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PubMed1988

Sodium current kinetics in intact rat papillary muscle: measurements with the loose-patch-clamp technique.

1. Rapid inward sodium current (INa) was studied on intact rat papillary muscles and trabeculae excised from right or left ventricle using the loose-patch-clamp technique. All experiments were carried out at 25 degrees C. 2. Currents were recorded from patches with a large current density of mean 5.9 +/- 0.5 mA/cm2. 3. The current was reduced by tetrodotoxin (TTX) in a dose-dependent manner. The concentration of TTX producing half-maximal blockade of INa was 6.3 +/- 0.8 mumol/l. 4. Na+ current appeared upon depolarization at a threshold potential of about -55 mV and reached its maximum at about -20 mV. 5. Kinetic data were evaluated using the Hodgkin-Huxley model. 6. Time constants of activation (tau m) were estimated using single-pulse and tail-current measurements. They had a maximum of about 0.4 ms near the threshold potential and declined at more positive and at more negative potentials to values near 0.1 ms. 7. Two time constants were necessary to describe inactivation. Both time constants had their maximal values of 135 +/- 8.1 and 29.1 +/- 5.9 ms at about -80 mV and decreased towards 4 and 0.5 ms at potentials positive to -20 mV.

Action Potentials↗

PubMed2003

New illumination technique for IR-video guided patch-clamp recording from neurons in slice cultures on biomembrane.

Slice cultures on biomembrane are the method of choice for studying Ca2+-dependent plastic changes occurring over several days to weeks. Using IR-differential interference contrast, good visualization of neurons in biomembrane slice cultures has been achieved despite a negative optical effect of the biomembrane, but epifluorescence imaging requires removal of a Wollaston prism and the analyzer. Here, we describe a novel illumination method to overcome this problem. Using optic fiber illumination at a shallow angle from the top of the slice culture, with or without additional illumination from the bottom, we obtained good cellular resolution of neurons in biomembrane slice cultures as well as in acute slices with an infrared-video camera. With this technique, we demonstrate visually guided whole-cell patch-clamp recording of Na+- and K+-currents as well as combination of whole-cell recording with fluorescence imaging of hippocampal and entorhinal cortex neurons in biomembrane slice cultures. Our inexpensive method should prove very useful for studying in vitro effects of long-term manipulations on membrane currents and intracellular Ca2+-signaling.

Animals↗

PubMed1994

Activation of exocytosis by GTP analogues in adrenal chromaffin cells revealed by patch-clamp capacitance measurement.

The role of GTP-binding proteins in exocytosis in bovine adrenal chromaffin cells was examined using patch-clamp capacitance measurement. Internal dialysis with the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate and xanthosine triphosphate (XTP) activated a capacitance increase. Exocytosis triggered by XTP was blocked by guanosine 5'-[beta-thio]diphosphate (GDP beta S) but Ca(2+)-induced exocytosis was unaffected. The capacitance increase due to XTP could not be explained by Ca2+ mobilisation since Ins(1,4,5)P3 and caffeine did not mimic the response. Chromaffin cells appear to possess a Ca(2+)-independent pathway for exocytosis that involves GTP-binding proteins. The magnitude of the response to XTP suggested that GTP analogues stimulate both exocytosis and recruitment of secretory granules.

Adenosine Triphosphate↗

PubMed1993

Change in K+ current of HeLa cells with progression of the cell cycle studied by patch-clamp technique.

The K+ channel of HeLa S3 cells in metaphase was analyzed by inside-out and whole cell patch-clamp techniques. The channel had the characteristics of strong inward rectification, small conductance (22 pS at -100 mV), and dependence on intracellular Ca2+. We investigated the cell cycle dependency of the channel, using cells synchronized by harvesting them at the mitotic stage. The cell capacitance increased gradually with increases in the cell volume toward the S phase. The inward K+ currents through the channel at fixed membrane potentials were highest in early G1 and then decreased with time to a minimum in the S phase, increasing again in the M phase. The permeabilities at fixed membrane potentials were also highest in early G1, decreased to minima in the S phase, and increased again toward the next mitosis. In contrast, mean amplitude and the open probability of the single channel at a fixed membrane potential (-60 mV) did not change significantly during the cell cycle. Therefore the capacitance increases with progression of the cell cycle, whereas the permeability decreases from early G1 to an apparent minimum in the S phase. These changes may be caused by cell cycle-dependent changes in the number of channels.

Calcium↗

PubMed1992

Patch-clamp recording in myenteric neurons of guinea pig small intestine.

The results of our research established the feasibility of applying patch-clamp methods in the study of the cellular neurophysiology of myenteric neurons enzymatically dissociated from adult guinea pig small intestine. Recording in current-clamp mode revealed two populations of neurons. One population discharged repetitively during depolarizing current pulses and displayed anodal-break excitation reminiscent of S/type 1 myenteric neurons. In the second population, spike discharge was limited to one or two spikes at the onset of depolarizing pulses and was similar to the behavior of AH/type 2 neurons. Recording in voltage-clamp mode revealed a complex of overlapping inward and outward whole cell currents. Fast and slow components of inward current were interpreted as sodium and calcium currents, respectively. Outward currents were blocked by cesium and consisted of components with properties of delayed rectifier current and A-type potassium current.

Animals↗

PubMed1997

Existence of voltage-dependent Ca2+ channels in vestibular dark cells: cytochemical and whole-cell patch-clamp studies.

To determine whether functional Ca2+ channels are present in vestibular dark cells, changes in intracellular Ca2+ concentration ([Ca2+]i) due to K+ applications were measured using the Ca(2+)-sensitive dye (fura-2) and patchclamp whole-cell recordings were made in dark cells isolated from the ampullae of the semicircular canal of the guinea pig. Exchange of the external solution with a buffer medium containing a high K+ concentration (80 mM K+ or 150 mM K+) caused a concentration-dependent increase in [Ca2+]i in vestibular dark cells. Application of 1 microM nifedipine as a Ca2+ channel antagonist completely blocked the increase in [Ca2+]i. Further treatment with 10 microM BAY K 8644 as a Ca2+ channel agonist caused an increase in [Ca2+]i. In the patch-clamp whole-cell recordings a 1-s depolarizing pulse given into the dark cell in the presence of a high barium concentration (50 mM Ba2+) induced an inward current. In determining the current-voltage relationship, a current was detected at a potential that depolarized at-50 mV and was maximal at +10 mV. This inward current was completely blocked by 1 mM La3+ as a Ca2+ channel antagonist. These findings suggest the presence of voltage-dependent Ca2+ channels in dark cells, which have a presumed function in the regulation of [Ca2+]i in the vestibular endolymph.

Animals↗

PubMed2005

ATP and vasopressin activate a single type of store-operated Ca2+ channel, identified by patch-clamp recording, in rat hepatocytes.

Hepatocytes are highly polarised epithelial cells that mediate a large number of metabolic pathways, the transcellular movement of numerous ions and metabolites, and the secretion of proteins from both basal and canalicular membrane regions. Hormone-induced changes in the concentration of intracellular Ca2+ play a central role in regulating these functions. Store-operated Ca2+ channels (SOCs) and other Ca2+-permeable channels in the plasma membrane which are activated by hormones are essential for regulating the amount of Ca2+ in the hepatocyte in order to allow these Ca2+ signalling processes to occur. However, the properties of hormone-activated Ca2+ channels in hepatocytes and in other epithelial cells are not well defined. In this study, we have investigated SOCs in cultured rat hepatocytes by patch-clamp recording using IP3 and hormones as activators. We show that IP3 activates a single type of SOC, which, on the basis of its high selectivity for Ca2+ over Na+, inhibition by La3+ and 2-aminoethyl diphenylborate (2-APB), and the time course of fast inactivation, is very similar to CRAC channel in mast cells and lymphocytes. Moreover, a current (ISOC) with properties identical to those of the IP3-activated current can be activated by physiological concentrations of ATP and vasopressin. It is concluded that SOCs with properties similar to those of CRAC channel are present in hepatocytes, highly differentiated primary cells, and these channels can be activated by hormones under conditions close to physiological.

Adenosine Triphosphate↗

PubMed2006

Controlling desensitized states in ligand-receptor interaction studies with cyclic scanning patch-clamp protocols.

Ligand-gated ion channels are important control elements in regulation of cellular activities, and increasing evidence demonstrates their role as therapeutic targets. The receptors display complex desensitization kinetics, occurring on vastly different time scales. This is not only important in biology and pharmacology but might also be of technological significance since populations of receptors under microfluidic control can function analogously to DRAM memory circuits. Using a novel microfluidic method, and computer modeling of the receptor state distributions, we here demonstrate that GABAA receptor populations can be controlled to display high or low EC50 values, depending on input function (i.e., the exact pattern of agonist application). The sensitivity of the receptors can be tuned up to 40-fold (beta-alanine) by the particular agonist exposure pattern. By combining patch-clamp experiments with computer modeling of receptor state distributions, we can control the assembly of receptors in desensitized states. The technique described can be used as an analytical tool to study the effect of desensitization on the activity of ion channel effectors. We describe the differential blocking effect of the competitive antagonist bicuculline on the high- and low-EC50 GABAA receptor preparations and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. Furthermore, we show that both GABA and beta-alanine, two agonists with different affinity but similar efficacy, induce the same type of desensitization behavior and memory effects in GABAA receptors.

Animals↗

PubMed1989

ATP-sensitive K-channels in HIT T15 beta-cells studied by patch-clamp methods, 86Rb efflux and glibenclamide binding.

ATP-sensitive K-channels in the cloned beta-cell line HIT T15 were studied by patch-clamp methods; by measurement of 86Rb efflux; and by [3H]glibenclamide binding to isolated membrane preparations. In inside-out patches a 50 pS K-channel was found which was blocked by ATP or tolbutamide applied to the intracellular membrane surface. A minimum estimate of about 500 channels per beta-cell was obtained by combining whole-cell and single-channel data. The rate of efflux of 86Rb from 86RbCl-loaded HIT cells was markedly increased by intracellular ATP-depletion; 86Rb-efflux was progressively inhibited by increasing concentrations of glibenclamide or tolbutamide. In non-ATP-depleted cells, diazoxide elicited a concentration-dependent stimulation of 86Rb-efflux which was completely blocked by 1 microM glibenclamide. Isolated membranes showed dose-dependent saturable binding of [3H]glibenclamide to both high (Kd = 1.12 nM) and low (Kd = 136 nM) affinity binding sites. We estimate about 5000 high-affinity binding sites per cell. [3H]-glibenclamide binding was inhibited by tolbutamide (IC50 = 125 microM) but was not affected by diazoxide. ADP (0.5 or 1.0 mM) markedly reduced binding; other nucleotides tested were ineffective.

Adenosine Diphosphate↗

PubMed1996

Functional and molecular analysis of glutamate-gated channels by patch-clamp and RT-PCR at the single cell level.

In the central nervous system (CNS) rapid excitatory neurotransmission is mainly mediated by ligand gated, cationic channels activated by glutamate. Three main subtypes of glutamate-gated channels have been characterized by pharmacological studies. They have been named according to their preferred agonist, N-methyl-D-aspartate (NMDA), high affinity kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). Furthermore, a large diversity within each class of glutamate-gated channels has been revealed by the molecular cloning of multiple subunits and their spliced and edited variants (for review see Wisden and Seeburg, 1993). These subunits can potentially form different oligomeric complexes with diverging properties. A crucial question is therefore to determine the actual subunit composition of naturally occurring glutamate receptors. We have combined patch-clamp recording, reverse transcription (RT) and PCR to correlate, at the single cell level, the pattern of subunits expression with the functional properties of native glutamate receptors. We describe here results obtained on the AMPA receptors of hippocampal neurones and on the NMDA receptors of cerebellar granule cells which show that the subunit composition of these two types of receptors explains some of their functional properties. Furthermore, our data also indicate that the expression of NMDA receptor subunits during the postnatal development of cerebellar granule cells is regulated by an activity-dependent mechanism.

Animals↗

PubMed2005

Orexin A modulates mitral cell activity in the rat olfactory bulb: patch-clamp study on slices and immunocytochemical localization of orexin receptors.

Orexin A and B are involved in feeding behaviors, and recently fibers containing these peptides were found in the rat olfactory bulb. These fibers, which originate from the lateral and posterior hypothalamus and the perifornical area, are distributed in the glomerular, mitral cell, and granule cell layers. Orexin receptors are mainly expressed by mitral cells. In the present study, RT-PCR experiments were done to determine orexin receptor expression during the early postnatal life of rats, and immunocytochemical experiments were performed to further clarify the structural and ultrastructural localization of orexin receptors in the olfactory bulb. Furthermore, a functional electrophysiological approach examined the action of orexin A on mitral cell excitability and spontaneous activity using in vitro patch-clamp techniques. RT-PCR results show that mRNA of the two type receptors, type 1 orexin receptors and type 2 orexin receptors, are expressed in the olfactory bulb of rat from 10 d to the adult stage. At the same ages, immunocytochemical data show that orexin 1 receptors are localized in the cell bodies of periglomerular, mitral/tufted, and granule cells. Immunoreactivity was also demonstrated in mitral/tufted cell dendrites arborizing in the glomerulus and mitral/tufted and granule cell processes running in the external plexiform layer. Functionally, orexin A produced either a direct, tetrodotoxin-insensitive depolarization in one group of mitral cells (7%), or, in another group (30%), an indirect, tetrodotoxin-sensitive hyperpolarization. Both actions were mediated by type 1 orexin receptors because the response was antagonized by SB-334867-A, a selective antagonist. Mitral cell recordings performed under bicuculline [gamma-aminobutyric acid (GABA)A receptor antagonist], indicate that the orexin-induced indirect hyperpolarization was partly mediated through GABA(A) receptors. Because granule cells and periglomerular cells express orexin receptors and are GABAergic cells, they could be both involved in this hyperpolarization. Other mechanisms, which could support an indirect hyperpolarization of mitral cells through dopamine interneuron solicitation, are proposed. Our results provide data that should allow us to better understand neural communication and regulation mechanisms between the hypothalamic feeding centers and the olfactory bulb.

Age Factors↗

PubMed2004

Patch clamp study of the UNC-105 degenerin and its interaction with the LET-2 collagen in Caenorhabditis elegans muscle.

Degenerins have emerged from genetic studies in Caenorhabditis elegans as candidate mechanically gated amiloride-sensitive ion channels for transducing mechanical stimuli into cellular responses. In C. elegans muscle, the existence of a genetic interaction between the unc-105 degenerin gene and let-2, a gene encoding an alpha2(IV) collagen, raised the possibility that UNC-105 may function as a mechanically gated channel in a stretch receptor complex. However, to date, ion channel activity of UNC-105 has only been recorded in a gain-of-function mutant form in heterologous expression systems. In this study we investigated the in situ properties of UNC-105 using the whole cell configuration of the patch clamp technique on body wall muscle cells from acutely dissected C. elegans. Amiloride was found to be without effect on membrane potential of wild-type muscle cells, suggesting that the UNC-105 degenerin is electrically silent in resting muscle. Hypo-osmotic shocks induced a reversible depolarization of muscle cells but which was not affected by amiloride. Deformation of the cells by applying tension to the filamentous complex on which muscle cells remained attached or by ejecting external solution under pressure failed to induce any change of membrane potential. In gain-of-function unc-105(n506) mutant cells, an amiloride-sensitive inward Na(+) current was found to be constitutively active, leading to maintained muscle depolarization. An associated mutation in the alpha2(IV) collagen LET-2 led to the closure of the mutant UNC-105(n506) channel while a collagenase treatment of these double mutant cells caused it to re-open, giving evidence for a functional interaction between LET-2 collagen and mutant UNC-105 channel.

Amiloride↗

PubMed2006

Q-method for high-resolution, whole-cell patch-clamp impedance measurements using square wave stimulation.

High-resolution measurements of cell impedance provide invaluable information on various cellular processes such as exocytosis, ion channel gating, or fertilization. The best recent techniques, although achieving impedance resolution at theoretical limits, have limited applicability due to their inherent constrains and high complexity. We report here a simple method of high-resolution impedance measurement, dubbed as the Q-method, based on measurement of a charge by integrating the cell current during square wave stimulation and on its decomposition into specific components related to segments of the voltage stimulus. Simple relations were derived allowing very fast and direct estimation of cell impedance parameters. The major advantages of the Q-method are its inherently low sensitivity to low-pass filtering, rejection of periodic interference signals, automatic on-the-fly adjustment of the stimulation frequency for the highest capacitance resolution, and simultaneous high-resolution low-crosstalk monitoring of membrane resistance, series resistance and parasitic capacitance in addition to membrane capacitance. Implementation of the Q-method is straightforward with any patch-clamp setup and any cell type. Theoretical grounds of the Q-method, including its resolution and the noise of individual parameters, are developed and experimentally verified.

Cell Membrane↗

PubMed1998

Mechanism of peptide-induced mast cell degranulation. Translocation and patch-clamp studies.

Substance P and other polycationic peptides are thought to stimulate mast cell degranulation via direct activation of G proteins. We investigated the ability of extracellularly applied substance P to translocate into mast cells and the ability of intracellularly applied substance P to stimulate degranulation. In addition, we studied by reverse transcription--PCR whether substance P-specific receptors are present in the mast cell membrane. To study translocation, a biologically active and enzymatically stable fluorescent analogue of substance P was synthesized. A rapid, substance P receptor- and energy-independent uptake of this peptide into pertussis toxin-treated and -untreated mast cells was demonstrated using confocal laser scanning microscopy. The peptide was shown to localize preferentially on or inside the mast cell granules using electron microscopic autoradiography with 125I-labeled all-D substance P and 3H-labeled substance P. Cell membrane capacitance measurements using the patch-clamp technique demonstrated that intracellularly applied substance P induced calcium transients and activated mast cell exocytosis with a time delay that depended on peptide concentration (delay of 100-500 s at concentrations of substance P from 50 to 5 microM). Degranulation in response to intracellularly applied substance P was inhibited by GDPbetaS and pertussis toxin, suggesting that substance P acts via G protein activation. These results support the recently proposed model of a receptor-independent mechanism of peptide-induced mast cell degranulation, which assumes a direct interaction of peptides with G protein alpha subunits subsequent to their translocation across the plasma membrane.

Animals↗

PubMed2006

Effect of temperature on the activation of myocardial KATP channel in guinea pig ventricular myocytes: a pilot study by whole cell patch clamp recording.

BACKGROUND: The myocardial ATP sensitive potassium channel (K(ATP) channel) has been known for more than two decades, the properties of this channel have been intensively investigated, especially the myocardial protection effect by opening this channel. Numerous studies, including hypothermic, using K(ATP) agonists to achieve a hyperpolarizing cardioplegic arrest, have shown a better myocardial protection than potassium arrest. However, there is no evidence showing that K(ATP) channel could be opened by its agonists under profound hypothermia. We investigated the effect of temperature on activation of myocardial K(ATP) channel by nicorandil. METHODS: Isolated ventricular myocytes were obtained by collagenase digestion of the hearts of guinea pigs and stored in KB solution at 4 degrees C. With a steady ground current, the myocytes were perfused with 1 mmol/L nicorandil until a steady IK(ATP) occurred. Then the cells were perfused with 1 mmol/L nicorandil plus 1 micromol/L glybenclamide. Currents signals were recorded on whole cells using patch clamp technique at several temperatures. The temperature of the bath solution around myocytes was monitored and was controlled at 4 degrees C, 10 degrees C, 20 degrees C, 25 degrees C and 35 degrees C respectively. About 10 cells were tested at each temperature, the cells were considered useful only when the outward current could be induced by nicorandil and blocked by glybenclamide. All data were analyzed using Graphpad PRISM 3.0 (Graphpad, San Diego, CA, USA). Nonlinear curve fitting was done in Clampfit (Axon) or Sigmaplot (SPSS). RESULTS: At 4 degrees C, 10 degrees C, 20 degrees C, 25 degrees C and 35 degrees C, the time needed to open the myocardial K(ATP) channel was (81.0 +/- 0) minutes, (50.5 +/- 11.7) minutes, (28.8 +/- 2.3) minutes, (9.4 +/- 10.2) minutes and (2.3 +/- 1.0) minutes respectively (P = 0.003). The linear relationship between temperature and time needed to open the channel was y (min) = (4348.790 - 124.277x)/60, where y (min) is time needed to open K(ATP) channel, x is temperature, correlation coefficient r = -0.942 (P = 0.00), regression coefficient b = -124.277 (P = 0.00). The current densities among different temperatures were statistically different (P = 0.022), the current density was greater after the activation of K(ATP) channel at higher temperatures. The lower the temperature, the fewer cells in which K(ATP) channels could be opened. At 4 degrees C, only one cell in which the K(ATP) channel could be opened, took a quite long time (81 minutes) and the I-V curve was quite untypical. CONCLUSIONS: K(ATP) channel activated by nicorandil is temperature dependent and the temperature linearly related to time needed to open K(ATP) channel; the lower the temperature, the longer the time needed to open channel and the smaller the current density. At profound hypothermia, it is difficult to activate K(ATP) channels.

Adenosine Triphosphate↗

PubMed2004

Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry.

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca(2+) ions. Rod CNG channels are directly inhibited by Ca(2+)-calmodulin (Ca(2+)/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca(2+)/CaM-dependent modulation. The molecular basis for Ca(2+)/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca(2+)/CaM to a site in the NH(2)-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH(2)-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca(2+)/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca(2+)/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH(2)- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca(2+)/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH(2)- and COOH-terminal regions underlies Ca(2+)/CaM-dependent inhibition.

Animals↗

PubMed1991

Separate mechanisms of long-term potentiation in two input systems to CA3 pyramidal neurons of rat hippocampal slices as revealed by the whole-cell patch-clamp technique.

Excitatory synaptic transmission from two input systems to hippocampal CA3 pyramidal neurons was investigated by the whole-cell patch-clamp technique for thin slice preparation, with special reference to long-term potentiation (LTP) in these systems. Excitatory postsynaptic currents (EPSCs) evoked by fimbrial stimulation consisted of two components; one was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the other was persistent at depolarized membrane potentials and blocked by D-2-amino-5-phosphonovalerate (D-AP5). The contribution of the D-AP5-sensitive component to EPSCs evoked by stimulation of mossy fibers was much less than that to fimbrial EPSCs. High-frequency stimulation of afferent fibers, under current-clamp conditions, elicited LTP. Bath application of D-AP5 blocked the induction of LTP in the fimbrial but not in the mossy fiber synapses. Induction of fimbrial LTP was completely blocked by 10 mM BAPTA applied intracellularly. In contrast, mossy fiber LTP was not blocked by 10 mM BAPTA. Furthermore, mossy fiber LTP, but not fimbrial LTP, was elicited by high-frequency stimulation under voltage-clamp (-80 mV) conditions. These results suggest that activation of NMDA receptors, increase in postsynaptic [Ca2+]i, and postsynaptic membrane depolarization are required for the induction of fimbrial but not for mossy fiber LTP.

2-Amino-5-phosphonovalerate↗

PubMed1996

Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events.

We present here a maximal likelihood algorithm for estimating single-channel kinetic parameters from idealized patch-clamp data. The algorithm takes into account missed events caused by limited time resolution of the recording system. Assuming a fixed dead time, we derive an explicit expression for the corrected transition rate matrix by generalizing the theory of Roux and Sauve (1985, Biophys. J. 48:149-158) to the case of multiple conductance levels. We use a variable metric optimizer with analytical derivatives for rapidly maximizing the likelihood. The algorithm is applicable to data containing substates and multiple identical or nonidentical channels. It allows multiple data sets obtained under different experimental conditions, e.g., concentration, voltage, and force, to be fit simultaneously. It also permits a variety of constraints on rate constants and provides standard errors for all estimates of model parameters. The algorithm has been tested extensively on a variety of kinetic models with both simulated and experimental data. It is very efficient and robust; rate constants for a multistate model can often be extracted in a processing time of approximately 1 min, largely independent of the starting values.

Algorithms↗