Whole-cell and single-channel calcium currents in guinea pig basal forebrain neurons

1994 ◽  
Vol 71 (6) ◽  
pp. 2359-2376 ◽  
Author(s):  
W. H. Griffith ◽  
L. Taylor ◽  
M. J. Davis
Keyword(s):  
Steady State ◽  
Guinea Pig ◽  
Single Channel ◽  
Low Voltage ◽  
Bay K 8644 ◽  
Calcium Currents ◽  
Voltage Step ◽  
Whole Cell ◽  
Multiple Channel ◽  
Dependent Inactivation

1. Whole-cell and single-channel patch-clamp recordings of calcium (Ca2+) currents were made in acutely dissociated neurons from the medial septum (MS) and nucleus of the diagonal band (nDB) of adult guinea pig. Barium (Ba2+) was used as the charge carrier across the Ca2+ channel and multiple channel types were identified in different cell types. 2. Both low-voltage-activated (LVA) and high-voltage-activated (HVA) currents were distinguished on the basis of steady-state voltage dependence, activation and inactivation properties, and pharmacological sensitivity. HVA currents had activation thresholds approximately 20 mV more positive than LVA currents. Steady-state inactivation of HVA currents was approximately 50% when the holding potential was shifted from -80 to -40 mV. 3. The dihydropyridines had consistent effects on HVA currents. The amplitude was increased and the activation threshold shifted by 10 mV in the hyperpolarizing direction in the presence of the agonist Bay K 8644 (2–5 microM). The antagonist nifedipine (10 microM) produced approximately 50% inhibition of HVA currents from a holding potential of -80 mV. 4. A second component of the HVA current was blocked by omega-conotoxin (omega-CTX) (300–700 nM). At a holding potential of -80 mV, omega-CTX inhibited 45% of the HVA current. 5. LVA currents were activated near -70 mV and displayed time-dependent inactivation during a 200- to 300-ms voltage step. Voltage-dependent inactivation of LVA currents was also observed and could be described by a single Boltzman relationship with a half-inactivation potential of -84 mV. LVA currents were not significantly changed by Bay K 8644 and were not blocked by low concentrations of nifedipine or omega-CTX. 6. Single voltage-gated Ca2+ channels were investigated using cell-attached patches. In these experiments, 100 mM Ba2+ was used in the patch pipette and the membrane potential was zeroed with isotonic potassium (K+)-aspartate. A low-conductance channel was activated at negative potentials and inactivated rapidly during a 200- to 300-ms voltage step. Unitary amplitudes were determined at different membrane potentials with single-channel conductances calculated to be 7.8 +/- 1.2 (SD) pS. These channels were not blocked by nifedipine (10 microM) and appeared similar to T channels previously reported in both peripheral and central neurons. Ensemble averages from cell-attached patches of T channels resembled LVA currents recorded in the whole-cell configuration.(ABSTRACT TRUNCATED AT 400 WORDS)

Propofol Inhibits Cardiac L-Type Calcium Current in Guinea Pig Ventricular Myocytes

1996 ◽  
Vol 84 (3) ◽  
pp. 626-635 ◽  
Author(s):  
Ching-Yue Yang ◽  
Chih-Shung Wong ◽  
Chuan-Cheng Yu ◽  
Hsiang-Ning Luk ◽  
Cheng-I Lin
Keyword(s):  
Steady State ◽  
Guinea Pig ◽  
Time Course ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Hill Coefficient ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Inactivation Curve ◽  
Whole Cell

Background Propofol may exert negative inotropic and chronotropic actions in the heart. Single-channel studies show that propofol affects the kinetics of opening and closing of cardiac L-type calcium channels (ICa(L)) without altering channel conductance. The aim of this study was to investigate the mechanisms of depressant effects of propofol on cardiac whole-cell ICa(L). Methods Single ventricular myocytes were freshly dissciated from guinea pig hearts using enzymatic isolation. One-suction electrode voltage-clamp technique (whole-cell mode) was used. LCa(L) was separated from other contaminated ionic currents. Propofol was applied in the commercial 10% Intralipid emulsion formula (Zeneca, UK). Results In isolated cardiomyocytes, propofol significantly inhibited whole-cell ICa(L) in a concentration-dependent manner (K D = 52.0 microM; Hill coefficient = 1.3). The solvent (Intralipid) did not affect ICa(L). Propofol decreased ICa(L) at all potentials tested along the voltage axis and reduced the slope conductance. The threshold potential for activation and the peak potential of the current-voltage relationship were not changed by propofol. The steady-state activation curves overlapped in the absence and the presence of 56 microM propofol. In contrast, the steady-state inactivation curve was shifted in the hyperpolarizing direction. The time course of the recovery from inactivation was delayed by 56 microM propofol. The blocking action on ICa(L) of propofol shows marked resting block and use-dependent block. Propofol caused more pronounced inhibition at a higher stimulation frequency. The effect of propofol on the inactivation process was even more clear on ICa(L). Conclusions The authors conclude tha propofol, at supratherapeutic concentrations, inhibits cardiac ICa(L). This inhibition is mainly due to a shift of inactivation curve and a reduction in slope conductance.

The Neuromuscular Junction Revisited: Ca2+ Channels and Transmitter Release in Cholinergic Neurones in Xenopus Nerve and Muscle Cell Culture

1990 ◽  
Vol 153 (1) ◽  
pp. 129-140 ◽  
Author(s):  
T. P. FENG ◽  
ZHENG-SHAN DAI
Keyword(s):  
Neuromuscular Junction ◽  
Transmitter Release ◽  
Single Channel ◽  
Calcium Currents ◽  
Patch Clamps ◽  
Whole Cell ◽  
Voltage Gated ◽  
Cholinergic Neurones ◽  
Ca2 Channels ◽  
Evoked Transmitter Release

Although the entry of calcium ions into the presynaptic nerve terminals through voltage-gated Ca2+ channels is now universally recognized as playing an essential role in evoked transmitter release at the neuromuscular junction (NMJ), and indeed in chemical synapses generally, we have as yet very little direct knowledge of the Ca2+ channels of the presynaptic terminals. In this work, making use of cocultured nerve and muscle cells from Xenopus embryos, we studied the NMJ formed between the soma of identified cholinergic neurones and myoball, which allowed the use of patch-clamps on both the pre- and postsynaptic components. Both whole-cell and single-channel recordings of Ca2+ channels in the presynaptic cell were made. We found only one type of voltage-gated Ca2+ channel with highvoltage activation and slow inactivation characteristics, allowing its classification either as the L or the N type. The channels were susceptible to block by metenkephalin but not to block by nifedipine or to enhancement by Bay K 8644. This combination of pharmacological properties favours their classification as the N type. Preliminary observations on the correlation between calcium currents and transmitter release disclosed a strikingly rapid run-down of the evoked release with unchanged calcium currents and spontaneous release during whole-cell recording, indicating a specific wash-out effect on some link between calcium entry and evoked transmitter release.

Gadolinium Reduces AMPA Receptor Desensitization and Deactivation in Hippocampal Neurons

2001 ◽  
Vol 86 (1) ◽  
pp. 173-182 ◽  
Author(s):  
Saobo Lei ◽  
John F. MacDonald
Keyword(s):  
Steady State ◽  
Ampa Receptor ◽  
Hippocampal Neurons ◽  
Time Course ◽  
Single Channel ◽  
Trivalent Cation ◽  
Receptor Desensitization ◽  
Whole Cell ◽  
Low Concentrations ◽  
Cultured Hippocampal Neurons

The actions of the trivalent cation Gd3+ on whole cell AMPA receptor-mediated currents were studied in isolated hippocampal neurons, in nucleated or outside-out patches taken from cultured hippocampal neurons, and on miniature excitatory postsynaptic currents (mEPSCs) recorded in cultured hippocampal neurons. Glutamate, AMPA, or kainate was employed to activate AMPA receptors. Applications of relatively low concentrations of Gd3+ (0.1–10 μM) substantially enhanced steady-state whole cell glutamate and kainate-evoked currents without altering peak currents, suggesting that desensitization was reduced. However, higher concentrations (>30 μM) depressed steady-state currents, indicating an underlying inhibition of channel activity. Lower concentrations of Gd3+also increased the potency of peak glutamate-evoked currents without altering that of steady-state currents. An ultrafast perfusion system and nucleated patches were then used to better resolve peak glutamate-evoked currents. Low concentrations of Gd3+ reduced peak currents, enhanced steady-state currents, and slowed the onset of desensitization, providing further evidence that this cation reduces desensitization. In the presence of cyclothiazide, a compound that blocks desensitization, a low concentration Gd3+ inhibited both peak and steady-state currents, indicating that Gd3+ both reduces desensitization and inhibits these currents. Gd3+ reduced the probability of channel opening at the peak of the currents but did not alter the single channel conductance calculated using nonstationary variance analysis. Recovery from desensitization was enhanced, and glutamate-evoked current activation and deactivation were slowed by Gd3+. The Gd3+-induced reduction in desensitization did not require the presence of the GluR2 subunit as this effect was seen in hippocampal neurons from GluR2 null-mutant mice. Gd3+ reduced the time course of decay of mEPSCs perhaps as a consequence of its slowing of AMPA receptor deactivation although an increase in the frequency of mEPSCs also suggested enhanced presynaptic release of transmitter. These results demonstrate that Gd3+ potently reduces AMPA receptor desensitization and mimics a number of the properties of the positive modulators of AMPA receptor desensitization such as cyclothiazide.

Low-Voltage Activated T-Type Calcium Currents Are Differently Expressed in Superficial and Deep Layers of Guinea PigPiriform Cortex

1998 ◽  
Vol 79 (2) ◽  
pp. 808-816 ◽  
Author(s):  
Jacopo Magistretti ◽  
Marco de Curtis
Keyword(s):  
Guinea Pig ◽  
Calcium Current ◽  
Low Voltage ◽  
Piriform Cortex ◽  
Neuronal Firing ◽  
Calcium Currents ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Deep Layers ◽  
Group 2

Magistretti, Jacopo and Marco de Curtis. Low-voltage activated T-type calcium currents are differently expressed in superficial and deep layers of guinea pig piriform cortex. J. Neurophysiol. 79: 808–816, 1998. A variety of voltage-dependent calcium conductances are known to control neuronal excitability by boosting peripheral synaptic potentials and by shaping neuronal firing patterns. The existence and functional significance of a differential expression of low- and high-voltage activated (LVA and HVA, respectively) calcium currents in subpopulations of neurons, acutely isolated from different layers of the guinea pig piriform cortex, were investigated with the whole cell variant of the patch-clamp technique. Calcium currents were recorded from pyramidal and multipolar neurons dissociated from layers II, III, and IV. Average membrane capacitance was larger in layer IV cells [13.1 ± 6.2 (SD) pF] than in neurons from layers II and III (8.6 ± 2.8 and 7.9 ± 3.1 pF, respectively). Neurons from all layers showed HVA calcium currents with an activation voltage range positive to −40 mV. Neurons dissociated from layers III and IV showed an LVA calcium current with the biophysical properties of a T-type conductance. Such a current displayed the following characteristics: 1) showed maximal amplitude of 11–16 pA/pF at −30 mV, 2) inactivated rapidly with a time constant of ∼22 ms at −30 mV, and 3) was completely steady-state inactivated at −60 mV. Only a subpopulation of layer II neurons (group 2 cells; circa 18%) displayed an LVA calcium current similar to that observed in deep layers. The general properties of layer II-group 2 cells were otherwise identical to those of group 1 neurons. The present study demonstrates that LVA calcium currents are differentially expressed in neurons acutely dissociated from distinct layers of the guinea pig piriform cortex.

scholarly journals Single calcium channel behavior in native skeletal muscle.

1995 ◽  
Vol 105 (2) ◽  
pp. 227-247 ◽  
Author(s):  
R T Dirksen ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Calcium Channels ◽  
Single Channel ◽  
Bay K 8644 ◽  
Dependent Manner ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Whole Cell ◽  
Macroscopic Properties ◽  
Voltage Dependent

The purpose of this study was to use whole-cell and cell-attached patches of cultured skeletal muscle myotubes to study the macroscopic and unitary behavior of voltage-dependent calcium channels under similar conditions. With 110 mM BaCl2 as the charge carrier, two types of calcium channels with markedly different single-channel and macroscopic properties were found. One class was DHP-insensitive, had a single-channel conductance of approximately 9 pS, yielded ensembles that displayed an activation threshold near -40 mV, and activated and inactivated rapidly in a voltage-dependent manner (T current). The second class could only be well resolved in the presence of the DHP agonist Bay K 8644 (5 microM) and had a single-channel conductance of approximately 14 pS (L current). The 14-pS channel produced ensembles exhibiting a threshold of approximately -10 mV that activated slowly (tau act approximately 20 ms) and displayed little inactivation. Moreover, the DHP antagonist, (+)-PN 200-110 (10 microM), greatly increased the percentage of null sweeps seen with the 14-pS channel. The open probability versus voltage relationship of the 14-pS channel was fitted by a Boltzmann distribution with a VP0.5 = 6.2 mV and kp = 5.3 mV. L current recorded from whole-cell experiments in the presence of 110 mM BaCl2 + 5 microM Bay K 8644 displayed similar time- and voltage-dependent properties as ensembles of the 14-pS channel. Thus, these data are the first comparison under similar conditions of the single-channel and macroscopic properties of T current and L current in native skeletal muscle, and identify the 9- and 14-pS channels as the single-channel correlates of T current and L current, respectively.

scholarly journals Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

1994 ◽  
Vol 104 (2) ◽  
pp. 357-373 ◽  
Author(s):  
S Koumi ◽  
R Sato ◽  
T Aramaki
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cl Channels ◽  
Inside Out

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

Whole cell calcium currents in acutely isolated olfactory bulb output neurons of the rat

1996 ◽  
Vol 75 (3) ◽  
pp. 1138-1151 ◽  
Author(s):  
X. Wang ◽  
J. S. McKenzie ◽  
R. E. Kemm
Keyword(s):  
Olfactory Bulb ◽  
Bay K 8644 ◽  
Calcium Currents ◽  
Neonatal Rat ◽  
Bathing Solution ◽  
Whole Cell ◽  
Peak Current ◽  
Maximum Peak ◽  
Slope Factor ◽  
Output Neurons

1. Voltage-gated whole cell Ca2+ currents have been investigated in olfactory bulb (OB) output neurons acutely isolated from neonatal rats. 2. Identification of OB output neurons, mitral or tufted cells, was based on morphology and size and validated by their retrograde labeling with rhodamine or Fast Blue. Of labeled neurons, 45% exhibited either phasic or nonphasic spontaneous firing that was blocked by 10(-7) M tetrodotoxin, 0.5 mM Cd2+, or 1 mM Co2+ in the bathing solution. 3. Whole cell Ca2+ currents displayed holding potential sensitivity indicative of low voltage-activated (LVA) and high voltage-activated (HVA) currents, which exhibited similar dependence on extracellular Ca2+ concentration and could be completely abolished by bathing in 500 microM Cd2+ or in Ca(2+)-free solution. 4. A T-type LVA Ca2+ current, detected in 65% of OB output neurons tested, was activated by depolarizing to -57 mV from holding potential -86 mV and fully inactivated at holding potentials more positive than -60 mV. It was permeated equally by 2.6 mM Ca2+, Sr2+ and Ba2+. The half-activation potential was -35 mV with a slope factor of 7 mV. Depolarizing to -26 mV from different holding potentials in a 2.6-mM Ca2+ solution gave a steady-state half-inactivation potential of -82 mV with a slope factor of 10.7 mV. This LVA current was not sensitive to 5 microM omega-conotoxin (omega-CgTx) or 5 microM Bay K 8644 and was resistant to block by 30 microM Cd2+, by 50 microM verapamil or by 5 microM nifedipine. 5. HVA Ca2+ currents, detected in 97% of OB output cells, activated at around -30 to -20 mV, with maximum peak current at approximately 4 mV in 2.6 mM Ca2+ external solution. They showed similar permeability to 2.6 mM Ca2+ and Sr2+, but the maximum peak current was increased 40% in 2.6 mM Ba2+. Depolarizing to 4 mV from different holding potentials yielded a half-inactivation potential of -67 mV with a slope factor of 13.2 mV. Two components, as suggested by their sensitivities to 5 microM Bay K 8644, nifedipine. omega-CgTx and to voltage, may resemble the L-type and N-type currents described in other neural preparations. However, 5 microM omega-CgTx seemed to block both components, being more effective at more positive potentials. There was a residual component of Cd(2+)-sensitive current not affected by cumulative addition of nifedipine and omega-CgTx. 6. omega-Agatoxin IVA (omega-Aga), a selective P-type Ca2+ channel blocker, had no detectable effect at 50 or 200 nM and 1 microM doses on whole cell Ca2+ currents elicited by 200-ms voltage steps to 4 mV from holding potential -86 mV. 7. We conclude that both LVA and HVA Ca2+ currents exist in neonatal rat OB output neurons, showing distinct kinetic and pharmacological characteristics. The HVA Ca2+ currents contain at least two components, probably resembling L- and N-type currents. Another fast-inactivating HVA component, insensitive to nifedipine, omega-CgTx and omega-Aga, could represent the newly established R-type Ca2+ current.

Patch-clamp study of single-channel and whole-cell K+ currents in guinea pig pancreatic acinar cells

1988 ◽  
Vol 255 (3) ◽  
pp. G275-G285 ◽  
Author(s):  
K. Suzuki ◽  
O. H. Petersen
Keyword(s):  
Patch Clamp ◽  
Guinea Pig ◽  
Single Channel ◽  
Pancreatic Acinar Cells ◽  
Intact Cells ◽  
Membrane Area ◽  
Pancreatic Acini ◽  
Whole Cell ◽  
Single Channel Currents ◽  
K Currents

K+ channels in the plasma membrane of isolated guinea pig pancreatic acini were studied by patch-clamp single-channel and whole-cell current recording techniques. Three types of K+-permeable pores were found in excised patch experiments: Ca2+-activated nonselective cation channels with a unit conductance of approximately 25 pS that could be inhibited by ATP acting on the membrane inside, and two kinds of Ca2+- and voltage-activated K+-selective channels with unit conductances (in symmetrical K+-rich solutions) of about 200 and 30 pS, respectively. In intact cells, pentagastrin activation of currents through the 30 pS K+-selective pores was demonstrated. In these experiments pentagastrin was added to the bath solution and had no direct contact with the electrically isolated membrane area from which the single-channel currents were recorded, suggesting that the activation is mediated via an intracellular messenger system. Pentagastrin stimulation of voltage-gated K+ currents was also observed in whole-cell recording experiments. Results from these experiments suggest that in the stimulated condition the membrane electrical properties were dominated by the 30 pS K+-selective channels.

Hypoxia modulates nitric oxide-induced regulation of NMDA receptor currents and neuronal cell death

1999 ◽  
Vol 277 (4) ◽  
pp. C673-C683 ◽  
Author(s):  
Muyiwa Gbadegesin ◽  
Stefano Vicini ◽  
Sandra J. Hewett ◽  
David A. Wink ◽  
Michael Espey ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Steady State ◽  
Nmda Receptor ◽  
Single Channel ◽  
Receptor Activation ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Nmda Channel

Nitric oxide (NO) released from a new chemical class of donors enhances N-methyl-d-aspartate (NMDA) channel activity. Using whole cell and single-channel patch-clamp techniques, we have shown that ( Z)-1-[ N-(3-ammoniopropyl)- N-( n-propyl)amino]-NO (PAPA-NO) and diethylamine NO, commonly termed NONOates, potentiate the glutamate-mediated response of recombinant rat NMDA receptors (NR1/NR2A) expressed in HEK-293 cells. The overall effect is an increase in both peak and steady-state whole cell currents induced by glutamate. Single-channel studies demonstrate a significant increase in open probability but no change in the mean single-channel open time or mean channel conductance. Reduction in oxygen levels increased and prolonged the PAPA-NO-induced change in both peak and steady-state glutamate currents in transfected HEK cells. PAPA-NO also enhanced cell death in primary cultures of rodent cortical neurons deprived of oxygen and glucose. This potentiation of neuronal injury was blocked by MK-801, indicating a critical involvement of NMDA receptor activation. The NO-induced increase in NMDA channel activity as well as NMDA receptor-mediated cell death provide firm evidence that NO modulates the NMDA channel in a manner consistent with both a physiological role under normoxic conditions and a pathophysiological role under hypoxic conditions.

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