Single-channel and whole-cell studies of calcium currents in young and aged rat hippocampal slice neurons

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.

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Whole-Cell and Single-Channel Analysis of P-Type Calcium Currents in Cerebellar Purkinje Cells of Leaner Mutant Mice

Journal of Neuroscience ◽

10.1523/jneurosci.18-19-07687.1998 ◽

1998 ◽

Vol 18 (19) ◽

pp. 7687-7699 ◽

Cited By ~ 92

Author(s):

Leonard S. Dove ◽

Louise C. Abbott ◽

William H. Griffith

Keyword(s):

Purkinje Cells ◽

Single Channel ◽

Calcium Currents ◽

Mutant Mice ◽

Whole Cell ◽

Single Channel Analysis ◽

Channel Analysis ◽

Cerebellar Purkinje Cells ◽

P Type

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Whole-cell and single-channel calcium currents of isolated smooth muscle cells from saphenous vein.

Circulation Research ◽

10.1161/01.res.60.4.523 ◽

1987 ◽

Vol 60 (4) ◽

pp. 523-533 ◽

Cited By ~ 103

Author(s):

A Yatani ◽

C L Seidel ◽

J Allen ◽

A M Brown

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Saphenous Vein ◽

Single Channel ◽

Muscle Cells ◽

Calcium Currents ◽

Whole Cell ◽

Isolated Smooth Muscle Cells

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Nonmodal gating of cardiac calcium channels as revealed by dihydropyridines.

The Journal of General Physiology ◽

10.1085/jgp.93.6.1243 ◽

1989 ◽

Vol 93 (6) ◽

pp. 1243-1273 ◽

Cited By ~ 60

Author(s):

A E Lacerda ◽

A M Brown

Keyword(s):

Calcium Channels ◽

Single Channel ◽

Calcium Currents ◽

Neonatal Rat ◽

Whole Cell ◽

Frequency Distributions ◽

Time Frequency ◽

Channel Current ◽

Open Time ◽

Open States

The hypothesis that dihydropyridine (DHP)-sensitive calcium channels have three distinct modes of gating has been examined. The major prediction is that the relative frequencies among modes depend on DHP concentration while the kinetics within a mode do not. We tested this by studying whole-cell and single-channel calcium currents in neonatal rat and adult guinea pig cardiac myocytes in different concentrations of several DHPs. In the absence of DHPs calcium currents declined with time but the kinetics, which are the focus of this study, were unchanged. Open-time frequency distributions had insignificant numbers of prolonged openings and were well fit by single tau's. Agonist DHP stereoisomers produced concentration-dependent changes in whole-cell tail current tau's. The frequency distribution of single calcium channel current open times became biexponential and the tau's were concentration dependent. The average number of openings per trace of channels with customary open times increased with increases in DHP concentration. Latencies to first opening for the customary openings and for prolonged openings were shorter in the presence of DHPs. A second larger conductance is another important feature of DHP-bound single calcium channels. Thus DHPs not only caused prolonged openings; they produced numerous changes in the kinetics of customary openings and increased channel conductance. It follows that these effects of DHPs do not support the hypothesis of modal gating of calcium channels. The mode model is not the only model excluded by the results; models in which DHPs are allowed to act only or mainly on open states are excluded, as are models in which the effects are restricted to inactivated states. We suggest a different type of model in which cooperative binding of DHPs at two sites produces the essential changes in kinetics and conductance.

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

Journal of Neurophysiology ◽

10.1152/jn.1994.71.6.2359 ◽

1994 ◽

Vol 71 (6) ◽

pp. 2359-2376 ◽

Cited By ~ 29

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)

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Patch and whole cell calcium currents recorded simultaneously in snail neurons.

The Journal of General Physiology ◽

10.1085/jgp.83.5.727 ◽

1984 ◽

Vol 83 (5) ◽

pp. 727-750 ◽

Cited By ~ 55

Author(s):

H D Lux ◽

A M Brown

Keyword(s):

Single Channel ◽

Calcium Currents ◽

Single Channels ◽

Ca Channels ◽

Maximum Slope ◽

Whole Cell ◽

Snail Neurons ◽

Tail Current ◽

Time Component ◽

Noise Spectra

The flow of Ca ions through single Ca channels has been examined. The gigaseal method was used on identifiable snail neurons that were voltage clamped using a two-microelectrode voltage clamp method. Average Ca patch currents and whole cell currents have similar time courses. They are affected similarly by changes in temperature. The differences in amplitude and inactivation between Ba and Ca whole cell currents were present in the patch records. The stationary noise spectra recorded from ensembles of multichannel patches have two components with fast and slow time constants equivalent to two components in the whole cell tail current relaxations. Elementary current amplitudes measured from the variance-mean relationship and from noise spectra gave values comparable to measurements from single channels. The single channel I-V relationship was curvilinear and the maximum slope conductance in 40 mM Cao was 7 pS. The amplitude of unitary currents was unchanged at long times when inactivation had occurred; hence depletion is not involved in this process. Channel density was approximately 3 microns-2 and was the same for Ba and Ca currents. The whole cell asymmetry currents gave very large values for the gating charge per channel. Changes in temperature from 29 to 9 degrees C had only a slight effect on the two Ca tail current tau's at potentials where turn-on of patch and whole cell currents was markedly slowed and the peak amplitudes were reduced by one-third. Single channel recordings were obtained at these two temperatures, and the mean open time and the fast component of the closed times were scarcely affected. Unit amplitudes were reduced by 30% and the slow closed time component was doubled. Therefore, peak currents and the slow closed time component was doubled. Therefore, peak currents were reduced partly as a result of the reduction in unit amplitude, but mainly as a result of a reduction in opening probability, the latter arising from an increase of the long closed times. It is concluded that the behavior of single Ca channels in membrane patches is the same as it is in whole cells. Cooling from 29 to 9 degrees C acts primarily on transitions among closed states and has little effect on the open to closed transition.

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