Deterministic modeling of single-channel and whole-cell currents

1. Single-channel properties of desensitizing glutamate-activated channels were analyzed in outside-out patch-clamp recordings from a motoneuron-enriched cell fraction from embryonic chick. A piezo-driven device was used to achieve fast solution exchange at the electrode tip, resulting in maximum activation within 2 ms. 2. Quisqualate/AMPA receptors, with a 13-pS conductance, desensitized rapidly; the desensitization rate depended on agonist concentration but not on membrane potential. When quisqualate was applied slowly, the quisqualate-activated channels desensitized without prior channel opening, indicating desensitization from the closed state. After a 10-ms refractory period, resensitization of all channels required up to 300 ms; resensitization rate did not depend on the duration of the preceding quisqualate application. 3. At agonist concentrations less than or equal to 1 mM, kainate receptors, with a 20-pS conductance, did not desensitize. At kainate concentrations greater than or equal to 1 mM, though, kainate receptors desensitized to a low steady-state conductance within approximately 200 ms. Resensitization of all channels required as long as 3 s, which could render kainate receptors inexcitable during high-frequency activation. 4. Desensitization rates of whole-cell currents were similar to those observed in outside-out mode. Glutamate- and quisqualate-activated responses were similar, suggesting that the rapidly desensitizing quisqualate-sensitive receptor type may dominate the kinetics of whole-cell excitatory postsynaptic currents (EPSCs) in this preparation. 5. It may be concluded that the efficacy of glutamate-mediated synaptic transmission is modulated by differences in the rates of desensitization and resensitization.

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Whole-cell and single-channel currents activated by GABA and glycine in granule cells of the rat cerebellum.

The Journal of Physiology ◽

10.1113/jphysiol.1995.sp020739 ◽

1995 ◽

Vol 485 (2) ◽

pp. 419-435 ◽

Cited By ~ 146

Author(s):

M Kaneda ◽

M Farrant ◽

S G Cull-Candy

Keyword(s):

Granule Cells ◽

Single Channel ◽

Whole Cell ◽

Rat Cerebellum ◽

Single Channel Currents ◽

Channel Currents

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

Journal of Experimental Biology ◽

10.1242/jeb.153.1.129 ◽

1990 ◽

Vol 153 (1) ◽

pp. 129-140 ◽

Cited By ~ 1

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.

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

Journal of Experimental Biology ◽

10.1242/jeb.177.1.201 ◽

1993 ◽

Vol 177 (1) ◽

pp. 201-221 ◽

Cited By ~ 2

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.

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Gadolinium Reduces AMPA Receptor Desensitization and Deactivation in Hippocampal Neurons

Journal of Neurophysiology ◽

10.1152/jn.2001.86.1.173 ◽

2001 ◽

Vol 86 (1) ◽

pp. 173-182 ◽

Cited By ~ 3

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.

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Actin filament organization is required for proper cAMP-dependent activation of CFTR

AJP Cell Physiology ◽

10.1152/ajpcell.1999.277.6.c1160 ◽

1999 ◽

Vol 277 (6) ◽

pp. C1160-C1169 ◽

Cited By ~ 42

Author(s):

Adriana G. Prat ◽

C. Casey Cunningham ◽

G. Robert Jackson ◽

Steven C. Borkan ◽

Yihan Wang ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Single Channel ◽

Fold Increase ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Whole Cell ◽

Excised Patches ◽

Cystic Fibrosis Transmembrane ◽

Molecular Nature

Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(−)]. cAMP stimulation of ABP(−) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl− currents. In ABP(−) cells expressing CFTR, cAMP was also without effect on Cl− conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl− currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(−)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl−channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl− channel activity in excised patches of ABP(−)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.

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Transient potassium currents in avian sensory neurons

Journal of Neurophysiology ◽

10.1152/jn.1990.63.4.725 ◽

1990 ◽

Vol 63 (4) ◽

pp. 725-737 ◽

Cited By ~ 5

Author(s):

S. K. Florio ◽

C. D. Westbrook ◽

M. R. Vasko ◽

R. J. Bauer ◽

J. L. Kenyon

Keyword(s):

Single Channel ◽

Outward Current ◽

Potassium Currents ◽

Delayed Rectifier ◽

Transient Outward Current ◽

Single Channels ◽

Patch Clamp Technique ◽

Small Component ◽

Whole Cell ◽

Outward Currents

1. We used the patch-clamp technique to study voltage-activated transient potassium currents in freshly dispersed and cultured chick dorsal root ganglion (DRG) cells. Whole-cell and cell-attached patch currents were recorded under conditions appropriate for recording potassium currents. 2. In whole-cell experiments, 100-ms depolarizations from normal resting potentials (-50 to -70 mV) elicited sustained outward currents that inactivated over a time scale of seconds. We attribute this behavior to a component of delayed rectifier current. After conditioning hyperpolarizations to potentials negative to -80 mV, depolarizations elicited transient outward current components that inactivated with time constants in the range of 8-26 ms. We attribute this behavior to a transient outward current component. 3. Conditioning hyperpolarizations increased the rate of activation of the net outward current implying that the removal of inactivation of the transient outward current allows it to contribute to early outward current during depolarizations from negative potentials. 4. Transient current was more prominent on the day the cells were dispersed and decreased with time in culture. 5. In cell-attached patches, single channels mediating outward currents were observed that were inactive at resting potentials but were active transiently during depolarizations to potentials positive to -30 mV. The probability of channels being open increased rapidly (peaking within approximately 6 ms) and then declined with a time constant in the range of 13-30 ms. With sodium as the main extracellular cation, single-channel conductances ranged from 18 to 32 pS. With potassium as the main extracellular cation, the single-channel conductance was approximately 43 pS, and the channel current reversed near 0 mV, as expected for a potassium current. 6. We conclude that the transient potassium channels mediate the component of transient outward current seen in the whole-cell experiments. This current is a relatively small component of the net current during depolarizations from normal resting potentials, but it can contribute significant outward current early in depolarizations from hyperpolarized potentials.

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Regulation of the Epithelial Na+ Channel by Membrane Tension

The Journal of General Physiology ◽

10.1085/jgp.112.2.97 ◽

1998 ◽

Vol 112 (2) ◽

pp. 97-111 ◽

Cited By ~ 64

Author(s):

Mouhamed S. Awayda ◽

Muthangi Subramanyam

Keyword(s):

Cytochalasin B ◽

Single Channel ◽

Direct Injection ◽

Expression System ◽

Cell Swelling ◽

Na Channel ◽

Bathing Solution ◽

Membrane Tension ◽

Whole Cell ◽

Epithelial Na Channel

The sensitivity of αβγ rat epithelial Na+ channel (rENaC) to osmotically or mechanically induced changes of membrane tension was investigated in the Xenopus oocyte expression system, using both dual electrode voltage clamp and cell-attached patch clamp methodologies. ENaC whole-cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100-mM KCl. Similarly, ENaC whole-cell currents were insensitive to osmotic cell swelling caused by a 33% decrease of bathing solution osmolarity. The lack of an effect of cell swelling on ENaC was independent of the status of the actin cytoskeleton, as ENaC remained insensitive to osmotic and mechanical cell swelling in oocytes pretreated with cytochalasin B for 2–5 h. This apparent insensitivity of ENaC to increased cell volume and changes of membrane tension was also observed at the single channel level in membrane patches subjected to negative or positive pressures of 5 or 10 in. of water. However, and contrary to the lack of an effect of cell swelling, ENaC currents were inhibited by cell shrinking. A 45-min incubation in a 260-mosmol solution (a 25% increase of solution osmolarity) caused a decrease of ENaC currents (at −100 mV) from −3.42 ± 0.34 to −2.02 ± 0.23 μA (n = 6). This decrease of current with cell shrinking was completely blocked by pretreatment of oocytes with cytochalasin B, indicating that these changes of current are not likely related to a direct effect of cell shrinking. We conclude that αβγ rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.

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