Changes In Current Kinetics Research Articles

NMDA receptors mediate synaptic depression, but not spine loss in the dentate gyrus of adult amyloid Beta (A\u03b2) overexpressing mice

Amyloid beta (Aβ)-mediated synapse dysfunction and spine loss are considered to be early events in Alzheimer’s disease (AD) pathogenesis. N-methyl-D-aspartate receptors (NMDARs) have previously been suggested to play a role for Amyloid beta (Aβ) toxicity. Pharmacological block of NMDAR subunits in cultured neurons and mice suggested that NMDARs containing the GluN2B subunit are necessary for Aβ-mediated changes in synapse number and function in hippocampal neurons. Interestingly, NMDARs undergo a developmental switch from GluN2B- to GluN2A-containing receptors. This indicates different functional roles of NMDARs in young mice compared to older animals. In addition, the lack of pharmacological tools to efficiently dissect the role of NMDARs containing the different subunits complicates the interpretation of their specific role. In order to address this problem and to investigate the specific role for Aβ toxicity of the distinct NMDAR subunits in dentate gyrus granule cells of adult mice, we used conditional knockout mouse lines for the subunits GluN1, GluN2A and GluN2B. Aβ-mediated changes in synaptic function and neuronal anatomy were investigated in several-months old mice with virus-mediated overproduction of Aβ and in 1-year old 5xFAD mice. We found that all three NMDAR subunits contribute to the Aβ-mediated decrease in the number of functional synapses. However, NMDARs are not required for the spine number reduction in dentate gyrus granule cells after chronic Aβ-overproduction in 5xFAD mice. Furthermore, the amplitude of synaptic and extrasynaptic NMDAR-mediated currents was reduced in dentate gyrus granule of 5xFAD mice without changes in current kinetics, suggesting that a redistribution or change in subunit composition of NMDARs does not play a role in mediating Amyloid beta (Aβ) toxicity. Our study indicates that NMDARs are involved in AD pathogenesis by compromising synapse function but not by affecting neuron morphology.

Direct Estimation of CaV1.2 Gating Parameters: Quantification of Voltage Sensor - Pore Transductions and their Modulation by FLP 64176.

Calcium agonists such as FPL 64174 increase macroscopic calcium channel currents and induce substantial changes in current kinetics. Their molecular mechanism of action is currently unknown. Here we propose a technique enabling the estimation of FPL 64174 effects on rate constants of the voltage sensing machinery and pore transitions from macroscopic CaV1.2 current kinetics making use of a hybrid stochastic-deterministic optimization procedure. Current traces of wild type CaV1.2, a channel construct with neutralized segment IIS4 (IIS4N) and a pore mutant (A780T) were fitted to a circular four-state (rest, activated, open, deactivated) channel model in control and FPL 64174 (1 µM). The estimated rate constants provided novel insights in how structural elements of the voltage sensing unit and the channel pore influence the action of FPL 64174. The new approach may be applicable for the analysis of drug effects on other ion channels as well as for quantification of VS transitions and rate constants of pore gating from macroscopic current kinetics.

The Kelch‐Like 1 Actin Binding Protein Modulates Neuronal Voltage Gated Calcium Channels

Kelch‐like 1 (KLHL1) is a neuronal actin‐binding protein first identified in humans as part of the genetic locus of the neurological movement disorder Spinocerebellar Ataxia type 8. KLHL1 is expressed in the cytosol, axons and dendrites. KLHL1 KO causes loss of post‐synaptic structures, loss of motor coordination and gait abnormalities. KLHL1 is an ion channel modulator and upregulates HVA and LVA calcium currents in vitro by increased recycling endosome activity and by alteration of the kinetics of τdeactivation. We analyzed the endogenous HVA and LVA calcium currents in neurons from WT and KLHL1 KO mice, and compared these data with the acute down‐regulation of KLHL1 using a knock‐down shRNA (KD) strategy. KLHL1 KD caused decreased HVA and LVA calcium current expression. In contrast, the KO had no differences in total LVA current density, yet changes in current kinetics suggest there is a down‐regulation of α1H and compensatory expression of α1I. HVA current density was also decreased in KO (40%) at 4–6 DIV but not at 7–10 DIV, suggesting this deficit was also compensated for after 7 DIV. Our data confirm that KLHL1 modulates LVA and HVA channel function in vivo; the compensatory modulation of currents in the KO may contribute to the mild motor phenotype seen in these mice.This material is based upon work supported by the National Science Foundation under Grant No. 1022075

Comparative effects of pentobarbital on spontaneous and evoked transmitter release from inhibitory and excitatory nerve terminals in rat CA3 neurons

Pentobarbital (PB) modulates GABA(A) receptor-mediated postsynaptic responses through various mechanisms, and can directly activate the channel at higher doses. These channels exist both pre- and postsynaptically, and on the soma outside the synapse. PB also inhibits voltage-dependent Na⁺ and Ca²⁺ channels to decrease excitatory synaptic transmission. Just how these different sites of action combine to contribute to the overall effects of PB on inhibitory and excitatory synaptic transmission is less clear. To compare these pre- and postsynaptic actions of PB, we used a 'synaptic bouton' preparation of isolated rat hippocampal CA3 pyramidal neurons where we could measure in single neurons the effects of PB on spontaneous and single bouton evoked GABAergic inhibitory and glutamatergic excitatory postsynaptic currents (sIPSCs, sEPSCs, eIPSCs and eEPSCs), respectively. Low (sedative) concentrations (3-10 μM) of PB increased the frequency and amplitude of sIPSCs and sEPSCs, and also presynaptically increased the amplitude of both eIPSCs and eEPSCs. There was no change in current kinetics at this low concentration. At higher concentrations (30-300 μM), PB decreased the frequency, and increased the amplitude of sIPSCs, and presynaptically decreased the amplitude of eIPSCs. The current decay phase of sIPSCs and eIPSCs was increased. An increase in both frequency and amplitude was seen for sEPSCs, while the eIPSCs was also decreased by a bicuculline-sensitive presynaptic effect. The results confirm the multiple sites of action of PB on inhibitory and excitatory transmission and demonstrate that the most sensitive site of action is on transmitter release, via effects on presynaptic GABA(A) receptors. At low concentrations, however, both glutamate and GABA release is similarly enhanced, making the final effects on neuronal excitability difficult to predict and dependent on the particular systems involved and/or on subtle differences in susceptibility amongst individuals. At higher concentrations, release of both transmitters is decreased, while the postsynaptic effects to increase IPSPs and decrease EPSCs would be expected to both results in reduced neuronal excitability.

N‐type Ca2+ channel regulation by the light chain 1 (LC1) of the microtubule associated protein B (MAP1B)

MAP1B is a protein complex consisting of heavy and light (LC) chains that play an important role in regulating neuronal morphogenesis and function. Recent studies have shown that LC1 interacts with NMDA and HT5 receptors at the postsynapsis regulating their trafficking and surface expression. However, much less is known regarding its role at the presynaptic level where voltage‐gated N‐type Ca2+ channels couple membrane depolarization to neurotransmitter release. Here, using a strategy that combines electrophysiology with biochemical and molecular biology techniques, we investigated whether LC1 interacts with the N‐type channel and whether this interaction has any physiological significance. Hence, using confocal immunofluorescence, co‐localization of these proteins was revealed in hippocampal cells from neonatal mouse, and their interaction was confirmed by pulldown assays using whole brain homogenates. Likewise, heterologous expression of recombinant N‐type channels and LC1 in HEK‐293 cells revealed a significant decrease in whole cell current density without apparent changes in current kinetics, suggesting a diminished number of functional channels in the cell surface. Furthermore, pulldown experiments as well as confocal microscopy showed an interaction between LC1 and the channel in the heterologous system. These results reveal a functional coupling between LC1 and the N‐type channel.This work was supported by Conacyt‐Conicyt binational cooperation program.

Ion Channel Phosphorylopathy: a link between genomic variations and human disease

Voltage gated ion channel (VGIC) regulate several important physiological events including muscle contraction, brain function and secretion. Mutations that inhibit or up-regulate VGIC activities can dramatically interfere with normal organ function and can often lead to unpredictable organ failure, and therefore poor quality of life, or even death. Many genomic variations that change amino acids in cytoplasmic domains of ion channels have been found associated with several diseases however, no mechanism have been proposed to link these mutations to ion channel malfunction. Phosphorylation exerts significant effects on the biophysical mechanisms of ion channels ranging from changes in current kinetics to changes in ion channel trafficking. We have observed that numerous mutations on VGIC associated with diseases can create or disrupt consensus sites for kinases. We call these events “PHOSPHORYLOPATHIES”. We show here that: A mutation on the Cav1.2 associated with Timothy syndrome creates a consensus site for CAMKII and leads to a change in ion channel behavior. Two mutations of the voltage gated potassium channel Kv11.1 associated with the cardiac arrhythmia Long-QT syndrome create or disrupt consensus sites for specific kinases, and therefore affect ion channel behavior. Understanding Phosphorylopathies is crucial in the process of designing an effective pharmacological strategy.

Dual action of a dinoflagellate-derived precursor of Pacific ciguatoxins (P-CTX-4B) on voltage-dependent K + and Na + channels of single myelinated axons

The effects of Pacific ciguatoxin-4B (P-CTX-4B, also named gambiertoxin), extracted from toxic Gambierdiscus dinoflagellates, were assessed on nodal K + and Na + currents of frog myelinated axons, using a conventional voltage-clamp technique. P-CTX-4B decreased, within a few minutes, both K + and Na + currents in a dose-dependent manner, without inducing any marked change in current kinetics. The toxin was more effective in blocking K + than Na + channels. P-CTX-4B shifted the voltage-dependence of Na + conductance by about 14 mV towards more negative membrane potentials. This effect was reversed by increasing Ca 2+ in the external solution. A negative shift of about 16 mV in the steady-state Na + inactivation-voltage curve was also observed in the presence of the toxin. Unmodified and P-CTX-4B-modified Na + currents were similarly affected by the local anaesthetic lidocaine. The decrease of the two currents by lidocaine was dependent on both the concentration and the membrane potential during pre-pulses. In conclusion, P-CTX-4B appears about four times more effective than P-CTX-1B to affect K + channels, whereas it is about 50 times less efficient to affect Na + channels of axonal membranes. These actions may be related to subtle differences between the two chemical structures of molecules.

Regulation of Nav1.7 and Nav1.8 peripheral nervous system sodium channels by auxiliary β subunits

Event Abstract Back to Event Regulation of Nav1.7 and Nav1.8 peripheral nervous system sodium channels by auxiliary β subunits Juan Zhao1, Cojen Ho2, Michael E. O'Leary2 and Mohamed Chahine1* 1 Le Centre de Recherche Universite Laval Robert-Giffard, Canada 2 Jefferson Medical College, United States The Nav1. 7 and Nav1. 8 Na channel isoforms are preferentially expressed in small-diameter sensory neurons of the dorsal root ganglia (DRG) where they are known to play a central role in nociception. The goal of these studies was to investigate the β subunit regulation of the Nav1.7 and Nav1.8 channels. The Na channel α and β subunits were heterologously expressed in HEK 293 cells and the functional properties assessed from whole-cell Na current recordings. Co-expressing the β3 subunit produced a hyperpolarizing shift in Nav1.7 activation (-5 mV) and slowed channel gating. The β1 subunit produced a depolarizing shift (+ 7 mV) in the midpoint of steady-state inactivation but no change in current kinetics. The β2 and β3 subunit did not alter Nav1.7 gating and none of the β subunits produced significant changes in Na current density. Co-expressing the β1 subunit increased the Nav1.8 current density 2-fold and produced hyperpolarizing shifts in both activation (-4 mV) and inactivation (-5 mV). This contrasted with the β3 subunit, which produced a 30% reduction in Nav1.8 current amplitude. The β2 and β3 subunits did not alter the gating or current density of the Nav1.8 channel. Co-expressing the β4 subunit produced hyperpolarizing shifts in the activation (-18 mV) and inactivation (-6 mV) of the Nav1.8 channel. To further investigate the contribution of the auxiliary subunits in vivo the transcripts encoding for β subunits were quantitatively measured in small-diameter sensory neurons isolated from the rat DRG. The β3 subunit is widely expressed in DRG nociceptors where it produces a hyperpolarizing shift in Nav1.7 activation and a reduction in Nav1.8 peak current amplitude. Although the β1 and β4 subunits differentially regulate the Nav1.7 and Nav1.8 channels these subunits are expressed at comparatively low levels in sensory neurons. Conference: 3rd Mediterranean Conference of Neuroscience , Alexandria, Egypt, 13 Dec - 16 Dec, 2009. Presentation Type: Oral Presentation Topic: Neurobiology of pain and depression Citation: Zhao J, Ho C, O'Leary ME and Chahine M (2009). Regulation of Nav1.7 and Nav1.8 peripheral nervous system sodium channels by auxiliary β subunits. Front. Neurosci. Conference Abstract: 3rd Mediterranean Conference of Neuroscience . doi: 10.3389/conf.neuro.01.2009.16.115 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Nov 2009; Published Online: 23 Nov 2009. * Correspondence: Mohamed Chahine, Le Centre de Recherche Universite Laval Robert-Giffard, Beauport, Qc G1J 2G3, Canada, Mohamed.Chahine@phc.ulaval.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Juan Zhao Cojen Ho Michael E O'Leary Mohamed Chahine Google Juan Zhao Cojen Ho Michael E O'Leary Mohamed Chahine Google Scholar Juan Zhao Cojen Ho Michael E O'Leary Mohamed Chahine PubMed Juan Zhao Cojen Ho Michael E O'Leary Mohamed Chahine Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Role of slow delayed rectifier K+‐current in QT prolongation in the alloxan‐induced diabetic rabbit heart

In diabetes mellitus, several cardiac electrophysiological parameters are known to be affected. In rodent experimental diabetes models, changes in these parameters were reported, but only limited relevant information is available in other species, having cardiac electrophysiological properties more resembling the human, including the rabbit. The present study was designed to analyse the effects of experimental type 1 diabetes on ventricular repolarization and the underlying transmembrane potassium currents in rabbit hearts. Diabetes was induced by a single injection of alloxan (145 mg kg(-1) i.v.). After the development of diabetes (3 weeks), electrophysiological studies were performed using whole cell voltage clamp and ECG measurements. The QT(c) interval in diabetic rabbits was moderately but statistically significantly longer than measured in the control animals (155 +/- 1.8 ms vs. 145 +/- 2.8 ms, respectively, n = 9-10, P < 0.05). This QT(c)-lengthening effect of diabetes was accompanied by a significant reduction in the density of the slow delayed rectifier K(+) current, I(Ks) (from 1.48 +/- 0.35 to 0.86 +/- 0.17 pA pF(-1) at +50 mV, n = 19-21, P < 0.05) without changes in current kinetics. No differences were observed either in the density or in the kinetics of the inward rectifier K(+) current (I(K1)), the rapid delayed rectifier K(+) current (I(Kr)), the transient outward current (I(to)) and the L-type calcium current (I(CaL)) between the control and alloxan-treated rabbits. It is concluded that type 1 diabetes mellitus, although only moderately, lengthens ventricular repolarization. Diabetes attenuates the repolarization reserve by decreasing the density of I(Ks) current, and thereby may enhance the risk of sudden cardiac death.

Oxidative modification of M-type K+ channels as a mechanism of cytoprotective neuronal silencing

Voltage-gated K(+) channels of the Kv7 family underlie the neuronal M current that regulates action potential firing. Suppression of M current increases excitability and its enhancement can silence neurons. We here show that three of five Kv7 channels undergo strong enhancement of their activity by oxidative modification induced by physiological concentrations of hydrogen peroxide. A triple cysteine pocket in the channel S2-S3 linker is critical for this effect. Oxidation-induced enhancement of M current produced a hyperpolarization and a dramatic reduction of action potential firing frequency in rat sympathetic neurons. As hydrogen peroxide is robustly produced during hypoxia-induced oxidative stress, we used an oxygen/glucose deprivation neurodegeneration model that showed neuronal death to be severely accelerated by M current blockade. Such blockade had no effect on survival of normoxic neurons. This work describes a novel pathway of M-channel regulation and suggests a role for M channels in protective neuronal silencing during oxidative stress.

Ca2+ Controls Functional Expression of the Cardiac K+ Transient Outward Current via the Calcineurin Pathway

The transient outward K+ current (Ito) modulates transmembrane Ca2+ influx into cardiomyocytes, which, in turn, might act on Ito. Here, we investigated whether Ca2+ modifies functional expression of Ito. Whole-cell Ito were recorded using the patch clamp technique in single right ventricular myocytes isolated from adult rats and incubated for 24 h at 37 degrees C in a serum-free medium containing various Ca2+ concentrations ([Ca2+]o). Increasing the [Ca2+]o from 0.5 to 1.0 and 2.5 mM produced a gradual decrease in Ito density without change in current kinetics. Quantitativereverse transcriptase-PCR showed that a decrease of the Kv4.2 mRNA could account for this decrease. In the acetoxymethyl ester form of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM)-loaded myocytes (a permeant Ca2+ chelator), Ito density increased significantly when cells were exposed for 24 h to either 1 or 2.5 mM [Ca2+]o. Moreover, 24-h exposure to the Ca2+ channel agonist, Bay K8644, in 1 mM [Ca2+]o induced a decrease in Ito density, whereas the Ca2+ channel antagonist, nifedipine, blunted Ito decrease in 2.5 mM [Ca2+]o. The decrease of Ito in 2.5 mM [Ca2+]o was also prevented by co-incubation with either the calmodulin inhibitor W7 or the calcineurin inhibitors FK506 or cyclosporin A. Furthermore, in myocytes incubated for 24 h with 2.5 mM [Ca2+]o, calcineurin activity was significantly increased compared with 1 mM [Ca2+]o. Our data suggest that modulation of [Ca2+]i via L-type Ca2+ channels, which appears to involve the Ca2+/calmodulin-regulated protein phosphatase calcineurin, down-regulates the functional expression of Ito. This effect might be involved in many physiological and pathological modulations of Ito channel expression in cardiac cells, as well other cell types.

Downregulation of voltage-gated sodium channels by dexamethasone in clonal rat pituitary cells

The effect of chronic dexamethasone (DEX) treatment (4–5 days) on Na + channel expression was examined in a clonal strain of rat pituitary cells secreting growth hormone (GH) and prolactin (GH3 cells). Using whole-cell patch clamp recording, we found that DEX (1 μM) induces an 80% decrease in Na + current density. No concomitant changes in current kinetics or voltage dependence of Na + channel function were detected. Instead, the decrease in current density was accompanied by a similar reduction in maximal Na + conductance, suggesting the loss of Na + channels from the plasma membrane. Accordingly, saxitoxin binding assays carried out on intact cells showed that the average number of Na + channels per cell is markedly decreased by DEX. Thus, this glucocorticoid inhibits the cell surface expression of Na + channels when chronically applied to GH3 cells.

Dopamine D4 Receptor Activation Inhibits Presynaptically Glutamatergic Neurotransmission in the Rat Supraoptic Nucleus

Oxytocin and vasopressin release from magnocellular neurons of the supraoptic nucleus is under the control of glutamate-dependent excitation. The supraoptic nucleus also receives a generalized dopaminergic input from hypothalamic sources. To determine if dopamine can influence this excitatory drive onto the magnocellular neurons, we used whole-cell patch clamp to record the effect of dopamine on evoked and miniature excitatory postsynaptic currents in rat hypothalamic slices. Dopamine exposure (30 microM to 1 mM) induced a large and reversible reduction in the amplitude of evoked excitatory postsynaptic current in nearly all magnocellular cells tested. D4 receptors appeared to mediate dopamine's activity, based on inhibition of the response with 50 microM clozapine, but not by SCH 23390 or sulpiride, and mimicry of dopamine's action with the D4 specific agonist, PD 168077. Analysis of paired-pulse experiments and miniature postsynaptic currents indicated that dopamine's action involved a presynaptic mechanism, since the frequency of miniature postsynaptic currents was reduced with dopamine exposure without any change in current kinetics or amplitude, while the paired-pulse ratio increased. We therefore have demonstrated for the first time a role for dopamine D4 receptors in the supraoptic nucleus in the presynaptic inhibition of glutamatergic neurotransmission onto magnocellular neurons.

Block of Voltage-dependent Calcium Channel by the Green Mamba Toxin Calcicludine

A number of peptide toxins derived from marine snails and various spiders have been shown to potently inhibit voltage-dependent calcium channels. Here, we describe the effect of calcicludine, a 60 amino-acid peptide isolated from the venom of the green mamba (Dendroaspis angusticeps), on transiently expressed high voltage-activated calcium channels. Upon application of calcicludine, L-type (alpha(1)(C)) calcium channels underwent a rapid, irreversible decrease in peak current amplitude with no change in current kinetics, or any apparent voltage-dependence. However, even at saturating toxin concentrations, block was always incomplete with a maximum inhibition of 58%, indicating either partial pore block, or an effect on channel gating. Block nonetheless was of high affinity with an IC(50) value of 88 nm. Three other types of high voltage activated channels tested (alpha(1)(A), alpha(1)(B), and alpha(1)(E)) exhibited a diametrically different response to calcicludine. First, the maximal inhibition observed was around 10%, furthermore, the voltage-dependence of channel activation was shifted slightly towards more negative potentials. Thus, at relatively hyperpolarized test potentials, calcicludine actually upregulated current activity of (N-type) alpha(1)(B) channels by as much as 50%. Finally, the use of several chimeric channels combining the major transmembrane domains of alpha(1)(C) and alpha(1)(E) revealed that calcicludine block of L-type calcium channels involves interactions with multiple structural domains. Overall, calcicludine is a potent and selective inhibitor of neuronal L-type channels with a unique mode of action.

Developmental changes in voltage-activated potassium currents of rat retinal ganglion cells

Ca 2+-independent voltage-activated potassium currents were investigated during the differentiation of rat retinal ganglion cells. Whole-cell patch-clamp recordings of Ca 2+-independent voltage-activated potassium currents and their individual current components, i.e. a sustained, tetraethylammonium-sensitive current, a transient, 4-aminopyridine-sensitive current, and a slowly decaying current that was blocked by Ba 2+, revealed distinct ontogenetic modifications in current densities and in activation and inactivation parameters. All three current types were expressed simultaneously at embryonic day 17/18 and were present in all retinal ganglion cells thereafter without showing any significant changes until the end of the first postnatal week. Ca 2+-independent voltage-activated potassium current densities then increased strongly from postnatal day 8 onwards. Tetraethylammonium-sensitive current density increased about eightfold from 74 pA/pF in embryonic stages to 586 pA/pF in adult cells, whereas the transient potassium currents blocked by 4-aminopyridine increased only about 2.5-fold from 174 pA/pF to 442 pA/pF. The Ba 2+-sensitive current increased simultaneously from 35 pA/pF to 332 pA/pF. The much higher increase in the sustained current components during retinal ganglion cell differentiation accounted for the changes in decay kinetics of Ca 2+-independent voltage-activated potassium current observed in later postnatal stages. Alterations in current densities were paralleled by pronounced changes in current kinetics. From postnatal day 8 onwards, activation of Ca 2+-independent voltage-activated potassium current was right-shifted for about 10 mV owing to a shift in tetraethylammonium-sensitive current-activation, whereas activation of other K + components remained unaltered. Tetraethylammonium-sensitive current steady-state inactivation was incomplete at all developmental stages. About 50% of the tetraethylammonium-sensitive current elicited by a depolarization to +36 mV did not inactivate after prepulse potentials positive to −10 mV. In contrast, transient potassium current blocked by 4-aminopyridine almost fully inactivated during embryonic stages, whereas in adult retinal ganglion cells about 40% of this current component did not inactivate after prepulse potentials positive to −20 mV. Parallel investigation of the resting membrane potential during retinal ganglion cells differentiation showed an exponential increase from −3 mV at embryonic day 15/16 when no voltage-activated ion currents were expressed to a final value of −58 mV at postnatal day 8. These results show that fundamental potassium current modifications occur relatively late in retinal ganglion cell development and only after the resting potential is at its final value.

Inhibition of nicotinic acetylcholine response by atropine in frog isolated sympathetic neurons

The effect of atropine on nicotinic acetylcholine (ACh) response was studied in frog isolated sympathetic ganglion cells using a ‘concentration clamp’ technique which combines intra-cellular perfusion with a rapid external solution change within 3 ms. When atropine (more than 6 × 10 −7 M) was simultaneously applied to neurons with ACh (6 × 10 −6 M), the current amplitude was instantaneously reduced in a dose-dependent fashion. Further decreases in the current amplitude were observed in a time-dependent manner during about 20 min after the start of drug-application. Thereafter the current amplitude gradually restored toward the control level in the following 90–120 min in spite of the continuous presence of atropine. However, because d-tubocurarine greatly inhibited the ‘restored’ current, the last-mentioned is suggested be also mediated by nicotinic ACh receptor-ionophore complex. Therefore, the inhibitory action of atropine on the peak amplitude of ACh response was examined at 15–20 min after adding the agent. It was dose-dependent but not voltage-dependent. Respective concentrations of atropine and d-tubocurarine causing half the maximum inhibition (IC 50) of the peak current evoked by ACh (6 × 10 −6 M) were 1.8 × 10 −5 M and 1.8 × 10 −6 M. Thus, the inhibitory potency was 10 times less tha of d-tubocurarine, an antagonist of nicotinic ACh receptors. The blockade of ACh response by d-tubocaririne was competitive while that by atropine was non-competitive. The current elicited by Ach consisted of two (fast and low) exponentiald components plus a steady0state one in the control period. Atropine (3 × 10 −6 M) greatly reduced the amplitude of exponential components but it exerted a small effect on the steady-state one. Therefore, the changes in current kinetics during the application of atropine may be due to a different sensitivity of each component to atropine.