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At least 703 records · Page 39Linked to original sources
PubMed1993

Fluctuation analysis of patch-clamp or whole-cell recordings containing many single channels.

A novel analytical method for characterizing single-channel currents from recordings containing many identical, independent channels is described. The method is based on the assumption that the opening and closing of each single channel contributing to the summed current can be represented as a first-order, discrete-time, binary Markov chain and that the variance of the quiescent channel noise is known. Utilizing the first 3 moments of the record, and its power spectrum, all relevant single-channel parameters can be estimated. This includes the number of channels, the open current amplitude of a single channel, the mean open and closed durations and the probability of a channel being in the open state. In addition, the magnitude of the shot noise resulting from the flux of ions across the membrane can be estimated. Using fictitious multi-channel recordings generated by summing 2-990 independent binary Markov chains together with additive white noise, we have tested the reliability of the method in estimating the statistics of single channels. Finally, we discuss how the technique may be extended to cope with data which has been low-pass filtered, and also suggest further experiments which the technique now makes possible.

Electrophysiology↗
PubMed1989

The patch-clamp technique in the study of secretion.

One of the basic cellular functions of virtually every cell type is the exocytotic release of molecules synthesized, stored and packaged into intracellular vesicles or granules. Over decades much effort has been concentrated on elucidating the chain of events leading to exocytosis. Unfortunately, the nature of the process that ultimately induces membrane fusion is not known, nor has it been established definitively whether or not the final steps in the secretory cascade are identical in different cells. Although the fusion between vesicle and plasma membrane has been neatly documented by electron micrographs, it was only recently that the technique of time-resolved membrane capacitance measurement has provided a more detailed insight into mechanistic aspects of exocytosis, both in terms of the fusion event and the steps involved in stimulus-secretion coupling.

Adrenal Glands↗
PubMed1994

Recording and control of ion channel currents in bilayer and patch-clamp experiments.

A program was developed for the control and recording of ion-channel activity in lipid bilayers and cell membranes. The software runs on a PC under DOS. Stimulation waveforms may be applied simultaneously with recording at a sample rate of 10 kHz and at 12-bit resolution. Comments may be stored with the recordings, also during timed repetitive pulse applications. The time critical parts and graphics are written in C and assembler whereas the I/O and control is written in Quick-Basic 4.5 (QB). A nested 16-level macro facility allows the use of complex protocols. Array-buffer arithmetic is supported. There is mouse support for cursor measurements and parabolic offset subtractions. DOS applications such as editing macro files may be executed without halting the program. The program runs in the QB interactive environment with the foreign language routines in a library and is open to customization. The library of drawing subroutines support up to 1024 x 768 resolution.

Cell Membrane↗
PubMed1993

Patch-clamp and chloride channels in Ascaris suum.

Single-channel electrophysiology is an invaluable tool fo the study of ion channels. However, it is a technique that has failed to attract widespread use by parasitologists. Here, Diane Dixon and Richard Martin outline the principles undelrying single channel recording and highlight its uses in the discovery of a new and unusual chloride channel in the musculature of Ascaris suum.

Journal Article↗
PubMed1994

Dip patch clamp currents suggest electrodiffusive transport of the polyelectrolyte DNA through lipid bilayers.

Planar lipid bilayers formed from monolayers of diphytanoyl lecithin (DPhL) were found to interact with plasmid DNA (5.6 kbp; M(r) = 3.7 x 10(6)) leading to an increase in the conductance of the membrane. The association of DNA with a lipid bilayer greatly facilitates the transport of the small ions of the main salt KCl. The appearance of long-lived current levels, for instance, of 27.6 pA at Vm = +60 mV membrane voltage, where the actual contact (adsorption) is electrophoretically enhanced, suggests a locally conductive DNA/lipid interaction zone where parts of the DNA strand may be transiently inserted in the bilayer, leaving other parts of the DNA probably protruding out from the outer surface of the bilayer. At Vm = -60 mV, where DNA can be electrophoretically moved away from the membrane, the membrane current is practically zero. This current asymmetry is initially also observed at higher voltages, for instance at 200 mV. However, if the voltage sign (Vm = +200 mV) is changed after a transient positive current (approximately 15 pA) was observed, there is also now (at Vm = -200 mV) a finite negative current at the negative membrane voltage. Thus, it appears that at Vm = +200 mV the adsorbed parts of the polyelectrolyte DNA are not only transiently inserted in, but actually also electrophoretically pulled through, the porous zones onto the other membrane side leaving the bilayer structure basically intact. These data provide direct electric evidence for the electrophoretic transport of a highly charged and hydrated macromolecule, probably together with the associated gegen-ions, through the thin hydrophobic film of the lipid bilayer.

Diffusion↗
PubMed1984

Patch-clamp study of the tetrodotoxin-resistant sodium current in group C sensory neurones.

Conditions were devised to isolate in cranial sensory neurones transfer of Na ions: K and Ca were omitted from the extracellular medium, and simultaneously cells were intracellularly loaded with 120 mM caesium and 20 mM TEA at [Ca]i = 10(-8) M. A tetrodotoxin (TTX)-resistant current was shown to be elicited by step depolarization from -25 MV upwards. This current successively activates and inactivates at increasing rates on further depolarization and at 0 mV (where peak amplitude is reached) its time course is of 20-50 ms. Absence of TTX-sensitivity (up to 15 microM), slow time course and an activation curve shifted by 15 mV towards the depolarized potentials differentiate this current from the more classical fast Na current which can be elicited on the same cells. Inactivation was provoked by a prepulse of varying amplitude and duration: with a prepulse command to -20 mV, inactivation was of 50% within a delay of 300 ms and almost 100% in about 1 min. After complete inactivation by command to 0 mV for 300 ms, recovery by holding the potential at -80 mV was of 50% in 205 ms, and of 100% after 1-4 s. It is concluded that a charge transfer of Na accounts for most of the hump which prolongs the action potential of these sensory neurones, and thus it can be proposed that spike duration as modulated by neurotransmitters may also involve Na in addition to Ca.

Animals↗
PubMed1991

Identification and dissociation of cardiovascular neurons from the medulla for patch clamp analysis.

This study describes a preparation that will enable us to study, using voltage clamp techniques, ionic currents from dissociated cardiovascular neurons that have retained their anatomical and functional identity of the intact animal. To identify dispersed preganglionic cardiac motoneurons various fluorescent dyes (rhodamine, fluorogold, microspheres, bizbenzimide and dextrans) were examined to determine which can be absorbed by preganglionic cardiac motorneuron nerve terminals (without surgical penetration of cardiac tissue), transported retrogradely to their soma in the medulla and retained during dissociation of the neurons. Rhodamine fulfilled these criteria. Dissociated preganglionic cardiac motorneurons had resting membrane potentials of -52.4 +/- 3 mV and input resistances of 236 +/- 71 M omega (mean +/- S.E.M., n = 10). Depolarizing voltage steps to -50 mV or above evoked a tetrodotoxin (TTX) sensitive inward sodium current followed by a biphasic outward current.

Animals↗
PubMed1989

The firing patterns of rat melanotrophs recorded using the patch clamp technique.

Cell-attached and whole-cell recordings were made from adult rat melanotrophs maintained in vitro by standard cell culture techniques. In cell-attached recordings the cells showed small biphasic currents which reflected spontaneous cell firing. Single channel currents often had distinct relaxations and depolarizing currents through single channels could trigger the discharge of an action potential in the cell; both observations are consistent with the high input resistance (1-10 G omega) measured in the whole-cell configuration. The discharge of action potentials occurring either spontaneously or by current injection was eliminated by tetrodotoxin or by removing Na from the external medium. A Na-dependent plateau depolarization which activated near the spike threshold was also seen. In cells exposed to tetrodotoxin and K-channel blocking agents it was possible to evoke a long-lasting (up to 20 s) action potential which was enhanced and reduced, respectively, by Ba and Cd and thus appeared to reflect currents through voltage-activated Ca channels. Small amplitude Ca-dependent depolarizations could also be evoked at membrane potentials as low as -40 mV. In cell-attached and whole-cell recordings 10 mM Ba caused the discharge of tetrodotoxin-insensitive action potentials prior to a maintained depolarization of the membrane. The low threshold for Ca-dependent depolarizations suggest that Ca influx might occur in these cells even at the resting potential. Additionally, both a Ca current and the current underlying the Na-dependent plateau depolarization may influence the rate of cell firing and in doing so further increase Ca influx through voltage-activated channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1989

Diffusion into the patch-clamp recording pipette of a factor necessary for muscarinic current response.

Ca-dependent K and Cl currents were measured in isolated cells from rat lacrimal glands using the tight-seal whole-cell recording method. Upon application of acetylcholine (ACh), both K and Cl-selective currents were activated. The size of the ACh-activated currents declined after a few minutes of whole-cell recording. The rundown curve was composed of an initial stable period followed by a rather rapid decline. Both the length of the initial plateau and the speed of the falling phase were dependent on cell size and recording pipette resistance. The results suggest that the rundown was due to washout of an unknown cytosolic substance. Another manifestation of washout was an increase in the delay of the response. Plots of the inverse of the delay as a function of time in whole-cell recording showed again an initial plateau and a falling phase, but the stable period lasted less than in amplitude plots. Analysis of the washout time course suggested that the cytosolic substance has a diffusion coefficient of 5.4 x 10(-6) cm2/s, corresponding to a molecular weight of 350. Washed-out cells were insensitive to GTP-gamma-S, but responded normally to an internal application of inositol-trisphosphate (InsP3), introduced through the pipette. Thus, the step of the response which is sensitive to washout is closely related to the production of InsP3. Addition of various exogenous water soluble substances failed to halt washout. Among the inactive substances were GTP (or a combination of Mg and GTP) and small water soluble precursors of InsP3. The results imply that the production of InsP3 by muscarinic agonists in exocrine glands requires the presence of a small molecular weight, water soluble substance. It is suggested that this substance is an unknown co-factor of phospholipase C or of Gp, the GTP binding protein governing the production of InsP3.

Acetylcholine↗
PubMed1991

Measurement of voltage dependence of capacitance of planar bilayer lipid membrane with a patch clamp amplifier.

The voltage dependence of capacitance was measured by using the setup which was almost the same as that for the study of ion channels. The coefficient which represents the voltage dependence of capacitance itself also changes as a function of the duration of voltage application if hexadecane is contained in bilayer lipid membrane (BLM). The method of Alvarez, O., and R. Latorre (1978. Biophys. J. 21:1-17) was extended to treat BLM with hexadecane.

Alkanes↗
PubMed1988

Kinetic modeling for the channel gating process from single channel patch clamp data.

Allosteric interactions play a crucial role in the regulation of protein-ligand binding. The single-channel recordings of Na+ channels revealed deviations from the Hodgkin-Huxley mechanism, that may be explained in the context of protein co-operativity. In this paper, we present a Na+ channel gating model based on the allosteric nature of the channel proteins. The model contains a co-operative parameter c and a subunit interaction parameter s, in addition to four Hodgkin-Huxley kinetic parameters. Thus, in our model a deviation from the Hodgkin-Huxley mechanism is measured by these two parameters (i.e., whether they are different from unity). We then discuss how to estimate the kinetic parameters in the model from the single channel recordings. Our method of estimating the parameters is tested with Monte Carlo runs with very good results.

Kinetics↗