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S D Silberberg

Publications and source records attributed to S D Silberberg.

At least 19 recordsLinked to original sources
PubMed2001

Two-dimensional kinetic analysis suggests nonsequential gating of mechanosensitive channels in Xenopus oocytes.

Xenopus oocytes express mechanosensitive (MS(XO)) channels that can be studied in excised patches of membrane with the patch-clamp technique. This study examines the steady-state kinetic gating properties of MS(XO) channels using detailed single-channel analysis. The open and closed one-dimensional dwell-time distributions were described by the sums of 2-3 open and 5-7 closed exponential components, respectively, indicating that the channels enter at least 2-3 open and 5-7 closed kinetic states during gating. Dependency plots revealed that the durations of adjacent open and closed intervals were correlated, indicating two or more gateway states in the gating mechanism for MS channels. Maximum likelihood fitting of two-dimensional dwell-time distributions to both generic and specific models was used to examine gating mechanism and rank models. A kinetic scheme with five closed and five open states, in which each closed state could make a direct transition to an open state (two-tiered model) could account for the major features of the single-channel data. Two-tiered models that allowed direct transitions to subconductance open states in addition to the fully open state were also consistent with multiple gateway states. Thus, the gating mechanism of MS(XO) channels differs from the sequential (linear) gating mechanisms considered for MS channels in bacteria, chick skeletal muscle, and Necturus proximal tubule.

Animals↗
PubMed2001

Genomic structure, developmental distribution and functional properties of the chicken P2X(5) receptor.

We report here the cloning of a chicken cDNA (402 aa) showing high sequence similarity to the previously cloned rat and human P2X(5) receptors (67 and 69%, respectively). The chicken P2X(5) subunit is encoded by a gene composed of 12 translated exons, which shows conserved genomic structure with mammalian P2X genes. In HEK-293 cells heterologously expressing chicken P2X(5) receptors, ATP activates a current that desensitizes in a way that is dependent on the presence of extracellular divalent cations. ATP and 2-methylthio ATP are equipotent agonists (EC(50) approximately 2 microM) and suramin and pyridoxal 5-phosphate-6-azophenyl-2',4'-disulfonic acid are potent antagonists. Additionally, reversal potential measurements indicate that chicken P2X(5) is permeable not only to cations but also to chloride (P(Cs+)/P(Cl-) approximately 1.9), as has been described for native P2X receptor mediated responses in embryonic chicken skeletal muscle. mRNA distribution of chicken P2X(5) was determined by in situ hybridization analysis in both whole embryos and on tissue slices of heart and skeletal muscle. Our results suggest that chicken P2X(5) receptors are expressed in developing muscle and might play a role in early muscle differentiation.

Amino Acid Sequence↗
PubMed2000

Modulation of the voltage-dependent anion channel (VDAC) by glutamate.

The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is a large channel permeable to anions, cations, ATP, and other metabolites. VDAC was purified from sheep brain synaptosomes or rat liver mitochondria using a reactive red-agarose column, in addition to the hydroxyapatitate column. The red-agarose column allowed further purification (over 98%), concentration of the protein over ten-fold, decreasing Triton X-100 concentration, and/or replacing Triton X-100 with other detergents, such as Nonidet P-40 or octylglucoside. This purified VDAC reconstituted into planar-lipid bilayer, had a unitary maximal conductance of 3.7 +/- 0.1 nS in 1 M NaCl, at 10 mV and was permeable to both large cations and anions. In the maximal conducting state, the permeability ratios for Na(+), acetylcholine(+), dopamine,(+) and glutamate(-), relative to Cl(-), were estimated to be 0.73, 0.6, 0.44, and 0.4, respectively. In contrast, in the subconducting state, glutamate(-) was impermeable, while the relative permeability to acetylcholine(+) increased and to dopamine(+) remained unchanged. At the high concentrations (0.1-0.5 M) used in the permeability experiments, glutamate eliminated the bell shape of the voltage dependence of VDAC channel conductance. Glutamate at concentrations of 1 to 20 mM, in the presence of 1 M NaCl, was found to modulate the VDAC channel activity. In single-channel experiments, at low voltages (+/-10 mV), glutamate induced rapid fluctuations of the channel between the fully open state and long-lived low-conducting states or short-lived closed state. Glutamate modification of the channel activity, at low voltages, is dependent on voltage, requiring short-time (20-60 sec) exposure of the channel to high membrane potentials. The effect of glutamate is specific, since it was observed in the presence of 1 M NaCl and it was not obtained with aspartate or GABA. These results suggest that VDAC possesses a specific glutamate-binding site that modulates its activity.

Acetylcholine↗
PubMed1999

Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels.

The patch-clamp technique allows currents to be recorded through single ion channels in patches of cell membrane in the tips of glass pipettes. When recording, voltage is typically applied across the membrane patch to drive ions through open channels and to probe the voltage-sensitivity of channel activity. In this study, we used video microscopy and single-channel recording to show that prolonged depolarization of a membrane patch in borosilicate pipettes results in delayed slow displacement of the membrane into the pipette and that this displacement is associated with the activation of mechanosensitive (MS) channels in the same patch. The membrane displacement, approximately 1 micrometer with each prolonged depolarization, occurs after variable delays ranging from tens of milliseconds to many seconds and is correlated in time with activation of MS channels. Increasing the voltage step shortens both the delay to membrane displacement and the delay to activation. Preventing depolarization-induced membrane displacement by applying positive pressure to the shank of the pipette or by coating the tips of the borosilicate pipettes with soft glass prevents the depolarization-induced activation of MS channels. The correlation between depolarization-induced membrane displacement and activation of MS channels indicates that the membrane displacement is associated with sufficient membrane tension to activate MS channels. Because membrane tension can modulate the activity of various ligand and voltage-activated ion channels as well as some transporters, an apparent voltage dependence of a channel or transporter in a membrane patch in a borosilicate pipette may result from voltage-induced tension rather than from direct modulation by voltage.

Animals↗
PubMed1999

Extracellular sodium regulates airway ciliary motility by inhibiting a P2X receptor.

The mucociliary system is responsible for clearing inhaled particles and pathogens from the airways. This important task is performed by the beating of cilia and the consequent movement of mucus from the lungs to the upper airways. Because ciliary motility is enhanced by elevated intracellular calcium concentrations, inhibition of calcium influx could lead to disease by jeopardizing mucociliary clearance. Several hormones and neurotransmitters stimulate ciliary motility, one of the most potent of which is extracellular ATP (ATP0), which acts by releasing calcium ions from internal stores and by activating calcium influx. Here we show that, in airway ciliated cells, extracellular sodium ions (Na+(0)) specifically and competitively inhibit an ATP0-gated channel that is permeable to calcium ions, and thereby attenuate ATP0-induced ciliary motility. Our finding points to a physiological role for Na+(0) in ciliary function, and indicates that mucociliary clearance might be improved in respiratory disorders such as chronic bronchitis and cystic fibrosis by decreasing the sodium concentration of the airway surface fluid in which the cilia are bathed.

Adenosine Triphosphate↗
PubMed1999

Ca2+-dependent gating mechanisms for dSlo, a large-conductance Ca2+-activated K+ (BK) channel.

The Ca2+-dependent gating mechanism of cloned BK channels from Drosophila (dSlo) was studied. Both a natural variant (A1/C2/E1/G3/IO) and a mutant (S942A) were expressed in Xenopus oocytes, and single-channel currents were recorded from excised patches of membrane. Stability plots were used to define stable segments of data. Unlike native BK channels from rat skeletal muscle in which increasing internal Ca2+ concentration (Cai2+) in the range of 5 to 30 microM increases mean open time, increasing Cai2+ in this range for dSlo had little effect on mean open time. However, further increases in Cai2+ to 300 or 3000 microM then typically increased dSlo mean open time. Kinetic schemes for the observed Ca2+-dependent gating kinetics of dSlo were evaluated by fitting two-dimensional dwell-time distributions using maximum likelihood techniques and by comparing observed dependency plots with those predicted by the models. Previously described kinetic schemes that largely account for the Ca2+-dependent kinetics of native BK channels from rat skeletal muscle did not adequately describe the Ca2+ dependence of dSlo. An expanded version of these schemes which, in addition to the Ca2+-activation steps, permitted a Ca2+-facilitated transition from each open state to a closed state, could approximate the Ca2+-dependent kinetics of dSlo, suggesting that Ca2+ may exert dual effects on gating.

Animals↗
PubMed1999

Membrane-pipette interactions underlie delayed voltage activation of mechanosensitive channels in Xenopus oocytes.

To investigate the mechanism for the delayed activation by voltage of the predominant mechanosensitive (MS) channel in Xenopus oocytes, currents were recorded from on-cell and excised patches of membrane with the patch clamp technique and from intact oocytes with the two-electrode voltage clamp technique. MS channels could be activated by stretch in inside-out, on-cell, and outside-out patch configurations, using pipettes formed of either borosilicate or soft glass. In inside-out patches formed with borosilicate glass pipettes, depolarizing voltage steps activated MS channels in a cooperative manner after delays of seconds. This voltage-dependent activation was not observed for outside-out patches. Voltage-dependent activation was also not observed when the borosilicate pipettes were either replaced with soft glass pipettes or coated with soft glass. When depolarizing voltage steps were applied to the whole oocyte with a two-electrode voltage clamp, currents that could be attributed to MS channels were not observed. Yet the same depolarizing steps activated MS channels in on-cell patches formed with borosilicate pipettes on the same oocyte. These observations suggest that the delayed cooperative activation of MS channels by depolarization is not an intrinsic property of the channels, but requires interaction between the membrane and patch pipette.

Animals↗
PubMed1998

Extracellular ATP directly gates a cation-selective channel in rabbit airway ciliated epithelial cells.

1. A membrane conductance activated by extracellular ATP was identified and characterized in freshly dissociated rabbit airway ciliated cells using the whole-cell and outside-out patch configurations of the patch-clamp technique. 2. In solutions designed to maximize currents through voltage-gated calcium channels, there were no indications of voltage-gated Ba2+ currents. 3. Extracellular ATP (but not UTP or ADP) activated a membrane conductance which remained activated for several minutes in the presence of ATP. The conductance was permeable to monovalent and divalent cations with approximate relative permeabilities (P) for PBa : PCs : PTEA of 4 : 1 : 0.1. Permeability to Cl- was negligible. 4. Including GDP-beta-S in the intracellular solution did not inhibit the effects of ATP, nor did GTP-gamma-S irreversibly activate the conductance. 5. In outside-out membrane patches, with GDP-beta-S in the pipette solution, ATP activated ion channels which had a chord conductance of approximately 6 pS in symmetrical 150 mM CsCl solutions at -120 mV. 6. Suramin (100 microM) inhibited the whole-cell currents activated by ATP (200 microM) by 93 +/- 3 %. Similar effects of suramin were observed on ATP-activated channels in outside-out membrane patches. 7. Extracellular ATP had a priming action on the response to subsequent exposure to ATP. At -40 mV, the time to half-maximal current activation (t1/2) was 46 +/- 9 s during the first exposure to 200 microM ATP and decreased to 5 +/- 3 s during a second exposure to the same concentration of ATP. The priming action of ATP was not inhibited by including GDP-beta-S in the intracellular solution. 8. The initial rate of activation increased with the concentration of ATP, and was voltage sensitive. During the first exposure to 200 microM ATP, t1/2 at +40 mV was 4-fold longer than t1/2 at -40 mV. 9. Half-maximal activation of the conductance shifted from 210 +/- 30 to 14 +/- 4 microM added ATP when CaCl2 in the extracellular solution was reduced from 1.58 to 0. 01 mM. The Hill coefficient for ATP was 1.2 in both solutions.10. These observations suggest that a form of ATP uncomplexed with divalent cations directly gates an ion channel (P2X receptor) in rabbit airway ciliated cells, which serves as a pathway for Ca2+ influx. This purinoceptor may contribute to sustained ciliary activation during prolonged exposures to ATP.

Adenosine Diphosphate↗
PubMed1997

Voltage-induced slow activation and deactivation of mechanosensitive channels in Xenopus oocytes.

1. The relationship between stretch and voltage activation of mechanosensitive (MS) channels from Xenopus oocytes was studied in excised patches of membrane using the patch clamp technique. 2. As is characteristic of MS channels to oocytes, stretching the membrane by applying negative pressure to the patch pipette at -50 mV activated the MS channels rapidly. The channels then deactivated rapidly when the stretch was removed. The stretch-activated MS channels entered a main conductance level (45 pS) and one or more subconductance levels in the range of about 75-90% of the main conductance level. 3. In the absence of stretch, a depolarizing step from -50 to +50 mV activated apparent MS channels after long delays of typically 1-20 s (range, 100 ms to 6 min). Upon repolarization, the channels deactivated slowly with a single exponential (mean time constant of 4 s) or double exponential (mean time constants of 0.8 and 3 s) time course. 4. Delayed activation with depolarization and slow deactivation upon repolarization were also observed for apparent MS channels in on-cell patches. 5. The voltage-activated channels were cation selective and had the same selectivity and conductance levels as the stretch activated MS channels. Applying stretch during voltage-induced channel activity did not activate any additional channels, and the same maximal number of channels were typically activated by either stretch or by voltage. These observations suggest that voltage activates the same MS channels that are activated by stretch. 6. The opening of MS channels following steps to +50 mV occurred in an apparently co-operative manner in 70% of the excised patches containing multiple MS channels. 7. In the absence of stretch, the opening frequency and open probability of MS channels increased with depolarization in the examined voltage range of -60 to -20 mV. 8. Applying a brief stretch during the delay to activation at +50 mV activated the MS channels rapidly, which then remained active when the stretch was removed. In contrast, applying a brief stretch during the slow deactivation induced by stepping from +50 to -50 mV abruptly terminated the voltage induced channel activity upon release of the stretch and inhibited subsequent depolarization-induced activity. 9. Depolarizing steps from -50 to +50 mV inhibited any spontaneous channel activity that was present before the depolarizing step. If the potential was stepped back to -50 mV before the channels activated at +50 mV, a delayed activation could occur at -50 mV, followed by normal deactivation, indicating that the depolarizing step initiated activation processes that were initially masked by inhibition. 10. These observations suggest that voltage and stretch can induce different functional gating configurations of MS channels with associated structures, and that these different gating configurations can interconvert.

Animals↗
PubMed1996

Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes.

Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.

Animals↗
PubMed1996

Wanderlust kinetics and variable Ca(2+)-sensitivity of dSlo [correction of Drosophila], a large conductance CA(2+)-activated K+ channel, expressed in oocytes.

Cloned large conductance Ca2+-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.

Animals↗
PubMed1993

Preventing errors when estimating single channel properties from the analysis of current fluctuations.

The conductance, number, and mean open time of ion channels can be estimated from fluctuations in membrane current. To examine potential errors associated with fluctuation analysis, we simulated ensemble currents and estimated single channel properties. The number (N) and amplitude (i) of the underlying single channels were estimated using nonstationary fluctuation analysis, while mean open time was estimated using covariance and spectral analysis. Both excessive filtering and the analysis of segments of current that were too brief led to underestimates of i and overestimates of N. Setting the low-pass cut-off frequency of the filter to greater than five times the inverse of the effective mean channel open time (burst duration) and analyzing segments of current that were at least 80 times the effective mean channel open time reduced the errors to < 2%. With excessive filtering, Butterworth filtering gave up to 10% less error in estimating i and N than Bessel filtering. Estimates of mean open time obtained from the time constant of decay of the covariance, tau obs, at low open probabilities (Po) were much less sensitive to filtering than estimates of i and N. Extrapolating plots of tau obs versus mean current to the ordinate provided a method to estimate mean open time from data obtained at higher Po, where tau obs no longer represents mean open time. Bessel filtering gave the least error when estimating tau obs from the decay of the covariance function, and Butterworth filtering gave the least error when estimating tau obs from spectral density functions.

Analysis of Variance↗
PubMed1992

A potassium current activated by lemakalim and metabolic inhibition in rabbit mesenteric artery.

K+ channels which are inhibited by intracellular ATP (ATPi) (KATP channels) are thought to be the physiological target site of the K+ channel opening drugs (2) and to underlie a variety of physiological phenomena including hypoxia induced vasodilation. However, electrophysiological evidence for ATPi-regulated K+ currents in smooth muscle is scarce. We, therefore, investigated the effects of one K+ channel opener, lemakalim, and metabolic inhibition on the membrane conductance of freshly dissociated rabbit mesenteric artery smooth muscle cells, using the perforated-patch whole cell recording technique. The cells were metabolically inhibited with 1 mM iodoacetic acid and 50 microM dinitrophenol. Both lemakalim (0.1-3 microM) and metabolic inhibition activated a time-independent and glyburide sensitive K+ current at physiological membrane potentials. The similarities between the lemakalim and metabolic inhibition activated currents suggest that a single class of channels underlies both currents. These results are the first whole-cell current recordings to demonstrate the activation of a smooth muscle membrane conductance by metabolic inhibition, lending support to the view that hypoxia induced vasodilation arises from the activation of KATP channels.

Animals↗
PubMed1991

Ca2(+)-activated K+ channels in airway smooth muscle are inhibited by cytoplasmic adenosine triphosphate.

Large-conductance Ca2(+)-activated K+ channels were studied in membranes of cultured rabbit airway smooth muscle cells, using the patch-clamp technique. In cell-attached recordings, channel openings were rare and occurred only at very positive potentials. Bradykinin (10 microM), an agonist which releases Ca2+ from the sarcoplasmic reticulum, transiently increased channel activity. The metabolic blocker 2,4-dinitrophenol (20 microM), which lowers cellular adenosine triphosphate (ATP) levels, induced a sustained increase of channel activity in cell-attached patches. In excised patches, these channels had a slope conductance of 155 pS at 0 mV, were activated by depolarization and by increasing the Ca2+ concentration at the cytoplasmic side above 10(-7) mol/l. ATP, applied to the cytoplasmic side of the patches, dose-dependently decreased the channel's open-state probability. An inhibition constant (Ki) of 0.2 mmol/l was found for the ATP-induced inhibition. ATP reduced the Ca2+ sensitivity of the channel, shifting the Ca2+ activation curve to the right and additionally reducing its steepness. Our results demonstrate that cytoplasmic ATP inhibits a large-conductance Ca2(+)-activated K+ channel in airway smooth muscle. This ATP modulation of Ca2(+)-activated K+ channels might serve as an important mechanism linking energy status and the contractile state of the cells.

Adenosine Triphosphate↗
PubMed1991

Contraction of reptile, amphibian, and fish blood vessels by endothelin-1.

Endothelin-1, a 21 amino acid peptide originally isolated from porcine endothelial cells, has been reported to contract arteries and veins from several mammalian species. We studied the effect of endothelin-1 on blood vessels removed from turtle (Pseudymes scripta), frog (Rana pipiens), and catfish (Amiurus melas). The vessels were suspended for isometric recording in thermally controlled organ baths and were exposed cumulatively to endothelin-1 (0.5-100 nM). All vessels showed a concentration-dependent increase in isometric force. The half-maximal concentrations (EC50) of endothelin-1 were all in the nanomolar range: turtle left systemic arch (17.9 +/- 1.2 nM), frog systemic arch (5.5 +/- 1.1 nM), catfish mesenteric artery (6.3 +/- 1.3 nM), and catfish posterior cardinal vein (2.5 +/- 1.4 nM). Vessels from frog and catfish developed more tension when exposed to 100 nM endothelin-1 than to 80 mM KCl (ET/KCl tension ratio). Our observations that endothelin-1 potently contracts blood vessels from reptiles, amphibians, and fish suggest that endothelin-1 may be a vertebrate peptide that has been conserved during evolution.

Animals↗
PubMed1990

An ATP, calcium and voltage sensitive potassium channel in porcine coronary artery smooth muscle cells.

There is increasing interest in the roles played by potassium channels of smooth muscle in protecting against ischemic and anoxic insults. Hence, potassium-selective channels were studied in freshly dispersed porcine coronary artery smooth muscle cells using the inside-out variant of the patch-clamp technique. The most abundant potassium channel had a conductance of 148 pS in a 5.4/140 mM K+ gradient, at 0 mV, and was regulated by cytoplasmic ATP (0.05-3.0 mM), cytoplasmic Ca2+ (0.1-10 microM) and voltage. ATP and AMP-PNP (0.5 mM) reduced the probability of channel opening (Po) by 87 and 92%, respectively. This inhibition was partially reversed by the addition of 0.5 mM ADP. ADP on its own (2 mM) reduced Po by 46%. It appears, therefore, that this channel shares properties with both the ATP-sensitive and the calcium-regulated potassium channels, raising the possibility that it plays a central role in the regulation of coronary blood flow.

Adenosine Diphosphate↗
PubMed1990

ATP inhibits smooth muscle Ca2(+)-activated K+ channels.

There has been much recent interest in the roles played by smooth-muscle K+ channels in protecting cells against ischemic and anoxic insults and in therapeutic vaso- and bronchodilation (Buckingham 1990; Longmore & Weston 1990). A K+ channel, which is uniquely sensitive to cytoplasmic ATP (KATP), has been identified as a likely candidate for mediating these important functions (Standen et al. 1989). We now show, by using electrophysiological techniques in three different types of smooth muscle, that a large-conductance voltage and Ca2(+)-sensitive channel, otherwise indistinguishable from the the large-conductance Ca2(+)-activated K+ channel (BK channel), is also sensitive to cytoplasmic ATP and cromakalim. ATP, in a dose-dependent manner, decreased the probability of channel opening (Po) of rabbit aortic, rabbit tracheal and pig coronary artery BK channels with a Ki of 0.2-0.6 mM. Cromakalim, 10 microM, partially reversed the ATP induced inhibition and increased Po. Our observations raise the possibility that the ubiquitous BK channel may play a role during pathophysiological events.

Adenosine Triphosphate↗