Dwell-time Histograms Research Articles

Short and long-range correlations in single-channel currents from inwardly rectifying K[formula omitted] channels

Ion channels mediate the transport of ions through the cell membrane and are involved in key processes such as muscle contraction, nerve excitability and hormone secretion. For decades, the main focus of the standard techniques for the analysis and modeling of single-channel currents has been to characterize the gating of ionic channels by analyzing the current amplitudes and dwell-time histograms, thus neglecting the possibility that pore fluctuations (open and closed), usually considered to be stochastic noise, might contain valuable information about the functional aspects of ion channels. Based on the analysis of a deterministic model indicating that the stochastic gating of ion channels could be produced by correlated pore fluctuations, in this work, we analyzed experimental recordings of single channel currents of an inwardly rectifying K+ channel in order to demonstrate the presence of both short and long range correlations in the open and closed pore fluctuations

2D-dwell-time analysis with simulations of ion-channel gating using high-performance computing

Single-channel patch-clamp recordings allow observing the action of a single protein complex in real time and hence the deduction of the underlying conformational changes in the ion-channel protein. Commonly, recordings are modeled using hidden Markov chains, connecting open and closed states in the experimental data with protein conformations. The rates between states denote transition probabilities that could be modified by membrane voltage or ligand binding. Modeling algorithms have to deal with limited recording bandwidth and a very noisy background. It was previously shown that the fit of two-dimensional (2D)-dwell-time histograms with simulations is very robust in that regard. Errors introduced by the low-pass filter or noise cancel out to a certain degree when comparing experimental and simulated data. In addition, the topology of models (that is, the chain of open and closed states) could be inferred from 2D-histograms. However, the 2D-fit was never applied to its full potential. A major reason may be the extremely time-consuming and often unreliable fitting process, due to the stochastic variability in the simulations. We have now solved these issues by introducing a message-passing interface (MPI) allowing massive parallel computing on a high-performance computing (HPC) cluster and obtaining ensemble solutions. With ensembles, we have demonstrated how important ranked solutions are for difficult tasks related to a noisy background, fast gating events beyond the corner frequency of the low-pass filter, and topology estimation of the underlying Markov model. Finally, we have shown that, by combining the objective function of the 2D-fit with the deviation of the current amplitude distributions, automatic determination of the current level of the conducting state is possible, even with an apparent current reduction due to low-pass filtering. Making use of an HPC cluster, the power of 2D-dwell-time analysis can be used to its fullest with minor input of the experimenter.

Energetic destabilization of shut states underpins the membrane stretch dependent activation of Piezo1.

Piezo1 is a non-selective cation channel activated by mechanical stimuli such as cell membrane stretch. It has been shown that Piezo1 conforms to a curved, upright, bowl-like structure in its non-conductive (“shut”) state. Stretching of the cell membrane is thought to flatten this curved structure of Piezo1 to open its ion-conducting pore. Yet, how stretching of the cell membrane modulates the functional states of Piezo1 to open the channel is not known. Therefore, we collected single-channel Piezo1 recordings at various steady-state pressure-induced membrane stretch to study how the membrane stretch alters functional states of Piezo1. One-dimensional single-channel dwell time histograms reveal that Piezo1 has a minimum of two shut (long-lived and short-lived) and two open (long-lived and short-lived) functional states. Two-dimensional dwell time histograms and Markov modeling show that membrane stretch increases the rate of microscopic shut-to-open transitions, with relatively smaller effects on closure rates. Critically, increasing membrane stretch up to saturation of open probability correlates with a ∼25- and ∼50-fold decrease of mean dwell times for the short-lived and long-lived shut states, respectively. In contrast, the mean dwell times for the short-lived and long-lived open states only increased by ∼3- and ∼2-fold, respectively. Taken together, these results indicate that membrane stretch predominantly acts by energetically destabilizing Piezo1 shut states.

The Joy of Markov Models—Channel Gating and Transport Cycling Made Easy

ABSTRACT The behavior of ion channels and transporters is often modeled using discrete state continuous-time Markov models. Such models are helpful for the interpretation of experimental data and can guide the design of experiments by testing specific predictions. Here, we describe a computational tool that allows us to create Markov models of chosen complexity and to calculate the predictions on a macroscopic scale, as well on a single-molecule scale. The program calculates steady-state properties (current, state probabilities, and cycle frequencies), deterministic macroscopic and stochastic time courses, gating currents, dwell-time histograms, and power spectra of channels and transporters. In addition, a visual simulation mode allows us to follow the time-dependent stochastic behavior of a single channel or transporter. After a basic introduction into the concept of Markov models, real-life examples are discussed, including a model of a simple K+ channel, a voltage-gated sodium channel, a 3-state ligand-gated channel, and an electrogenic uniporter. In this manner, the article has a modular architecture, progressing from basic to more advanced topics. This illustrates how the MarkovEditor program can serve students to explore Markov models at a basic level but is also suited for research scientists to test and develop models on the mechanisms of protein function.

A User-Friendly Computational Tool for Markov Modelling Channel Gating and Transport Cycling

The behavior of ion channels and transporters is often modelled using discrete-state continuous-time Markov models. Such models are helpful for the interpretation of experimental data and can guide the design of experiments by testing specific predictions. Here, we present a computational tool that allows to create Markov models of chosen complexity, and to calculate their predictions on a macroscopic as well on a single-molecule scale. The program (https://github.com/mikpusch/MarkovEditor) calculates steady-state properties (current, state-probabilities, cycle frequencies), deterministic macroscopic and stochastic time courses, gating currents, dwell time histograms, and power spectra of channels and transporters. In addition, a visual simulation mode allows to follow the time-dependent stochastic behavior of a single channel or transporter. After a basic introduction into the concept of Markov models, real-life examples are discussed, including a model of a simple K+ channel, a voltage-gated sodium channel, a three-state ligand-gated channel, and an electrogenic uniporter. The MarkovEditor program can serve students to explore Markov models at a basic level, but is also suited for research scientists to test and develop models on the mechanisms of protein function.

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions.

Single-molecule Förster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act togetherwith interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail. Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates. By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Quantitative Protein Kinetics from sm-FRET Time Traces

Single molecule time traces (from fluorescence or force spectroscopy, nanopores, ion channels, etc.) provide unique information on the kinetic state sequence of the system under study.However, single molecule Foerster resonance energy transfer (smFRET) data is often used to uncover equilibrium distributions only, leaving the kinetic information unexploited. This is because of experimental limitations associated with smFRET, such as photo-bleaching, inter-fluorophore variation and the limited time window. Therefore, analysis tools beyond dwell-time histograms are required for smFRET time traces.Here we present a new maximum likelihood-based approach custom-tailored for experimental smFRET data. Our analysis tool handles multiple input traces, experimental inter-fluorophore variation, truly hidden states, and performs unbiased model selection. We demonstrate the robustness of rate determination and model selection on the basis of experimental and simulated data.Finally, we solve the kinetic state model for the complex conformational dynamics of the heat shock protein Hsp90 with unprecedented accuracy. This reveals novel insights into the effects of drug candidates, nucleotides and co-chaperones on Hsp90's kinetics.

Single-molecule imaging and kinetic analysis of cooperative cofilin-actin filament interactions.

The actin filament-severing protein actin depolymerizing factor (ADF)/cofilin is ubiquitously distributed among eukaryotes and modulates actin dynamics. The cooperative binding of cofilin to actin filaments is crucial for the concentration-dependent unconventional modulation of actin dynamics by cofilin. In this study, the kinetic parameters associated with the cooperative binding of cofilin to actin filaments were directly evaluated using a single-molecule imaging technique. The on-rate of cofilin binding to the actin filament was estimated to be 0.06 µM(-1)⋅s(-1) when the cofilin concentration was in the range of 30 nM to 1 µM. A dwell time histogram of cofilin bindings decays exponentially to give an off-rate of 0.6 s(-1). During long-term cofilin binding events (>0.4 s), additional cofilin bindings were observed in the vicinity of the initial binding site. The on-rate for these events was 2.3-fold higher than that for noncontiguous bindings. Super-high-resolution image analysis of the cofilin binding location showed that the on-rate enhancement occurred within 65 nm of the original binding event. By contrast, the cofilin off-rate was not affected by the presence of prebound cofilin. Neither decreasing the temperature nor increasing the viscosity of the test solution altered the on-rates, off-rates, or the cooperative parameter (ω) of the binding. These results indicate that cofilin binding enhances additional cofilin binding in the vicinity of the initial binding site (ca. 24 subunits), but it does not affect the off-rate, which could be the molecular mechanism of the cooperative binding of cofilin to actin filaments.

Single-molecule studies of disulfide bond reduction pathways used by human thioredoxin

Disulfide bond reduction pathways used by human thioredoxin (hTrx) are studied at the single molecule level using a recombinant protein (I27SS)8. (I27SS)8 contains eight tandem repeats of identical immunoglobulin-like modules with one disulfide bond in each module. Single (I27SS)8 molecules are stretched at constant force applied by a cantilever in a force-clamp mode of atomic force microscopy (FC-AFM). Disulfide reduction events are accurately detected from stepwise increases in the end-to-end length of (I27SS)8. Earlier FC-AFM studies observed one disulfide reduction pathway used by hTrx and suggested an additional electron tunneling mechanism. Here, a very large set of unbiased FC-AFM data is collected in a range of clamping forces. By analyzing the data using exponential fits and dwell time histograms two disulfide reduction pathways used by hTrx are resolved. Based on previous studies one of these pathways is attributed to force-dependent Michaelis–Menten catalysis. The latter reduction pathway is weakly force-inhibited and occurs sporadically. Bimolecular nucleophilic substitutions (SN2) and electron tunneling (ET) mechanisms are discussed to explain the second pathway. Direct SN2 and ET mechanisms cannot be discounted, but a hypothetical E2–SN2 mechanism involving a hydride reducing a disulfide bond provides an interesting alternative, which needs to be verified in future experiments.

Nicotinic Receptor Transduction Zone: Invariant Arginine Couples to Multiple Electron-Rich Residues

Gating of the muscle-type acetylcholine receptor (AChR) channel depends on communication between the ACh-binding site and the remote ion channel. A key region for this communication is located within the structural transition zone between the ligand-binding and pore domains. Here, stemming from β-strand 10 of the binding domain, the invariant αArg209 lodges within the hydrophobic interior of the subunit and is essential for rapid and efficient channel gating. Previous charge-reversal experiments showed that the contribution of αArg209 to channel gating depends strongly on αGlu45, also within this region. Here we determine whether the contribution of αArg209 to channel gating depends on additional anionic or electron-rich residues in this region. Also, to reconcile diverging findings in the literature, we compare the dependence of αArg209 on αGlu45 in AChRs from different species, and compare the full agonist ACh with the weak agonist choline. Our findings reveal that the contribution of αArg209 to channel gating depends on additional nearby electron-rich residues, consistent with both electrostatic and steric contributions. Furthermore, αArg209 and αGlu45 show a strong interdependence in both human and mouse AChRs, whereas the functional consequences of the mutation αE45R depend on the agonist. The emerging picture shows a multifaceted network of interdependent residues that are required for communication between the ligand-binding and pore domains.

Psalmotoxin-1 Docking to Human Acid-sensing Ion Channel-1

Acid-sensing ion channel-1 (ASIC-1) is a proton-gated ion channel implicated in nociception and neuronal death during ischemia. Recently the first crystal structure of a chicken ASIC was obtained. Expanding upon this work, homology models of the human ASICs were constructed and evaluated. Energy-minimized structures were tested for validity by in silico docking of the models to psalmotoxin-1, which potently inhibits ASIC-1 and not other members of the family. The data are consistent with prior radioligand binding and functional assays while also explaining the selectivity of PcTX-1 for homomeric hASIC-1a. Binding energy calculations suggest that the toxin and channel create a complex that is more stable than the channel alone. The binding is dominated by the coulombic contributions, which account for why the toxin-channel interaction is not observed at low pH. The computational data were experimentally verified with single channel and whole-cell electrophysiological studies. These validated models should allow for the rational design of specific and potent peptidomimetic compounds that may be useful for the treatment of pain or ischemic stroke.

Conformational Changes in a Pore-lining Helix Coupled to Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ATP-binding cassette transporters in that it functions as an ion channel. In CFTR, ATP binding opens the channel, and its subsequent hydrolysis causes channel closure. We studied the conformational changes in the pore-lining sixth transmembrane segment upon ATP binding by measuring state-dependent changes in accessibility of substituted cysteines to methanethiosulfonate reagents. Modification rates of three residues (resides 331, 333, and 335) near the extracellular side were 10-1000-fold slower in the open state than in the closed state. Introduction of a charged residue by chemical modification at two of these positions (resides 331 and 333) affected CFTR single-channel gating. In contrast, modifications of pore-lining residues 334 and 338 were not state-dependent. Our results suggest that ATP binding induces a modest conformational change in the sixth transmembrane segment, and this conformational change is coupled to the gating mechanism that regulates ion conduction. These results may establish a structural basis of gating involving the dynamic rearrangement of transmembrane domains necessary for vectorial transport of substrates in ATP-binding cassette transporters.

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times of Cy3-EDA-ATP (2'(3')-O-[[2-[[6-[2-[3-(1-ethyl-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-5-sulfo-3H-indolio]-1-oxohexyl]amino]ethyl]carbamoyl]ATP) but reduced the number of Cy3-signals per field of view, approaching 50% at phosphate concentrations of 1-2 mM. Apparently, inhibition of movement does not result from binding of phosphate to an actomyosin-ADP intermediate as proposed by Hooft and coworkers (Hooft, A. M., Maki, E. J., Cox, K. K., and Baker, J. E. (2007) Biochemistry 46, 3513-3520) but, rather, from forming a strong-binding actomyosin-phosphate intermediate.

Characterisation of large-conductance calcium-activated potassium channels (BK Ca) in human NT2-N cells

Large-conductance calcium-activated potassium (BK Ca) channels were studied in inside-out patches of human NTERA2 neuronal cells (NT2-N). In symmetrical (140 mM) K + the channel mean conductance was 265 pS, the current reversing at ∼ 0 mV. It was selective ( P K / P Na = 20:1) and blocked by internal paxilline and TEA. The open probability–voltage relationship for BK Ca was fitted with a Boltzmann function, the V ½ being 76.3 mV, 33.6 mV and − 14.1 mV at 0.1 μM, 3.3 μM and 10 μM [Ca 2+] i , respectively. The relationship between open probability and [Ca 2+] i was fitted by the Hill equation (Hill coefficient 2.7, half maximal activation at 2.0 μM [Ca 2+] i ). Open and closed dwell time histograms were fitted by the sum of two and three voltage-dependent exponentials, respectively. Increasing [Ca 2+] i produced both an increase in the longer open time constant and a decrease in the longest closed time constant, so increasing mean open time. “Intracellular” ATP evoked a concentration-dependent increase in NT2-N BK Ca activity. At + 40 mV half-maximum activation occurred at an [ATP] i of 3 mM (30 nM [Ca 2+] i ). ADP and GTP were less potent, and AMP-PNP was inactive. This is the first characterisation of a potassium channel in NT2-N cells showing that it is similar to the BK Ca channel of other preparations.

Intracellular Mg2+ Influences Both Open and Closed Times of a Native Ca2+-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg2+ on a native Ca(2+)-and voltage-sensitive large-conductance K+ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca2+ ([Ca2+](i)) of 10(-5)-10(-4) M, addition of 1-10 mM: Mg2+ increased the open probability (P(o)) of the channel, which shifted the P(o) -membrane potential (V(m)) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg(2+)-induced increase in P(o) was suppressed at a relatively low [Ca2+](i) (10(-5.5)-10(-6) M). Dwell-time histograms have revealed that addition of Mg2+ mainly increased P(o) by extending open times at 10(-5) M Ca2+ and extending both open and closed times simultaneously at 10(-5.5) M Ca2+. Since our data showed that raising the [Ca2+](i) from 10(-5) to 10(-4) M increased P(o) mainly by shortening the closed time, extension of the closed time at 10(-5.5) M Ca(2+) would result from the Mg(2+)-inhibited Ca(2+)-dependent activation. At a constant V(m), adding Mg2+ enhanced the sigmoidicity of the P(o)-[Ca2+](i) relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg2+ on this channel is to elevate P(o) by lengthening the open time, while extension of the closed time at a relatively low [Ca2+](i) results from a lowering of the sensitivity to Ca2+ of the channel by Mg2+, which causes the increase in the Hill coefficient.

Model-Based Fitting of Single-Channel Dwell-Time Distributions

Single-channel recordings provide unprecedented resolutions on kinetics of conformational changes of ion channels. Several approaches exist for analysis of the data, including the dwell-time histogram fittings and the full maximal-likelihood approaches that fit either the idealized dwell-time sequence or more ambitiously the noisy data directly using hidden Markov modeling. Although the full maximum likelihood approaches are statistically advantageous, they can be time-consuming especially for large datasets and/or complex models. We present here an alternative approach for model-based fitting of one-dimensional and two-dimensional dwell-time histograms. To improve performance, we derived analytical expressions for the derivatives of one-dimensional and two-dimensional dwell-time distribution functions and employed the gradient-based variable metric method for fast search of optimal rate constants in a model. The algorithm also has the ability to allow for a first-order correction for the effects of missed events, global fitting across different experimental conditions, and imposition of typical constraints on rate constants including microscopic reversibility. Numerical examples are presented to illustrate the performance of the algorithm, and comparisons with the full maximum likelihood fitting are discussed.

Voltage Dependence and pH Regulation of Human Polycystin-2-mediated Cation Channel Activity

Polycystin-2, the product of the human PKD2 gene, whose mutations cause autosomal dominant polycystic kidney disease, is a large conductance, Ca(2+)-permeable non-selective cation channel. Polycystin-2 is functionally expressed in the apical membrane of the human syncytiotrophoblast, where it may play a role in the control of fetal electrolyte homeostasis. Little is known, however, about the mechanisms that regulate polycystin-2 channel function. In this study, the role of pH in the regulation of polycystin-2 was assessed by ion channel reconstitution of both apical membranes of human syncytiotrophoblast and the purified FLAG-tagged protein from in vitro transcribed/translated material. A kinetic analysis of single channel currents, including dwell time histograms, confirmed two open and two close states for spontaneous channel behavior and a strong voltage dependence of the open probability of the channel (P(o)). A reduction of cis pH (pH(cis)) decreased P(o) and shifted the voltage dependence of channel function but had no effect on the single channel conductance. An increase in pH(cis), in contrast, increased NP(o) (channel number times P(o)). Elimination of the H(+) chemical gradient did not reverse the low pH(cis) inhibition of polycystin-2. Similar findings confirmed the pH effect on the in vitro translated, FLAG-tagged purified polycystin-2. The data indicate the presence of an H(+) ion regulatory site in the channel protein, which is accessible from the cytoplasmic side of the protein. This protonation site controls polycystin-2 cation-selective channel activity.

Gating kinetics and ligand sensitivity modified by phosphorylation of cardiac ryanodine receptors.

The effects of protein-kinase- (PKA-) dependent phosphorylation on the stationary gating kinetics of single ryanodine receptor (RyR) channels was defined. The single-channel activity from canine cardiac RyR was reconstituted into planar lipid bilayers. Exogenously applied PKA increased the single-channel open probability ( P(o)) of both native and purified cardiac RyR channels, after preincubation with ATP and Mg2+. The action of PKA on the RyR channel occurred only in the presence of ATP and adenosine 5'- O-(3-thiotriphosphate) (ATPgammaS), but not in the presence of 5'-adenylimidodiphosphate (AMP-PCP). Thus, the action of PKA requires the presence of a hydrolyzable ATP analog. PKA-induced channel activation was blocked by specific PKA inhibitors. All these results confirmed that the RyR channel can be phosphorylated by exogenous protein kinase. The gating kinetics of single RyR channels before PKA treatment were significantly altered by ATP and Mg2+ as physiological ligands. In contrast, after PKA treatment, neither ATP nor Mg2+ significantly alters the gating kinetics of these channels. PKA-dependent phosphorylation thus decreases the ATP and Mg2+ apparent sensitivity in most of the gating parameters of single RyR channels. The phosphorylated RyR channels open and close more frequently, stay open for longer, and stay closed for shorter periods. The dwell-time histograms obtained demonstrate that the phosphorylated and the dephosphorylated channels have strikingly different open and closed kinetics at physiological cytoplasmic concentrations of Mg and ATP.

Analysis of single K(ATP) channels in mammalian dentate gyrus granule cells.

ATP-sensitive potassium (K(ATP)) channels are heteromultimer complexes of subunits from members of the inwardly rectifying K(+) channel and the ATP-binding cassette protein superfamilies. K(ATP) channels couple metabolic state to membrane excitability, are distributed widely, and participate in a variety of physiological functions. Understood best in pancreatic beta cells, where their activation inhibits insulin release, K(ATP) channels have been implicated also in postischemia cardio- and neuroprotection. The dentate gyrus (DG) is a brain region with a high density of K(ATP) channels and is relatively resistant to ischemia/reperfusion-induced cell death. Therefore we were interested in describing the characteristics of single K(ATP) channels in DG granule cells. We recorded single K(ATP) channels in 59/105 cell-attached patches from DG granule cells in acutely prepared hippocampal slices. Single-channel openings had an E(K) close to 0 mV (symmetrical K(+)) and were organized in bursts with a duration of 19.3 +/- 1.6 (SE) ms and a frequency of 3.5 +/- 0.8 Hz, a unitary slope conductance of 27 pS, and a low, voltage-independent, probability of opening (P(open), 0.04 +/- 0.01). Open and closed dwell-time histograms were fitted best with one (tau(open) = 1.3 +/- 0.2 ms) and the sum of two (tau(closed,fast) = 2.6 +/- 0.9 ms, tau(closed,slow) = 302.7 +/- 67. 7 ms) exponentials, respectively, consistent with a kinetic model having at least a single open and two closed states. The P(open) was reduced ostensibly to zero by the sulfonylureas, glybenclamide (500 nM, 2/6; 10 microM,11/14 patches) and tolbutamide (20 microM, 4/6; 100 microM, 4/4 patches). The blocking dynamics for glybenclamide included transition to a subconductance state (43.3 +/- 2.6% of control I(open channel)). Unlike glybenclamide, the blockade produced by tolbutamide was reversible. In 5/5 patches, application of diazoxide (100 microM) increased significantly P(open) (0.12 +/- 0.02), which was attributable to a twofold increase in the frequency of bursts (8.3 +/- 2.0 Hz). Diazoxide was without effect on tau(open) and tau(closed,fast) but decreased significantly tau(closed,slow) (24.4 +/- 2.6 ms). We observed similar effects in 6/7 patches after exposure to hypoxia/hypoglycemia, which increased significantly P(open) (0.09 +/- 0.03) and the frequency of bursts (7.1 +/- 1.7 Hz) and decreased significantly tau(closed,slow) (29.5 +/- 1.8 ms). We have presented convergent evidence consistent with single K(ATP) channel activity in DG granule cells. The subunit composition of K(ATP) channels native to DG granule cells is not known; however, the characteristics of the channel activity we recorded are representative of Kir6.1/SUR1, SUR2B-based channels.

Micromolar concentrations of steroids and of aldosterone antagonists inhibit the outwardly rectifying chloride channel with different kinetics.

We used the patch-clamp technique to analyse the open/close kinetics of single, outwardly rectifying, intermediate-conductance (ORIC) Cl- channels from cultured epithelial cells under control conditions and in presence of different inhibitors. As observed previously in excised inside/out patches under control conditions, the switching kinetics were characterized by one open-state time constant (tau0 is approximately 30 ms) and three closed-state time constants (tau(cl)is approximately = to 0.2 ms, tau(c2) is approximately = 2 ms and tau(c3) is approximately = 60 ms). Aldosterone, six further steroids and two aldosterone antagonists inhibited channel open probability (NPo) concentration dependently with the potency at 10 micromol/l increasing in the sequence: hydrocortisone, corticosterone, P-oestradiol, cortisone, aldosterone, testosterone, progesterone, canrenone, spironolactone. Although all substances decreased tau(o), neither the steroids nor the aldosterone antagonists affected tau(cl), tau(c2) or tau(c3) or induced additional transitions with additional time constants. Instead, the steroids increased the prevalence of tau(c2) in the dwell-time histograms and the aldosterone antagonists increased the prevalence of tau(c3), both in a concentration-dependent manner. These observations may be explained by a model in which one open state leads to one of three closed states with rate constants alpha, beta and gamma, and in which beta or gamma increase under the influence of steroids or aldosterone antagonists, respectively. Cytosol, which contains a Cl- channel inhibitor of unknown molecular structure, (Krick et al., Pflügers Arch 418:491, 1991) was also tested, but the results did not conform to the blocker mechanisms described above. This shows that there are even further modes of channel inhibition and argues against the cytosolic Cl- channel inhibitor being a steroid.