Ionic Channels of the Sugar Beet Tonoplast are Regulated by a Multi-ion Single-file Permeation Mechanism

Ionic channels of the sugar beet tonoplast were studied using the patch-clamp technique. At micromolar concentrations of cytosolic calcium, several (at least four) distinct single-channel current levels were routinely identified. On the basis of channel voltage dependence, kinetic properties and conductance of single openings, the largest channel (103 ± 2 pS in symmetric 150 mm KCl) corresponds to the slow vacuolar (SV) channel already identified by Hedrich and Neher (1987). The majority of the whole-vacuole current was ascribed to this time-dependent slow-activating channel elicited by positive vacuolar potentials. The channel of intermediate amplitude (41 ± 1 pS in 150 mm KCl) did not show any voltage dependence and delay in the activation upon the application of voltage steps to both positive and negative transmembrane potentials. Owing to its voltage independence this channel was denominated FV1. The opening probability of the SV-type channel increased by increasing the cytoplasmic calcium concentration, while the activity of the FV1 channel did not increase appreciably by changing the calcium concentration in the range from 6 μm to 1 mm. All the channels identified showed a linear current-voltage characteristic in the range ±100 mV and at least the three most conductive ones displayed potassium selectivity properties. Substitution of potassium with tetramethylammonium (TMA) on the cytosolic side demonstrated that both the SV and FV1 channels are impermeable to TMA influx into the vacuole and support the potassium selectivity properties of these two channels. Moreover, the single channel conductances of all the channels identified increased as a function of the potassium concentration and reached a maximum conductivity at [K⁺] ∼0.5 m. This behavior can be explained by a multi-ion occupancy single-file permeation mechanism. Received: 26 December 1995/Revised: 10 July 1996     

Reconstitution in Lipid Bilayers of an ATP-Sensitive K+ Channel from Pig Coronary Smooth Muscle

A K⁺ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid bilayers. This channel was identified as an ATP-sensitive K⁺ channel (K_ATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na₂. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0 mV and with a 5/250 KCl gradient, the main conductance of the K_ATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels. In general, K_ATP channels were characterized by brief openings followed by long closings (open probability, P _o ≈ 0.02); only occasionally (3 out of 12 experiments) did the K_ATP channels have a high open probability (P _o ≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl₂ to the internal side of the channel. Received: 7 November 1995/Revised: 10 June 1996     

Ion Channel Phenotype of Melanoma Cell Lines

Melanoma cells are transformed melanocytes of neural crest origin. K⁺ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify voltage-gated (K_V), Ca²⁺-activated (K_Ca), and inwardly rectifying (K_IR) K⁺ channels; swelling-sensitive Cl⁻ channels (Cl_swell); voltage-gated Ca²⁺ channels (Ca_V) and Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores (CRAC); and voltage-gated Na⁺ channels (Na_V). The presence of Ca²⁺ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca²⁺] _i . The expression of K⁺ channels varied widely between different cell lines and was also influenced by culture conditions. K_IR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for K_IR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). K_Ca channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). K_Ca channels in C8146 and SK28 cells were sensitive to CTX (K _d = 4 nm), but were unaffected by apamin. K_V channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed CRAC channels and a novel Cl_swell channel. Cl_swell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1 in symmetrical Cl⁻). We conclude that different melanoma cell lines express a diversity of ion channel types. Received: 2 April 1996/Revised: 22 August 1996     

An L-type Calcium Channel in Renal Epithelial Cells

A voltage-activated Ca⁺⁺ channel has been identified in the apical membranes of cultured rabbit proximal tubule cells using the patch-clamp technique. With 105 mm CaCl₂ solution in the pipette and 180 NaAsp in the bath, the channel had a conductance of 10.4 ± 1.0 pS (n= 8) in on-cell patches, and 9.8 ± 1.1 pS (n= 8) in inside-out patches. In both on-cell and inside-out patches, the channel is active by membrane depolarization. For this channel, the permeation to Ba⁺⁺ and Ca⁺⁺ is highly selective over Na⁺ and K⁺ (P_Ca(Ba):P_Na(K) >200:1). The sensitivity to dihydropyridines is similar to that for L-type channels where the channel was blocked by nifedipine (10 μm), and activated by Bay K 8644 (5 μm). When activated by Bay K 8644, the channel showed subconductance levels. Treatment with forskolin (12.5 μm), phorbol ester (1 μm), or stretching (40 cm water) did not activate this channel. These results indicate that this Ca⁺⁺ channel is mostly regulated by membrane voltage, and appears to be an epithelial class of L-type Ca⁺⁺ channel. As such, it may participate in calcium reabsorption during periods of enhanced sodium reabsorption, or calcium signaling in volume regulation, where membrane depolarization occurs for prolonged periods. Received: 1 April 1996/Revised: 5 August 1996     

A Large-Conductance Chloride Channel in Pigmented Ciliary Epithelial Cells Activated by GTPγS

A large-conductance (or maxi-) chloride channel was identified in bovine pigmented ciliary epithelial (PCE) cells using inside-out excised patch clamp recording. The channel had a mean conductance of 293 pS when excised patches were bathed in symmetrical 130 mm NaCl although the conductance decreased to 209 pS when the solution bathing the cytoplasmic face of the patch contained only 33 mm NaCl. The channel was highly selective for chloride, with a P _Cl/P _Na= 24. A flickery, reversible block was produced by the diuretic stilbene 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), while 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) produced a permanent block. The channel was rarely active in cell-attached patches and usually required several minutes of polarization before activity could be detected in excised patches, a process known as metagenesis. Once activated, the channel was voltage-dependent and was mainly open within the voltage range −30 to +30 mV closing when the membrane was polarized to larger values. GTPγS (100 μm) activated the channel with a latency of 170 sec when applied to the cytoplasmic face of patches. This activation was not reversible upon return to control solution within the duration of the experiment. We assess the available evidence and suggest a role for this channel in volume regulation. Received: 24 June 1996/Revised: 18 February 1997     

Activation of Maxi-K Channels by Parathyroid Hormone and Prostaglandin E2 in Human Osteoblast Bone Cells

Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuration (CAP) no channel activity was observed in 2 mm Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon addition of 50 nm parathyroid hormone (PTH), 5 nm prostaglandin E₂ (PGE₂) or 0.1 mm dibutyryl cAMP + 1 μm forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that in symmetrical solutions (140 mm K) the channel has a conductance of 246 ± 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (P_K/P_Na) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability (P _o ) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the P _o vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mm ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE₂ or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (⁴⁵Ca⁺⁺) by MG-63 cells was stimulated in the presence of PTH and PGE₂, an effect inhibited by Nitrendipine (10 μm). Second, whereas PGE₂ stimulated the calcium-activated maxi-K channel in 2 mm Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE₂ did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mm Ca Ringer and 10 μm Nitrendipine in CAP configuration, PGE₂ (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of this channel, it is concluded that activation of this channel by PTH, PGE₂ or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx. Received: 17 September 1995/Revised: 7 December 1995     

Properties of Chicken Lens MIP Channels Reconstituted into Planar Lipid Bilayers

Membrane fractions highly enriched in chicken lens MIP (MIP28) were found to form ion channels when incorporated into planar lipid bilayers. The channels displayed prominent unitary conductances of about 60 and 290 pS in symmetric 150 mm KCl solution and were slightly anion selective. For both depolarizing and hyperpolarizing voltages, voltage sensitivity of the MIP28-induced conductance could be fit by a Boltzmann relation, symmetric around zero mV, with V ₀ = 18.5 mV, n= 4.5 and g _min/g _max= 0.17. Channel properties were not appreciably altered by pH in the range of 5.8 to 7, although channel incorporation was observed to occur more frequently at lower pH values. Calcium, at millimolar concentrations, decreased the channel mean open time. Partial proteolysis of MIP28 to yield MIP21 did not appreciably affect single-channel conductance or voltage sensitivity of the reconstituted channels. MIP28 was not phosphorylated by cAMP dependent protein kinase (PKA). Although unitary conductance and selectivity of the chicken MIP channel are similar to those reported for the bovine MIP (MIP26), the voltage sensitivity of MIP28 was higher than that of the bovine homologue, and voltage sensitivity of MIP28 was not modulated by treatments previously shown to affect MIP26 voltage gating (partial proteolysis and protein phosphorylation by PKA: (Ehring et al., 1990). The existence of such strikingly different functional properties in highly homologous channel isoforms may provide a useful system for exploration of the structure-function relations of MIP channels. Received: 27 March 1996/Revised: 5 August 1996     

A9 Fibroblasts Transfected with the m3 Muscarinic Receptor Clone Express a Ca2+ Channel Activated by Carbachol,GTP and GDP

Muscarinic m3 receptor-mediated changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_l) occur by activation of Ca²⁺ release channels present in the endoplasmic reticulum membrane and Ca²⁺ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation of a voltage-insensitive, cation-selective channel of low conductance (3.2 ± 0.6 pS; 25 mm Ca²⁺ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side), suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised inside-out membrane patches from nonstimulated A9m3 cells GTP (10 μm) and GDP (10 μm) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mm Ca²⁺ as charge carrier) respectively. In contrast, ATP (10 μm), UTP (10 μm) or CTP (10 μm) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the activation of a cation channel that conducts calcium. Received: 14 November 1996/Revised: 4 April 1997     

Hyperpolarization-activated Ca2+-permeable Channels in the Plasma Membrane of Tomato Cells

The hyperpolarization of the electrical plasma membrane potential difference has been identified as an early response of plant cells to various signals including fungal elicitors. The hyperpolarization-activated influx of Ca²⁺ into tomato cells was examined by the application of conventional patch clamp techniques. In both whole cell and single-channel recordings, clamped membrane voltages more negative than −120 mV resulted in time- and voltage-dependent current activation. Single-channel currents saturated with increasing activities of Ca²⁺ and Ba²⁺ from 3 to 26 mm and the single channel conductance increased from 4 pS to 11 pS in the presence of 20 mm Ca²⁺ or Ba²⁺, respectively. These channels were 20–25 and 10–13 times more permeable to Ca²⁺ than to K⁺ and to Cl⁻, respectively. Channel currents were strongly inhibited by 10 μm lanthanum and 50% inhibited by 100 μm nifedipine. This evidence suggests that hyperpolarization-activated Ca²⁺-permeable channels provide a mechanism for the influx of Ca²⁺ into tomato cells. Received: 13 February 1996/Revised: 12 August 1996     

Associated Proteins and Renal Epithelial Na+ Channel Function

The hypothesis that amiloride-sensitive Na⁺ channel complexes immunopurified from bovine renal papillary collecting tubules contain, as their core conduction component, an ENaC subunit, was tested by functional and immunological criteria. Disulfide bond reduction with dithiothreitol (DTT) of renal Na⁺ channels incorporated into planar lipid bilayers caused a reduction of single channel conductance from 40 pS to 13 pS, and uncoupled PKA regulation of this channel. The cation permeability sequence, as assessed from bi-ionic reversal potential measurements, and apparent amiloride equilibrium dissociation constant (K ^amil _i ) of the Na⁺ channels were unaltered by DTT treatment. Like ENaC, the DTT treated renal channel became mechanosensitive, and displayed a substantial decrease in K ^amil _i following stretch (0.44 ± 0.12 μm versus 6.9 ± 1.0 μm). Moreover, stretch activation induced a loss in the channel's ability to discriminate between monovalent cations, and even allowed Ca²⁺ to permeate. Polyclonal antibodies generated against a fusion protein of αbENaC recognized a 70 kDa polypeptide component of the renal Na⁺ channel complex. These data suggest that ENaC is present in the immunopurified renal Na⁺ channel protein complex, and that PKA sensitivity is conferred by other associated proteins. Received: 5 June 1995/Revised: 29 September 1995     

Block of Voltage-dependent Calcium Channel by the Green Mamba Toxin Calcicludine

A number of peptide toxins derived from marine snails and various spiders have been shown to potently inhibit voltage-dependent calcium channels. Here, we describe the effect of calcicludine, a 60 amino-acid peptide isolated from the venom of the green mamba (Dendroaspis angusticeps), on transiently expressed high voltage-activated calcium channels. Upon application of calcicludine, L-type (α_{1 C} ) calcium channels underwent a rapid, irreversible decrease in peak current amplitude with no change in current kinetics, or any apparent voltage-dependence. However, even at saturating toxin concentrations, block was always incomplete with a maximum inhibition of 58%, indicating either partial pore block, or an effect on channel gating. Block nonetheless was of high affinity with an IC₅₀ value of 88 nm. Three other types of high voltage activated channels tested (α_{1 A} , α_{1 B} , and α_{1 E} ) exhibited a diametrically different response to calcicludine. First, the maximal inhibition observed was around 10%, furthermore, the voltage-dependence of channel activation was shifted slightly towards more negative potentials. Thus, at relatively hyperpolarized test potentials, calcicludine actually upregulated current activity of (N-type) α_{1 B} channels by as much as 50%. Finally, the use of several chimeric channels combining the major transmembrane domains of α_{1 C} and α_{1 E} revealed that calcicludine block of L-type calcium channels involves interactions with multiple structural domains. Overall, calcicludine is a potent and selective inhibitor of neuronal L-type channels with a unique mode of action. Received: 22 September 1999/Revised: 1 December 1999     

Effect of Calcium and Calcium Channel Blockers on Transient Outward Current of F76 and D1 Neuronal Soma Membranes in the Subesophageal Ganglia of Helix aspersa

Twin-electrode voltage-clamp techniques were used to study the effect of calcium and calcium channel blockers on the transient outward current in isolated F76 and D1 neurones of Helix aspersa subesophageal ganglia in vitro (soma only preparation with no cell processes). On lowering extracellular Ca²⁺ concentration from 10 to 2 mm or removing extracellular calcium from the bathing medium, the threshold for this current shifted in a negative direction by 11.5 and 20 mV, respectively. On the other hand, increasing the extracellular Ca²⁺ concentration from 10 to 20 and to 40 mm shifted the steady-state inactivation curves in positive directions on the voltage axis by 7 and 15 mV, respectively. Upon application of calcium channel blockers, Co²⁺, La³⁺, Ni²⁺ and Cd²⁺, transient potassium current amplitude was reduced in a voltage-dependent manner, being more effective at voltages close to the threshold. The current was elicited even at a holding potential of −34 mV. The specific calcium channel blockers, amiloride and nifedipine did not shift the activation and steady-state inactivation curves and did not reduce the transient outward current amplitude. It was concluded that the transient outward current is not dependent on intracellular Ca²⁺ but that it is modulated by Ca²⁺ and di- and trivalent ions extracellularly. The effects of these ions are very unlikely to be due to a surface charge effect because the addition of La³⁺ (200 μm) completely reverses the shift in a hyperpolarizing direction when the extracellular Ca²⁺ concentration was reduced from 10 to 1 mm and additionally shifts the kinetics further still in a depolarizing direction. The responses seen here are consistent with a specific effect of di- and trivalent ions on the transient outward current channels leading to a modification of gating. Received: 30 March 1999/Revised: 5 October 1999     

Regulation of calcium signalling by 1-O-alkylglycerols in human Jurkat T lymphocytes

We studied the role of natural occurring 1-O-alkylglycerols on the calcium signalling in Jurkat T-cells. Alkylglycerols evoked an increase in free intracellular calcium concentration [Ca2+]i, in a dose-dependent manner. When the experiments were performed in calcium-free buffer, the alkylglycerol response on the rise of [Ca2+]i was wholly abolished compared with the one in calcium-containing buffer, suggesting that these etherlipids induce a calcium influx by the opening of Ca2+ channels. We further employed inhibitors of voltage-gated calcium channels. We observed that omega-conotoxin, a blocker of N-type voltage-activated Ca2+ channels, but not verapamil, a blocker of L-type voltage-activated Ca2+ channels, curtailed significantly the calcium rise evoked by the lipid agents. Alkylglycerols also induced plasma membrane depolarisation, known to be involved in the opening of the voltage-gated calcium channels. Our study shows that alkylglycerols increase [Ca2+]i influx in human Jurkat T-cells possibly by modulating the permeability of calcium channels.     

GABA Concentration Sets the Conductance of Delayed GABAA Channels in Outside-out Patches from Rat Hippocampal Neurons

GABA_A channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches). The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased with GABA concentration from less than 10 pS (0.5 μm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC ₅₀ value of 33 μm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of 1 μm diazepam, the GABA EC ₅₀ decreased to 0.2 μm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 μm GABA the bicuculline IC ₅₀ was 19 μm. The effect of GABA on channel conductance shows that the role of the ligand in GABA_A receptor channel function is more complex than previously thought. Received: 23 October 2000/Revised: 27 February 2001     

The Ach-evoked Ca2+-activated K+ Current in Mouse Mandibular Secretory Cells. Single Channel Studies

Although acetylcholine (ACh) is able to activate voltage- and Ca²⁺-sensitive K⁺ (BK) channels in mouse mandibular secretory cells, our recent whole cell studies have suggested that these channels, like those in sheep parotid secretory cells, do not contribute appreciably to the conductance that carries the ACh-evoked whole cell K⁺ current. In the present study, we have used cell-attached patch clamp methods to identify and characterize the K⁺ channel type responsible for carrying the bulk of this current. When the cells were bathed in a NaCl-rich solution the predominant channel type activated by ACh (1 μmol/l or 50 nmol/l) had a conductance only of 40 pS; it was not blocked by TEA but it was sensitive to quinine and it conducted Rb⁺ to an appreciable extent. BK channels, which could be seen in some but not all patches from resting cells, also showed increased activity when ACh was added to the bath, but they were much less conspicuous during ACh stimulation than the 40-pS channels. When the cells were bathed in a KCl-rich rather than a NaCl-rich solution, a small-conductance K⁺ channel, sensitive to quinine but not to TEA, was still the most conspicuous channel to be activated by ACh although its conductance was reduced to 25 pS. Our studies confirm that the ACh-evoked whole-cell K⁺ current is not carried substantially by BK channels and show that it is carried by a small-conductance K⁺ channel with quite different properties. Received: 28 September 1995/Revised: 26 December 1995     

Characterization of the Ryanodine Receptor-Ca2+ Release Channel from the Thoracic Tissues of the Lepidopteran Insect Heliothis virescens

The existence of invertebrate forms of the RyR has recently been confirmed (Takeshima et al., 1994, Puente et al., 2000). However, information on the functional properties of this insect RyR is still limited. We report the functional characterization of a RyR from the thoracic muscle of H. virescens (Scott-Ward et al., 1997). A simple purification protocol produced membranes from homogenized prefrozen H. virescens thoracic muscle with a [³H]-ryanodine binding activity of 1.19 ± 0.21 pmol/mg protein (mean ±se; n= 4). [³H]-Ryanodine binding to the H. virescens receptor was dependent on the ryanodine concentration in a hyperbolic fashion with a K _D of 3.82 nm (n= 4). [³H]-ryanodine binding was dependent on [Ca²⁺] in a biphasic manner and was stimulated by 1 mm ATP. Millimolar caffeine did not stimulate [³H]-ryanodine binding to H. virescens membranes in the presence of either nanomolar or micromolar Ca²⁺. A protein of at least 400 KDa was recognized in H. virescens membrane proteins by a specific anti-H. virescens RyR antibody. Discontinuous density sucrose gradient fractionation of microsomal membranes produced vesicles suitable for single-channel studies. Ca²⁺-sensitive, Ca²⁺-permeable channels were successfully inserted into artificial lipid bilayers from H. virescens membrane vesicles. The H. virescens RyR-channel displayed a Ca²⁺ conductance of ∼110 pS and underwent a persistent and characteristic modification of ion handling and gating following addition of 100 nm ryanodine. The gating of H. virescens channels was sensitive to ATP and ruthenium red in a manner similar to mammalian RyR. This is the first report to describe the single channel and [³H]-ryanodine binding properties of a native insect RyR. Received: 3 July 2000/Revised: 17 October 2000     

Gating Kinetics of E. coli Poly-3-Hydroxybutyrate/Polyphosphate Channels in Planar Bilayer Membranes

Nonproteinaceous calcium channel complexes from Escherichia coli, composed of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP), exhibit two distinct gating modes (modes 1 and 2) in planar lipid bilayers. Here we report the kinetic characterization of the channel in mode 2, a mode characterized by two well-defined conductance levels, a fully open state (87 ± 3 pS), and a major subconductance state (56 ± 2 pS). Other subconductance states and full closures are rare (<0.5% of total time). Several kinetic properties of the channel showed asymmetric voltage-dependence indicating an asymmetry in the channel structure. Accordingly, single channels responded to potential change in one of two mirror-image patterns, postulated to arise from opposite orientations of the asymmetrical channel complex in the bilayer. The fraction of time spent in each conductance level was strongly voltage-sensitive. For channels reported in this study, presumably all oriented in the same direction, residence time in the fully open state increased as clamping potentials became more positive whereas residence time in the major subconductance state increased at more negative potentials. Analysis of open time distributions revealed existence of two kinetically distinct states for each level. The shorter time constants for both conductance states exhibited weak voltage-sensitivity; however, the longer time constants were strongly voltage-sensitive. A kinetic scheme, consistent with the complex voltage dependence of the channel, is proposed. Received: 1 February 1999/Revised: 2 April 1999     

Skeletal Muscle Ryanodine Receptor Channels Are Activated by the Fungal Metabolite, Gliotoxin

Interactions between the reactive disulfide fungal metabolite, gliotoxin (GTX), and rabbit skeletal ryanodine receptor (RyR) calcium release channels have been examined. RyRs in terminal cisternae vesicles formed a covalent complex with 100 μm ³⁵S-GTX, which was reversed by 1 mm dithiothreitol (DTT) or 1 mm glutathione. GTX (80–240 μm), added to either cytoplasmic (cis) or luminal (trans) solutions, increased the rate of Ca²⁺ release from SR vesicles and the frequency of opening of single RyR channels in lipid bilayers. Channel activation was reversed upon addition of 2 mm DTT to the cis solution, showing that the activation was due to an oxidation reaction (2 mm DTT added to the cis solution in the absence of GTX did not affect RyR activity). Furthermore, RyRs were not activated by trans GTX if the cis chamber contained DTT, suggesting that GTX oxidized a site in or near the membrane. In contrast to cis DTT, 2 mm DTT in the trans solution increased RyR activity when added either alone or with 200 μm trans GTX. The results suggest that (i) GTX increases RyR channel activity by oxidizing cysteine residues that are close to the membrane and located on RyR, or associated proteins, and (ii) a disulfide bridge or nitrosothiol, accessible only from the luminal solution, normally suppresses RyR channel activity. Some of the actions of GTX in altering Ca²⁺ homeostatsis might depend on its modification of RyR calcium channels. Received: 12 November 1999/Revised: 14 March 2000     

Permeabilization of Model Lipid Membranes by Bacillus sphaericus Mosquitocidal Binary Toxin and its Individual Components

The high larvicidal effect of Bacillus sphaericus (Bs), a mosquito control agent, originates from the presence of a binary toxin (Bs Bin) composed of two proteins (BinA and BinB) that work together to lyse gut cells of susceptible larvae. We demonstrate for the first time that the binary toxin and its individual components permeabilize receptor-free large unilamellar phospholipid vesicles (LUVs) and planar lipid bilayers (PLBs) by a mechanism of pore formation. Calcein-release experiments showed that LUV permeabilization was optimally achieved at alkaline pH and in the presence of acidic lipids. BinA was more efficient than BinB, BinB facilitated the BinA effect, and their stoichiometric mixture was more effective than the full Bin toxin. In PLBs, BinA formed voltage-dependent channels of ≈100–200 pS with long open times and a high open probability. Larger channels (≥400 pS) were also observed. BinB, which inserted less easily, formed smaller channels (≤100 pS) with shorter mean open times. Channels observed after sequential addition of the two components, or formed by their 1:1 mixture (w/w), displayed BinA-like activity. Bs Bin toxin was less efficient at forming channels than the BinA/BinB mixture, with channels displaying the BinA channel behavior. Our data support the concept of BinA being principally responsible for pore formation in lipid membranes with BinB, the binding component of the toxin, playing a role in promoting channel activity. Received: 29 March 2001/Revised: 20 July 2001     

Evidence That Calcium Release-activated Current Mediates the Biphasic Electrical Activity of Mouse Pancreatic β-Cells

The electrical response of pancreatic β-cells to step increases in glucose concentration is biphasic, consisting of a prolonged depolarization with action potentials (Phase 1) followed by membrane potential oscillations known as bursts. We have proposed that the Phase 1 response results from the combined depolarizing influences of potassium channel closure and an inward, nonselective cation current (I _CRAN) that activates as intracellular calcium stores empty during exposure to basal glucose (Bertram et al., 1995). The stores refill during Phase 1, deactivating I _CRAN and allowing steady-state bursting to commence. We support this hypothesis with additional simulations and experimental results indicating that Phase 1 duration is sensitive to the filling state of intracellular calcium stores. First, the duration of the Phase 1 transient increases with duration of prior exposure to basal (2.8 mm) glucose, reflecting the increased time required to fill calcium stores that have been emptying for longer periods. Second, Phase 1 duration is reduced when islets are exposed to elevated K⁺ to refill calcium stores in the presence of basal glucose. Third, when extracellular calcium is removed during the basal glucose exposure to reduce calcium influx into the stores, Phase 1 duration increases. Finally, no Phase 1 is observed following hyperpolarization of the β-cell membrane with diazoxide in the continued presence of 11 mm glucose, a condition in which intracellular calcium stores remain full. Application of carbachol to empty calcium stores during basal glucose exposure did not increase Phase 1 duration as the model predicts. Despite this discrepancy, the good agreement between most of the experimental results and the model predictions provides evidence that a calcium release-activated current mediates the Phase 1 electrical response of the pancreatic β-cell. Received: 5 June 1996/Revised: 15 August 1996