time, i.e. channels with a large unitary conductance had a longer mean open time. 7. The sensitivity to [Ca2+]i of the Ca(2+)-activated K+ channel in goldfish hair cells was one to two orders of magnitude lower than that of BK channels in rat muscle. Channels with a longer mean open time had a higher Ca2+ sensitivity. 8. The stability of the single Ca(2+)-activated K+ channel kinetics was studied by measuring the 'moving' mean duration of open and shut intervals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ryanodine- and thapsigargin-insensitive Ca2+-induced Ca2+ release is primed by lowering external Ca2+ in rabbit autonomic neurons

Rises in cytosolic Ca2+ induced by a high K+ concentration (30 or 60 mM) (K+-induced Ca2+ transient) were recorded by fluorimetry of Ca2+ indicators in cultured rabbit otic ganglion cells. When external Ca2+ ([Ca2+]o) was reduced to a micromolar (10-40 microM) or nanomolar (<10 nM) level prior to high-K+ treatment, K+-induced Ca2+ transients of considerable amplitude (50% of control) were generated in most cells, although those initiated at normal [Ca2+]o were reduced markedly or abolished by reducing [Ca2+]o during exposure to a high K+ concentration. Lowering [Ca2+]o alone occasionally caused a transient rise in cytosolic Ca2+. K+-induced Ca2+ transients at micromolar [Ca2+]o were repeatedly generated and propagated inwardly at a speed slower than that at normal [Ca2+]o, while those at nanomolar [Ca2+]o occurred only once. K+-induced Ca2+ transients at micromolar [Ca2+]o were not blocked by ryanodine (10 microM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 5 microM: at 20-22 degrees C but blocked at 31-34 degrees C) or thapsigargin (1-2 microM), but were blocked by Ni2+ (1 mM) or nicardipine (10 microM). Thus, there is a ryanodine-insensitive Ca2+-release mechanism in FCCP- and thapsigargin-insensitive Ca2+ stores in rabbit otic ganglion cells, which is primed by lowering [Ca2+]o and then activated by depolarization-induced Ca2+ entry. This Ca2+-induced Ca2+ release may operate when [Ca2+]o is decreased by intense neuronal activity.     

A voltage- and Ca2+-dependent big conductance K channel in cochlear spiral ligament fibrocytes

Evidence is accruing that spiral ligament fibrocytes (SLFs) play an important role in cochlear K(+) homeostasis, but little direct physiological data is available to support this concept. Here we report the presence and characterization of a voltage- and Ca(2+)-dependent big-conductance K (BK) channel in type I SLFs cultured from the gerbil cochlea. A single-channel conductance of 298+/-5.6 pS (n=28) was measured under symmetrical K(+). Membrane potentials for half-maximal open probability (P(o)) were -67, -45 and 85 mV with cytosolic free-Ca(2+) levels of 0.7 mM, 10 microM and 1 microM, respectively (n=8-14). The Hill coefficient for Ca(2+) affinity was 1.9 at a membrane potential of 60 mV (n=6). The BK channel showed very low activity (P(o)=0.0019, n=5) under normal physiological conditions, suggesting a low resting intracellular free [Ca(2+)]. Pharmacological results fit well with the profile of classic BK channels. The estimated half-maximal inhibitory concentration and Hill coefficient for tetraethylammonium were 0.086+/-0.021 mM and 0.99, respectively (n=4-9). In whole cell recordings, the voltage-activated outward K current was inhibited 85.7+/-4.5% (n=6) by 0.1 microM iberiotoxin. A steady-state kinetic model with two open and two closed stages best described the BK gating process (tau(o1) 0.23+/-0.08 ms, tau(o2) 1.40+/-0.32 ms; tau(c1) 0.26+/-0.09 ms, tau(c2) 3.10+/-1.2 ms; n=11). RT-PCR analyses revealed a splice variant of the BK channel alpha subunit in cultured type I SLFs and freshly isolated spiral ligament tissues. The BK channel is likely to play a major role in regulating the membrane potential of type I SLFs, which may in turn influence K(+) recycling dynamics in the mammalian cochlea.