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P H Barry

Publications and source records attributed to P H Barry.

18 recordsLinked to original sources
PubMed1992

A patch-clamp study of GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons.

GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons were studied by using patch-clamp techniques. Current-voltage relations for both agonists in symmetrical Cl- solutions showed outward rectification. Strychnine-sensitive glycine-activated currents have not been studied in detail before in post-natal tissue-cultured hippocampal neurons. Partial desensitization of whole-cell currents was observed in symmetrical Cl- solutions during bath application of GABA- and glycine. In F-/Cl- solutions, both agonists gave a PF/Pcl value of about 0.06. The reversal potentials in mixtures of Cl- and SO4(2-) solutions were close to the equilibrium potentials of Cl- in the presence of both GABA and glycine. Single channels in inside-out excised patches with 2 mumol l-1 GABA and 5 mumol l-1 glycine in the pipette showed outward current rectification. The dose-response curves for GABA and glycine gave Kd values of 52 and 61 mumol l-1, respectively, and Hill coefficients close to 2. GABA and glycine binding were competitively blocked by their respective antagonists (bicuculline and strychnine). The similarities between GABA- and glycine-activated currents and the response in a combination of saturating concentrations of both GABA and glycine implied that the two agonists activated comparable numbers of anatomically distinct channels with very similar permeation properties.

Animals↗
PubMed1992

Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily.

Amino Acid Sequence↗
PubMed1992

An analysis of Na+ currents in rat olfactory receptor neurons.

Na+ currents were observed in acutely-dissociated adult rat olfactory receptor neurons using the whole-cell recording techniques. The threshold for current activation was near -70 mV and currents were fully activated by -10 mV (midpoint: -45 mV). Steady-state inactivation was complete at potentials more positive than -70 mV and half complete at -110 mV (+/- less than 1, n = 8). Complete recovery from inactivation required one second at -100 mV (n = 7). The addition of 10 microM tetrodotoxin or 1 mM Zn2+ to the external solution was required to completely block the current. The current differs from those in amphibian and cultured neonatal rat olfactory neurons in its unusually negative voltage-dependence and slow recovery. Since mammalian olfactory neurons have very high input resistances, physiological resting potentials cannot usually be measured using whole-cell recording techniques. However, predominantly-capacitatively-coupled spikes activated by depolarisation were frequently observed in cell-attached patches. This indicates that the cells were excitable and implies that they must have had resting potentials more negative than -90 mV in order for this current to underlie the action potential.

Action Potentials↗
PubMed1991

Slowly activating K+ channels in rat olfactory receptor neurons.

Patch-clamp techniques were used to investigate slowly activating, Ca(2+)-insensitive K+ channels of isolated rat olfactory receptor neurons. These channels had a unitary conductance of 135 pS and were only found in a small proportion (less than 5%) of membrane patches. Upon depolarization to voltages more positive than -50 mV, the channels activated gradually over a period of at least 10 s. When hyperpolarized to negative voltages, channel activity deactivated in a slow but voltage-dependent manner. These channels may underlie a slowly activating K+ current that is observed in approximately 30% of whole-cell recordings. Similar single channels have been reported in smooth muscle cells, but this is the first demonstration of these channels in any type of neuron. The channels may contribute to the spike frequency adaptation and post-stimulus hyperpolarization that are observed during the excitatory response to odorants. They may also contribute to cell repolarization following large odorant-stimulated receptor currents.

Animals↗
PubMed1991

Inward rectification in rat olfactory receptor neurons.

Inwardly rectifying currents in enzymically dissociated olfactory receptor neurons of rat were studied by using patch-clamp techniques. Upon hyperpolarization to membrane potentials more negative than -100 mV, small inward-current relaxations were observed. Activation was described by a single exponential with a time constant that decreased e-fold for a 21 mV hyperpolarization. The current was not reduced by the external application of 5 mM Ba2+, but was abolished by the addition of 5 mM Cs+ to the bath solution. Increasing the external K+ concentration ([K+]o) to 25 mM dramatically enhanced the current without affecting the voltage range or the kinetics of activation. In 25 mM [K+]o, tail currents reversed at -26 mV, significantly more positive than the K+ equilibrium potential of -44 mV. These characteristics are consistent with those of a mixed Na+/K+ inward rectification that has been reported in several types of neuronal, cardiac and smooth muscle cells. The current may contribute to controlling cell excitability during the response to some odorants.

Action Potentials↗
PubMed1991

Properties of transient K+ currents and underlying single K+ channels in rat olfactory receptor neurons.

The transient potassium current, IK(t), of enzymatically dissociated rat olfactory receptor neurons was studied using patch-clamp techniques. Upon depolarization from negative holding potentials, IK(t) activated rapidly and then inactivated with a time course described by the sum of two exponential components with time constants of 22.4 and 143 ms. Single-channel analysis revealed a further small component with a time constant of several seconds. Steady-state inactivation was complete at -20 mV and completely removed at -80 mV (midpoint -45 mV). Activation was significant at -40 mV and appeared to reach a maximum conductance at +40 mV (midpoint -13 mV). Deactivation was described by the sum of two voltage-dependent exponential components. Recovery from inactivation was extraordinarily slow (50 s at -100 mV) and the underlying processes appeared complex. IK(t) was reduced by 4-aminopyridine and tetraethylammonium applied externally. Increasing the external K+ concentration ([K+]o) from 5 to 25 mM partially removed IK(t) inactivation, usually without affecting activation kinetics. The elevated [K+]o also hyperpolarized the steady-state inactivation curve by 9 mV and significantly depolarized the voltage dependence of activation. Single transient K+ channels, with conductances of 17 and 26 pS, were observed in excised patches and often appeared to be localized into large clusters. These channels were similar to IK(t) in their kinetic, pharmacological, and voltage-dependent properties and their inactivation was also subject to modulation by [K+]o. The properties of IK(t) imply a role in action potential repolarization and suggest it may also be important in modulating spike parameters during neuronal burst firing. A simple method is also presented to correct for errors in the measurement of whole-cell resistance (Ro) that can result when patch-clamping very small cells. The analysis revealed a mean corrected Ro of 26 G omega for these cells.

4-Aminopyridine↗
PubMed1990

PNSCROLL, a software package for graphical interactive analysis of single channel patch clamp currents and other binary file records: under mouse control.

The PNSCROLL program has been specifically developed for interactively scrolling through binary data files under mouse control, in order to analyse single channel current records and printout, or plot, records and histograms of currents. PNSCROLL requires the use of a Microsoft Mouse and runs on an IBM or IBM-compatible microcomputer operating under MS DOS, ideally with 256K EGA or VGA graphics card and colour display. It will print on Epson dot matrix printers and plot records and histograms on HP plotters and LaserJet laser printers.

Computer Graphics↗
PubMed1990

Membrane potential simulation program for IBM-PC-compatible equipment for physiology and biology students.

MEMPOT is an interactive graphic simulation program, written in Turbo C for IBM-PC-compatible equipment and designed to teach students in the physiological and biological sciences about the measurement and ion dependence of cell membrane potentials. Different external salt solution values can be chosen and an intracellular electrode can be moved on the screen to impale one of a group of cells and measure resting or action potential responses. A simulated voltage-to-frequency audio signal emulates normal experimental audio monitoring of the electrode potential, and a window displays a simulated oscilloscope trace (together with "electrical noise") of the resting or action potential response. The average value of each set of successful impalements is determined for each solution. Once measurements have been made over a suitable range of different solutions, the data can be reentered and plotted graphically on the screen and an interactive approach can be used to determine relative sodium-to-potassium permeabilities at rest or at the action-potential peak. A randomization routine varies permeability ratios within a small range between runs.

Biology↗
PubMed1989

Action potentials initiated by single channels opening in a small neuron (rat olfactory receptor).

Rat olfactory receptor neurons were enzymatically dissociated and studied with the cell-attached configuration of the patch-clamp technique. Biphasic current waveforms induced across the membrane patch by intracellular action potentials were observed in approximately 5% of cells studied. In one cell in particular, current injected by the opening of a single channel initiated an action potential in the remainder of the cell each time the channel opened. A conventional type of electrical model of the cell and patch allowed the accurate modeling of cell excitability. The same model was used to explain the shape of the action potential current waveforms induced across the patch. The analysis indicated that the whole cell resistance (Ro) was approximately 40 G omega and the membrane capacitance (Co) was close to the standard value of 1 microF.cm-2. In addition, the threshold potential change necessary to initiate an action potential (Vth) was approximately 13 mV and a minimum current injection of 1 pA was required to depolarize the cell to spike threshold. When the smaller size of mammalian receptors are taken into account, membrane electrical properties were found to be consistent with those of salamander cells investigated by others using whole-cell recording. The analysis also revealed possible errors in the determination of single-channel conductances and reversal potentials by cell-attached recording from small cells.

Action Potentials↗
PubMed1988

Ion permeation through single ACh-activated channels in denervated adult toad sartorius skeletal muscle fibres: effect of temperature.

The gigaohm seal technique was used to study the effects of temperature on ion permeation through acetylcholine-activated channels. This was done in cell-attached patches of the extrajunctional membrane of chronically-denervated, enzyme-treated cells from sartorius muscle of the toad Bufo marinus. The predominant extracellular cation in the pipette solution was Na+. Single channel current-voltage curves were measured at different temperatures and electrodiffusion and three-site-four-barrier rate theory models were used to characterize ion permeation through the channels and determine the effects of temperature on permeation parameters. The fitting of the experimental data to these models suggested the presence of at least three and probably more ion-selective sites within the channel. The most frequently occurring channel type (greater than 95% of channel openings) had a chord conductance of 25 pS at 11 degrees C and -70 mV and was classified as 'extrajunctional'. The single channel conductance of this channel had a low temperature-dependence (Q10 approximately equal to 1.3). The apparent activation enthalpy, Ea, for the conductance between 11 degrees C and 20 degrees C, did not appear to be significantly voltage-sensitive and had a value of about 17 +/- 2 kJ . mol-1 at a voltage of -70 mV. The Arrhenius plot of conductance appeared linear between 11 and 20 degrees C at all potentials examined. The data was consistent with a break in the slope of the Arrhenius plot at temperatures between 5 and 11 degrees C at all potentials examined, suggesting a possible phase transition of the membrane lipids. In contrast to the relative permeability, which was not very temperature sensitive, the relative binding constant was significantly affected by temperature. The relative Na/K binding constant sequence was: K5 degrees C greater than K20 degrees C greater than K15 degrees C much greater than K11 degrees C. In addition, the decrease in conductance observed at the most depolarized potentials was accentuated as the temperature was increased, suggesting a rate-limiting access step for ions from the intracellular solution into the channel.

Acetylcholine↗
PubMed1988

A temperature and solution control system for the measurement of single channel currents in excised membrane patches.

A technique is described which permits accurate temperature control and relatively rapid temperature changes (within about 2 min for 10 degrees C changes between 10 degrees and 40 degrees C) of the solution perfusing the exposed surface of excised membrane patches. The simultaneous exchange of temperature controlled solution is also possible. Using the "sleeve technique", patches excised from cells in standard tissue culture dishes are removed to a separate chamber where temperature and solution are accurately controlled. This avoids two common limitations of existing temperature or solution control systems: (1) test solution contamination of the tissue perfusion solution which may impair cell viability and (2) the use of specialised chambers which are unsuitable for use with cultured cells. In the system described, temperature control is possible over the range of at least 4-40 degrees C. Desired temperatures can be preset to within approximately +/- 1 degrees C, and can then be controlled and measured to an accuracy of +/- 0.1 degree C. At a constant temperature, the system enables rapid solution changes, the solution bathing the excised patch being exchanged in approximately 3 s.

Animals↗
PubMed1987

A simple technique for transferring excised patches of membrane to different solutions for single channel measurements.

A technical problem associated with the patch clamp technique has been the changing of solutions bathing the membrane patch. The simple technique described here solves this problem by means of a movable polythene sleeve placed on the shaft of the patch clamp pipette. The sleeve is initially placed so that the tip of the pipette is exposed. A gigaohm seal is formed using standard techniques. The patch is then excised and the sleeve is slipped down a few mm past the end of the tip of the pipette. When the pipette and sleeve is now removed from the solution, a small drop of solution covering the membrane patch is held in place at the end of the sleeve by surface tension. The pipette is then easily transferred to a different solution without passing the membrane patch through the air-water interface. The sleeve is then simply pulled back up the pipette shaft to expose the membrane patch to the new solution.

Cell Membrane↗
PubMed1987

Ion permeation through single channels activated by acetylcholine in denervated toad sartorius skeletal muscle fibers: effects of alkali cations.

The gigaohm seal technique was used to study ion permeation through acetylcholine-activated channels in cell-attached patches of the extrajunctional membrane of chronically denervated, enzyme-treated cells from the sartorius muscle of the toad Bufo marinus. The most frequently occurring channel type (greater than 95% of channel openings), provisionally classified as 'extrajunctional,' had a chord conductance of approximately 25 pS under normal conditions (-70 mV, 11 degrees C, Normal Toad Ringer's). The less frequently observed channel type (less than 5% of channel openings), classified as a 'junctional' type, had a conductance of 35 pS under the same conditions, and a similar null potential. In many patches, a small percentage (usually less than 2%) of openings of the extrajunctional channel displayed a lower conductance state. The shape of the I-V curves obtained for the extrajunctional channel depended on the predominant extracellular cation. For Cs and K, the I-V curves were essentially linear over the voltage range +50 to -150 mV across the patch, suggesting that the potential independent component of the energy profile within the channel was symmetrical. For Li, the I-V curve was very nonlinear, displaying a significant sublinearity at hyperpolarized potentials. Both an electrodiffusion and a symmetrical uniform four-barrier, three-site rate-theory model provided reasonable fits to the data, whereas symmetrical two-barrier, single-site rate-theory models did not. For the alkali cations examined, the relative permeability sequence was PCs greater than PK greater than PNa greater than PLi--a "proportional" selectivity sequence. This was different from the single channel conductance sequence which was found to be gamma K greater than gamma Cs greater than gamma Na greater than gamma Li implying that ions do not move independently through the channel. The relative binding constant sequence for the channel sites was found to be a "polarizability" sequence, i.e., KLi greater than KCs greater than KNa greater than KK. There was an inverse relationship between the relative binding constant and the relative mobility for the cations examined. Under conditions when the single-channel conductance was relatively high, the conductance at depolarized potentials was lower than that predicted by both electrodiffusion and rate theory models, suggesting that there was a rate-limiting access step for ions, from the intracellular compartment into the channel.

Acetylcholine↗
PubMed1979

Cation permeation of the amphibian motor end-plate.

Measurements of acetylcholine-induced single-channel conductance and null potentials at the amphibian motor end-plate in solutions containing Na, K, Li and Cs ions (Gage & Van Helden, 1979; J. Physiol. (London) (in press) were analyzed in terms of three models. Two of these models, the "neutral" site channel model and the "charged" site channel model were developed to cater for three cations. Both were shown to be able to explain the dependence of single-channel conductance on membrane potential and gave the following sequences of equilibrium constants and mobilities. KLi/KNa/KK/KCs = 7:1.1:1:0.9 and UCs/UK/UNa/ULi = 1.4:1:0.58:0.13 at 8 degrees C. Similar sequences were obtained at 20 degrees C. Although the neutral model fitted the data for relative conductances in Li-, Cs- and Na-solutions slightly better than the charged model, experiments done in normal [NaCl] and [NaCl]/2 solutions could only be fitted by the neutral model. In contrast, the third model, the Constant Field Equation, was unable to fit the conductance data in any of the above situations. The data available suggests that permeation is through "long" neutral channels, lined with high field-strength negative polar groups and including one or possibly more high resistance barriers for anions.

Amphibians↗
PubMed1979

End-plate channels behave as neutral site channels.

The voltage sensitivity and concentration dependence of single channel conductance at the motor end-plate in the presence of different alkali cations have been compared with the predictions of different permeation models. The models have been developed, using the diffusional approach, to cater for three cations and anion permeability has been assumed to be insignificant. It was found that a neutral site channel model provided the best fit to the experimental data. The analyses also implied a high field strength sequence for the equilibrium selectively of the sites.

Calcium↗