Whole cell and single channel properties of a new GABA receptor transiently expressed in the Hippocampus

1995 ◽  
Vol 73 (2) ◽  
pp. 902-906 ◽  
Author(s):  
M. Martina ◽  
F. Strata ◽  
E. Cherubini
Keyword(s):  
Postnatal Development ◽  
Hippocampal Neurons ◽  
Single Channel ◽  
Gamma Aminobutyric Acid ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Slow Kinetics ◽  
Inward Currents ◽  
Single Channel Currents ◽  
Channel Properties

1. The patch-clamp technique was used to characterize, in acutely dissociated CA3 rat hippocampal neurons, the whole cell and single channel properties of a novel response to gamma-aminobutyric acid (GABA) present only during a restricted period of postnatal development. 2. At postnatal days 0-10 (P0-P10), both GABA (100 microM) and isoguvacine (50 microM) evoked at a holding potential of -50 mV, in symmetrical chloride solution, whole cell inward currents. Bicuculline blocked the response to isoguvacine but only reduced the response to GABA (from 512 +/- 137 pA to 60 +/- 13 pA, mean +/- SE). After P12, bicuculline abolished the response to GABA. 3. The bicuculline-insensitive GABA currents were Cl- mediated and antagonized by picrotoxin. The desensitization rate was slower than the conventional bicuculline-sensitive response. The peak to plateau ratio induced by 0.1 or 1 mM of GABA shifted from 4.6 +/- 0.4 and 17.7 +/- 2.6 to 1.5 +/- 0.1 and 3.1 +/- 0.5 in the absence or in the presence of bicuculline, respectively. The recovery from desensitization was significantly faster for the bicuculline-insensitive responses. 4. In excised outside-out patches, GABA (20 microM) activated, in the presence of bicuculline (100 microM), single channel currents having conductances of 14, 22, and 31 pS. These values were similar to those obtained in the same preparation, in the absence of bicuculline. 5. These findings suggest that this new receptor type, which mediates bicuculline-insensitive responses with slow kinetics, may potentiate the depolarizing action of GABA during a critical period of postnatal development and therefore play a crucial role in synaptogenesis.

CALCIUM CHANNEL CURRENTS IN NEURONES FROM LOCUST (SCHISTOCERCA GREGARIA) THORACIC GANGLIA

1993 ◽  
Vol 177 (1) ◽  
pp. 201-221 ◽  
Author(s):  
H. A. Pearson ◽  
G. Lees ◽  
D. Wray
Keyword(s):  
Calcium Channel ◽  
Single Channel ◽  
Schistocerca Gregaria ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Transient Component ◽  
Thoracic Ganglia ◽  
Steady State Inactivation ◽  
Inward Currents ◽  
Channel Currents

1. Using the patch-clamp technique, Ca2+ channel currents were recorded from neurones freshly isolated from the thoracic ganglia of the desert locust Schistocerca gregaria. 2. In solutions containing 10 mmol l-1 Ba2+ we observed high-voltage-activated whole-cell inward currents with sustained and transient components, both of which had similar steady-state inactivation properties. 3. Substitution of Ca2+ for Ba2+ was found to reduce whole-cell currents, whereas removal of monovalent cations had no effect. 4. Cd2+ (1 mmol l-1) completely blocked the whole-cell current, but at 10 micromolar preferentially inhibited the sustained component without affecting the transient component. 5. Verapamil (1 micromolar) inhibited both current components but appeared to be more selective for the sustained component, whereas nitrendipine (1 micromolar) had no effect on either component. 6. A single-channel recording suggested that the transient component was carried by a low- conductance channel. 7. Certain compounds with insecticidal action (ryanodine, S-bioallethrin, deltamethrin and avermectin) did not affect calcium channel currents in these cells. 8. These data suggest that there are two types of Ca2+ channels present in locust neurones. These channel types have properties differing from the T-, L- and N-type channels found in vertebrates and, furthermore, were not targets for the insecticides we tested.

Potassium channels activated by GABAB agonists and serotonin in cultured hippocampal neurons

1994 ◽  
Vol 71 (6) ◽  
pp. 2570-2575 ◽  
Author(s):  
L. S. Premkumar ◽  
P. W. Gage
Keyword(s):  
Potassium Channels ◽  
Hippocampal Neurons ◽  
Single Channel ◽  
Gamma Aminobutyric Acid ◽  
Kinetic Behavior ◽  
Open Time ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Cultured Hippocampal Neurons

1. Single-channel currents were recorded in cell-attached patches on cultured hippocampal neurons in response to gamma-aminobutyric acid-B (GABAB) agonists or serotonin applied to the cell surface outside the patch area. 2. The channels activated by GABAB agonists and serotonin were potassium selective but had a different conductance and kinetic behavior. Channels activated by GABAB agonists had a higher conductance, longer open-time, and longer burst-length than channels activated by serotonin. 3. The kinetic behavior of channels activated by GABAB agonists varied with potential whereas channels activated by serotonin did not show voltage-dependent changes in kinetics. 4. In a few cell-attached patches, both types of channel were activated when the cell was exposed to GABA together with serotonin. 5. It was concluded that GABAB agonists and serotonin activate different potassium channels in the soma of cultured hippocampal neurons.

PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells

1998 ◽  
Vol 275 (2) ◽  
pp. C535-C543 ◽  
Author(s):  
C. A. Obejero-Paz ◽  
M. Auslender ◽  
A. Scarpa
Keyword(s):  
Okadaic Acid ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Whole Cell ◽  
A7r5 Cells ◽  
High Level ◽  
Single Channel Currents ◽  
Channel Currents

The possibility that protein kinase C (PKC) could control the activity of L-type Ca2+ channels in A7r5 vascular smooth muscle-derived cells in the absence of agonist stimulation was investigated using the patch-clamp technique. Consistent with the possibility that L-type Ca2+ channels are maximally phosphorylated by PKC under these conditions, we show that 1) activation of PKC with the phorbol ester phorbol 12,13-dibutyrate was ineffective in modulating whole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine or chelerythrine inhibited channel activity, 3) inhibition of protein phosphatases by intracellular dialysis of okadaic acid did not affect whole cell currents, and 4) the inhibitory effect of staurosporine was absent in the presence of okadaic acid. The inhibition of Ca2+ currents by PKC inhibitors was due to a decrease in channel availability and long open events, whereas the voltage dependence of the open probability and the single-channel conductance were not affected. The evidence suggests that in resting, nonstimulated A7r5 cells there is a high level of PKC activity that modulates the gating of L-type Ca2+ channels.

Multiple Mechanisms of Ketamine Blockade of N-methyl-D-aspartate Receptors 

1997 ◽  
Vol 86 (4) ◽  
pp. 903-917 ◽  
Author(s):  
Beverley A. Orser ◽  
Peter S. Pennefather ◽  
John F. MacDonald
Keyword(s):  
Hippocampal Neurons ◽  
Open Channel ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Open Time ◽  
Channel Opening ◽  
Single Channel Currents ◽  
A Site ◽  
Change In Mean

Background The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is blocked by ketamine, and this action likely contributes to ketamine's anesthetic and analgesic properties. Previous studies suggest that ketamine occludes the open channel by binding to a site located within the channel pore. This hypothesis was examined by investigating the effects of ketamine on single-channel currents from NMDA receptors. Methods The cell-attached and outside-out configurations of the patch clamp technique were used to study NMDA-activated currents recorded from cultured mouse hippocampal neurons. Results In cell-attached patches, NMDA evoked currents that had an apparent mean open time (tau o) of 3.26 ms. The probability of at least one channel being open (Po') was 0.058. The addition of ketamine (0.1 microM or 1 microM) to the pipette solution decreased Po' to 53% and 24% of control values, respectively. At 1 microM ketamine, this reduction was due to a decrease in both the frequency of channel opening and the mean open time (44% and 68% of control values, respectively). Ketamine did not influence channel conductance and no new components were required to fit the open- or closed-duration distributions. Ketamine (50 microM), applied outside the recording pipette, reduced the opening frequency of channels recorded in the cell attached configuration. This observation suggests that ketamine gained access to a binding site by diffusing across the hydrophobic cell membrane. In outside-out patches, ketamine potency was lower than that observed in cell-attached patches: 1 microM and 10 microM ketamine reduced Po' to 63% and 34% of control values, respectively, and this reduction was due primarily to a decrease in the frequency of channel opening with little change in mean open time. Conclusions These observations are consistent with a model whereby ketamine inhibits the NMDA receptor by two distinct mechanisms: (1) Ketamine blocks the open channel and thereby reduces channel mean open time, and (2) ketamine decreases the frequency of channel opening by an allosteric mechanism.

Ion channels activated by L-glutamate and GABA in cultured cerebellar neurons of the rat

1985 ◽  
Vol 224 (1236) ◽  
pp. 367-373 ◽  
Keyword(s):  
Single Channel ◽  
Noise Spectrum ◽  
Channel Noise ◽  
Single Channels ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Two Component ◽  
Single Channel Currents

Glutamate and GABA-receptor channels were investigated in explants of rat cerebellum grown in cell culture. The patch-clamp technique was used to examine neurons under whole cell clamp and the properties of channels were derived by analysis of glutamate and GABA-evoked current noise. In addition, single channel currents activated by glutamate were recorded from isolated outside-out patches of membrane. We found evidence for at least two types of glutamate receptor-channels in cerebellar cells. Some neurons exhibited a channel of 50 pS conductance with a Lorentzian noise spectrum of 5.9 ms time constant. Single channels were readily resolved both in whole cell clamp and excised patches. Other neurons possessed low conductance channels which produced two component spectra. Estimates of the single channel conductance gave a value of about 140 fS. GABA channel noise obtained from these cells was also fitted by two component spectra which gave single channel conductance of 16 pS.

Two types of kainate response in cultured rat hippocampal neurons

1991 ◽  
Vol 66 (1) ◽  
pp. 2-11 ◽  
Author(s):  
S. Ozawa ◽  
M. Iino ◽  
K. Tsuzuki
Keyword(s):  
Hippocampal Neurons ◽  
Single Channel ◽  
Pyramidal Cells ◽  
Nmda Receptor Antagonist ◽  
Inward Rectification ◽  
Type I ◽  
Type Ii ◽  
Induced Current ◽  
Patch Clamp Technique ◽  
Whole Cell

1. Two different types of kainate response were recorded in cultured rat hippocampal neurons with the use of the whole-cell and outside-out configurations of the patch-clamp technique. 2. There was an outward rectification in the current-voltage (I-V) plot of the kainate-induced current (type I response) in relatively large neurons bearing a morphological resemblance to young pyramidal cells. In smaller neurons with elliptical somata and fine neurites, the kainate response was characterized by a remarkable inward rectification in the I-V plot of the kainate-induced current and a significant permeability to Ca2+ (type II response). 3. Both type I and type II responses were negligible below 2 microM and almost saturated at 500 microM kainate. The concentrations producing half-maximal responses and the Hill coefficients were 68 microM and 1.76 and 56 microM and 1.21 for type I and type II responses, respectively. Both responses were suppressed similarly by the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). 4. The mean single-channel conductance (gamma) of the type II kainate response was estimated, from the relation between the whole-cell mean currents and current variances, to be 8.7 pS. The power spectrum for the current noise was fitted with the sum of two Lorentzians with cutoff frequencies (fc) of 61.1 +/- 1.4 and 327.8 +/- 10.5 Hz (n = 12).(ABSTRACT TRUNCATED AT 250 WORDS)

Single voltage-dependent potassium channels in cultured rat hippocampal neurons

1986 ◽  
Vol 56 (2) ◽  
pp. 481-493 ◽  
Author(s):  
M. A. Rogawski
Keyword(s):  
Hippocampal Neurons ◽  
Single Channel ◽  
Reversal Potential ◽  
Resting Potential ◽  
Moderate Degree ◽  
Patch Clamp Technique ◽  
Membrane Pore ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Single Channel Currents

Single-channel recordings using the gigohm seal patch-clamp technique were carried out on the somatic membranes of dissociated embryonic rat hippocampal neurons grown in cell culture. The recording medium contained tetrodotoxin to block the voltage-dependent Na+ conductance and Cd2+ to block Ca2+ and Ca2+-activated conductances. In the cell-attached configuration, depolarizing voltage steps activated outward directed single-channel currents with conductance 15-20 pS. The channel openings exhibited a moderate degree of flickering. The mean burst lifetimes ranged from 5 to 13 ms with a tendency to increase slightly at more depolarized potentials (T = 21-25 degrees C). Reversal potential measurements using excised membrane patches indicated that the channels behaved as expected of a K+-selective membrane pore. Channel opening occurred in Ca2+-free EGTA-containing solutions but was never observed in the presence of tetraethylammonium (TEA; 20 mM). The frequency of channel opening increased as the membrane was depolarized by up to 50 mV from resting potential; the fraction of time spent in the open state during the first 300 ms following a step depolarization increased e-fold for a 8-25 mV change in potential. First-latency histograms and simulations of the macroscopic current based on channel data obtained during repeated depolarizing voltage steps indicated that the probability of the channel being in the open state increases gradually with time after a step depolarization. During repeated depolarizing steps the channels appeared to randomly enter and exit a long-lived inactive state. It is concluded that these channels may underly the slowly activating, very slowly inactivating, TEA-sensitive voltage-dependent K+ current (IK) in cultured hippocampal neurons.

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

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