Heterogeneity of functional GABAA receptors in rat dentate gyrus neurons revealed by a change in response to drugs during the whole-cell current time-course

Physiological properties of GABAA receptors from acutely dissociated rat dentate granule cells. Study of fast, GABAA receptor-mediated, inhibitory postsynaptic currents (IPSCs) in hippocampal dentate granule cells has suggested that properties of GABAA receptors influence the amplitude and time course of the IPSCs. This study describes the physiological properties of GABAA receptors present on hippocampal dentate granule cells acutely isolated from 18- to 35-day-old rats. Rapid application of 1 mM GABA to outside-out macropatches excised from granule cells produced GABAA receptor currents with rapid rise time and biexponential decay of current after removal of GABA. After activation, granule cell GABAA receptor currents desensitized incompletely. During a 400-ms application of 1 mM GABA, peak current only desensitized ∼40%. In symmetrical chloride solutions there was no outward rectification of whole cell current. Activation rates and peak currents elicited by rapid application of GABA to macropatches were also similar at positive and negative holding potentials. However, deactivation of GABAA receptor currents was slower at positive holding potentials. When whole cell currents were recorded without ATP in the pipette, current run-down was not apparent for 30 min in 50% of neurons, but run-down appeared to start soon after access was established in the remaining neurons. When 2 mM ATP was included in the recording pipette no run-down was apparent in 30 min of recording. The efficacy and potency of GABA were lower in cells recorded with no ATP in the pipette and during run-down compared with those recorded with 2 mM ATP and no run-down.

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Voltage-dependent block by neomycin of the ATP-induced whole cell current of guinea-pig outer hair cells

Journal of Neurophysiology ◽

10.1152/jn.1993.70.4.1593 ◽

1993 ◽

Vol 70 (4) ◽

pp. 1593-1605 ◽

Cited By ~ 33

Author(s):

X. Lin ◽

R. I. Hume ◽

A. L. Nuttall

Keyword(s):

Steady State ◽

Guinea Pig ◽

Hair Cells ◽

Time Course ◽

Outer Hair Cells ◽

Current Noise ◽

Whole Cell ◽

Voltage Dependent ◽

Cell Current ◽

Whole Cell Current

1. The effects of externally applied ATP and neomycin on whole cell currents of isolated guinea pig cochlear outer hair cells (OHCs) were studied using the whole cell voltage-clamp technique. In OCHs held at -70 mV, ATP activated a large inward current. In the presence of neomycin, the ATP-induced whole cell current activated along a relatively unaltered time course, but the current then decreased to a reduced steady level. The neomycin inhibition of the ATP-induced current was dose dependent. The half-inhibitory concentration (IC50) of neomycin measured at steady state was estimated to be 90 microM. 2. Neomycin inhibition of the ATP response could not be reversed by increasing the concentration of ATP, indicating that the effect was noncompetitive. The inhibition was voltage dependent and was greatly reduced when OHCs were held at positive potentials. 3. Cells treated with 100 microM ATP gave maximal current responses. Addition of neomycin substantially increased membrane current noise of the 100 microM ATP responses. When neomycin concentration was varied from 10 to 500 microM, the current noise level peaked between 50 and 100 microM. The noise increase was observed at negative holding potentials but not at positive potentials. 4. The neomycin-induced whole cell current noise was used to estimate the size of the underlying elementary current. The ATP-induced single channel current of OHCs at -70 mV was estimated to be approximately 0.3 pA. The number of ATP-activated channels in a single OHC was estimated to be in the range of a few thousand. 5. The characteristics of the neomycin inhibition of ATP-induced currents were consistent with an open channel blocking mechanism. Analysis of the voltage dependence of the steady state neomycin inhibition suggested a neomycin binding site at an electrical distance of 0.3 from the extracellular side.

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Modulation of inhibitory and excitatory fast neurotransmission in the rat CNS by heavy water (D2O)

Journal of Neurophysiology ◽

10.1152/jn.00801.2014 ◽

2015 ◽

Vol 114 (2) ◽

pp. 1109-1118 ◽

Cited By ~ 1

Author(s):

Masahito Wakita ◽

Naoki Kotani ◽

Kiyomitsu Shoudai ◽

Toshitaka Yamaga ◽

Norio Akaike

Keyword(s):

Heavy Water ◽

Deuterium Oxide ◽

Dependent Manner ◽

General Anesthetics ◽

Concentration Dependent Manner ◽

Excitatory Postsynaptic Currents ◽

Whole Cell ◽

Postsynaptic Currents ◽

Cell Current ◽

Whole Cell Current

The effects of heavy water (deuterium oxide, D2O) on GABAergic and glutamatergic spontaneous and evoked synaptic transmission were investigated in acute brain slice and isolated “synaptic bouton” preparations of rat hippocampal CA3 neurons. The substitution of D2O for H2O reduced the frequency and amplitude of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in a concentration-dependent manner but had no effect on glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs). In contrast, for evoked synaptic responses in isolated neurons, the amplitude of both inhibitory and excitatory postsynaptic currents (eIPSCs and eEPSCs) was decreased in a concentration-dependent manner. This was associated with increases of synaptic failure rate ( Rf) and paired-pulse ratio (PPR). The effect was larger for eIPSCs compared with eEPSCs. These results clearly indicate that D2O acts differently on inhibitory and excitatory neurotransmitter release machinery. Furthermore, D2O significantly suppressed GABAA receptor-mediated whole cell current ( IGABA) but did not affect glutamate receptor-mediated whole cell current ( IGlu). The combined effects of D2O at both the pre- and postsynaptic sites may explain the greater inhibition of eIPSCs compared with eEPSCs. Finally, D2O did not enhance or otherwise affect the actions of the general anesthetics nitrous oxide and propofol on spontaneous or evoked GABAergic and glutamatergic neurotransmissions, or on IGABA and IGlu. Our results suggest that previously reported effects of D2O to mimic and/or modulate anesthesia potency result from mechanisms other than modulation of GABAergic and glutamatergic neurotransmission.

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Ionflux: A Microfluidic Approach to Ensemble Recording and Block of Whole-Cell Current from Voltage-Gated Ion Channels

Biophysical Journal ◽

10.1016/j.bpj.2009.12.1029 ◽

2010 ◽

Vol 98 (3) ◽

pp. 194a ◽

Cited By ~ 1

Author(s):

C. Ian Spencer ◽

Nianzhen Li ◽

Juliette Johnson ◽

Qin Chen ◽

Cristian Ionescu-Zanetti

Keyword(s):

Ion Channels ◽

Whole Cell ◽

Voltage Gated ◽

Cell Current ◽

Voltage Gated Ion Channels ◽

Whole Cell Current ◽

Gated Ion Channels

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The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action

International Journal of Molecular Sciences ◽

10.3390/ijms222212399 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12399

Author(s):

Hsin-Yen Cho ◽

Tzu-Hsien Chuang ◽

Sheng-Nan Wu

Keyword(s):

Single Channel ◽

Ionic Currents ◽

Reaction Scheme ◽

Gh3 Cells ◽

Whole Cell ◽

Type K ◽

Cell Current ◽

K Current ◽

Subsequent Addition ◽

Whole Cell Current

Solifenacin (Vesicare®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH3 cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K+ current (IK(M)) with effective EC50 value of 0.34 μM. The activation time constant of IK(M) was concurrently shortened in the SOL presence, hence yielding the KD value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of IK(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of IK(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated IK(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in IK(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K+ (KM) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH3 cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the IK(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with KM channels and hence in stimulating IK(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH3 or mHippoE-14 cells.

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[Ca2+]i oscillations in human sperm are triggered in the flagellum by membrane potential-sensitive activity of CatSper

Human Reproduction ◽

10.1093/humrep/deaa302 ◽

2020 ◽

Author(s):

Elis Torrezan-Nitao ◽

Sean G Brown ◽

Esperanza Mata-Martínez ◽

Claudia L Treviño ◽

Christopher Barratt ◽

...

Keyword(s):

Membrane Potential ◽

Large Scale ◽

Conflicts Of Interest ◽

Human Sperm ◽

Equilibrium Potential ◽

Current Clamp ◽

Whole Cell ◽

Cell Current ◽

Whole Cell Current

Abstract STUDY QUESTION How are progesterone (P4)-induced repetitive intracellular Ca2+ concentration ([Ca2+]i) signals (oscillations) in human sperm generated? SUMMARY ANSWER P4-induced [Ca2+]i oscillations are generated in the flagellum by membrane potential (Vm)-sensitive Ca2+-influx through CatSper channels. WHAT IS KNOWN ALREADY A subset of human sperm display [Ca2+]i oscillations that regulate flagellar beating and acrosome reaction. Although pharmacological manipulations indicate involvement of stored Ca2+ in these oscillations, influx of extracellular Ca2+ is also required. STUDY DESIGN, SIZE, DURATION This was a laboratory study that used >20 sperm donors and involved more than 100 separate experiments and analysis of more than 1000 individual cells over a period of 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS Semen donors and patients were recruited in accordance with local ethics approval from Birmingham University and Tayside ethics committees. [Ca2+]i responses and Vm of individual cells were examined by fluorescence imaging and whole-cell current clamp. MAIN RESULTS AND THE ROLE OF CHANCE P4-induced [Ca2+]i oscillations originated in the flagellum, spreading to the neck and head (latency of 1–2 s). K+-ionophore valinomycin (1 µM) was used to investigate the role of membrane potential (Vm). Direct assessment by whole-cell current-clamp confirmed that Vm in valinomycin-exposed cells was determined primarily by K+ equilibrium potential (EK) and was rapidly ‘reset’ upon manipulation of [K+]o. Pre-treatment of sperm with valinomycin ([K+]o = 5.4 mM) had no effect on the P4-induced [Ca2+] transient (P = 0.95; eight experiments), but application of valinomycin to P4-pretreated sperm suppressed activity in 82% of oscillating cells (n = 257; P = 5 × 10−55 compared to control) and significantly reduced both the amplitude and frequency of persisting oscillations (P = 0.0001). Upon valinomycin washout, oscillations re-started in most cells. When valinomycin was applied in saline with elevated [K+], the inhibitory effect of valinomycin was reduced and was dependent on EK (P = 10−25). Amplitude and frequency of [Ca2+]i oscillations that persisted in the presence of valinomycin showed similar sensitivity to EK (P < 0.01). The CatSper inhibitor RU1968 (4.8 and 11 µM) caused immediate and reversible arrest of activity in 36% and 96% of oscillating cells, respectively (P < 10−10). Quinidine (300 µM) which blocks the sperm K+ current (IKsper) completely, inhibited [Ca2+]i oscillations. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION This was an in-vitro study and caution must be taken when extrapolating these results to in-vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS [Ca2+]i oscillations in human sperm are functionally important and their absence is associated with failed fertilisation at IVF. The data reported here provide new understanding of the mechanisms that underlie the regulation and generation (or failure) of these oscillations. STUDY FUNDING/COMPETING INTEREST(S) E.T.-N. was in receipt of a postgraduate scholarship from the CAPES Foundation (Ministry of Education, Brazil). E.M-M received travel funds from the Programa de Apoyo a los Estudios de Posgrado (Maestria y Doctorado en Ciencias Bioquimicas-Universidad Autonoma de Mexico). SGB and CLRB are recipients of a Chief Scientist Office (NHS Scotland) grant TCS/17/28. The authors have no conflicts of interest.

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On the Contribution of the First Transmembrane Domain to Whole-Cell Current through an ATP-Gated Ionotropic P2X Receptor

Journal of Neuroscience ◽

10.1523/jneurosci.21-16-05885.2001 ◽

2001 ◽

Vol 21 (16) ◽

pp. 5885-5892 ◽

Cited By ~ 33

Author(s):

William R. Haines ◽

Mark M. Voigt ◽

Keisuke Migita ◽

Gonzalo E. Torres ◽

Terrance M. Egan

Keyword(s):

Transmembrane Domain ◽

P2x Receptor ◽

Whole Cell ◽

Cell Current ◽

Whole Cell Current

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Action of tetraethylammonium on calcium-activated potassium channels in pig pancreatic acinar cells studied by patch-clamp single-channel and whole-cell current recording

The Journal of Membrane Biology ◽

10.1007/bf01870780 ◽

1985 ◽

Vol 86 (2) ◽

pp. 139-144 ◽

Cited By ~ 51

Author(s):

N. Iwatsuki ◽

O. H. Petersen

Keyword(s):

Potassium Channels ◽

Patch Clamp ◽

Single Channel ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Whole Cell ◽

Cell Current ◽

Calcium Activated Potassium Channels ◽

Whole Cell Current

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Developmental Changes in the Electrophysiological Properties and Response Characteristics of Manduca Antennal-Lobe Neurons

Journal of Neurophysiology ◽

10.1152/jn.2002.87.6.2650 ◽

2002 ◽

Vol 87 (6) ◽

pp. 2650-2663 ◽

Cited By ~ 17

Author(s):

Alison R. Mercer ◽

John G. Hildebrand

Keyword(s):

Adult Development ◽

Action Potentials ◽

Antennal Lobe ◽

Electrical Excitability ◽

Whole Cell ◽

Electrophysiological Properties ◽

Response Characteristics ◽

Cell Current ◽

Whole Cell Current

Using whole cell patch-clamp recordings, we have examined changes in the electrophysiological properties and response characteristics of antennal lobe (AL) neurons associated with the metamorphic adult development of the sphinx moth, Manduca sexta. Whole cell current profiles and electrical excitability were examined in dispersed AL neurons in vitro, and in medial-group AL neurons in situ in semi-intact brain preparations. Around stages 2–4 of the 18 stages of metamorphic adult development, whole cell current profiles were dominated by large outward (K+) currents. Calcium-dependent action potentials could be elicited at this stage, but only a small percentage of cells exhibited sodium spikes. From stages 3 to 10, there was a rapid increase in the proportion of AL neurons exhibiting rapidly activating, transient sodium currents, and many cells in vitro exhibited spontaneous bursts of spike activity at this time. As development progressed, action-potential waveforms became shorter in duration and larger in amplitude. Cell-type–specific differences in the prevalence of spontaneous activity, and in the electrophysiological properties and response characteristics of AL neurons, were most apparent late in metamorphosis. While removal of antennal sensory input to the ALs early (stage 1–2) in metamorphosis had no detectable effect on the development of cell excitability, a significantly higher percentage of neurons in vitro from stage 4 pupae exhibited sodium-based action potentials following the addition of serotonin to the culture medium. Characteristic forms of electrical excitability in developing Manduca AL neurons, and their modulation by serotonin, seem likely to play a central role in the functional development of the ALs.

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