nAChR Currents Research Articles

L-type calcium channel ligands block nicotine-induced signaling to CREB by inhibiting nicotinic receptors

Nicotinic acetylcholine receptors (nAChRs) are inhibited by several drugs that are commonly thought to be specific for L-type calcium channels (LTCCs). In neurons, LTCCs are activated by nicotine-induced depolarization to engage downstream signaling events; however, the role of LTCC drug interactions with nAChRs in signaling has not been examined in detail. We investigated the effects of LTCC ligands on nAChR currents and downstream signaling in rat superior cervical ganglion (SCG) neurons. We found that 10 μM nicotine and 40 mM K + both reversibly depolarize SCG neurons to −20 mV, sufficient to activate LTCCs and downstream signaling, including induction of nuclear phospho-CREB (pCREB); this induction was blocked by LTCC antagonists. Interestingly, the effects of LTCC antagonists on nicotine-induced signaling to CREB are not mediated by their actions on LTCCs, but rather via inhibition of nAChRs, which prevents nicotine-induced depolarization. We show that this effect is sufficient to block pCREB induction in neurons expressing an antagonist-insensitive LTCC. Taken together, our data show that, at concentrations typically used to block LTCCs, these antagonists inhibit nAChR currents and downstream signaling. These findings serve as a caution in attributing a role for LTCCs when using these drugs experimentally or therapeutically.

Desensitized Nicotinic Receptors That, However, Afford Cytoprotection in Bovine Chromaffin Cells

Neuronal nicotinic receptors for acetylcholine (nAChRs) are among the ionotropic receptors that suffer the most desensitization upon prolonged exposure to their agonists. This is particularly true for the alpha7 subtype of nAChRs, although alpha3beta4 receptors also suffer quick desensitization. This study was planned to test the hypothesis that even after suffering desensitization, a given nAChR might still afford cell protection against a noxious stimulus. Of the many agonists developed for nAChRs, we selected the poorly desensitizing ligand dimethylphenylpiperazinium (DMPP) (Britt and Brenner, 1997) and the highly desensitizing agent epibatidine (EPB) (Marks et al., 1996). We have measured nAChR currents, catecholamine secretory responses, and changes of [Ca2+]c elicited by stimulation of nAChRs with DMPP or EPB. We have also investigated cytoprotection elicited by DMPP and EPB against the cytotoxic effects of veratridine in bovine chromaffin cells.

Antiemetics of the 5-Hydroxytryptamine 3A Antagonist Class Inhibit Muscle Nicotinic Acetylcholine Receptors

Antagonists of the serotonergic 5-hydroxytryptamine 3A receptor (5-HT(3A)R) and muscle nicotinic acetylcholine receptors (nAChR) are widely used in anesthesia practice. Both 5-HT(3A)R and nAChR are ligand-gated ion channels with known pharmacological overlap between some of their agonists and antagonists. We studied the actions of clinically used 5-HT(3A)R antagonist antiemetics and nondepolarizing muscle blockers on ionic currents elicited by the activation of mammalian 5-HT(3A)R and muscle nAChR, expressed in Xenopus laevis oocytes. Currents were recorded using a whole-cell two-electrode voltage clamp technique. Dolasetron, ondansetron, and granisetron reversibly inhibited 5-HT(3A)R function at nanomolar concentrations with 50% inhibitory concentrations (IC(50)) of 11.8, 6.4, and 0.2 nM; the rank order of inhibition correlated well with their clinical antiemetic potencies. The principal metabolite of dolasetron, hydrodolasetron, was 40 times more potent than the parent compound on 5-HT(3A)R (IC(50) = 0.29 nM). The potency of the nondepolarizing muscle blocker d-tubocurarine in blocking 5-HT(3A)R was similar to that of the antiemetics and significantly more than vecuronium and rapacuronium (IC(50) = 11.4 nM, 18.9 microM, 60.5 microM). Conversely, ondansetron, dolasetron, and granisetron also reversibly inhibited nAChR currents in a dose-dependent manner with IC(50)s of 14.2, 7.8, and 4.4 microM for the adult nAChR and 16.0, 18.6, and 13.9 microM for the embryonic nAChR. Again, hydrodolasetron showed significantly (10 times) more inhibitory potency on the adult nAChR than the parent compound dolasetron. These results indicate that drugs that target specific ligand-gated ion channels may also affect other ion channel types.

Nicotinic Acetylcholine Receptors at Glutamate Synapses Facilitate Long-Term Depression or Potentiation

The hippocampus is a center for learning and memory that receives abundant cholinergic innervation and richly expresses nicotinic acetylcholine receptors (nAChRs). Nicotinic mechanisms acting on the hippocampus influence attention, learning, and memory. During Alzheimer's dementia, nAChRs and cholinergic innervation decline in the hippocampus. Using mouse hippocampal slices, we examined the potential diversity of nAChR influences at the Schaffer collateral synapse onto CA1 pyramidal neurons. When nAChR currents were elicited locally at those excitatory synapses, various outcomes were possible depending on the relationship between the nAChR-mediated excitation and mild electrical stimulation. When mild presynaptic stimulation coincided with or preceded nAChR-induced action potentials by 1-5 s, then long-term potentiation was induced. However, if the nAChR-induced action potentials fell within 1 s before the electrical stimulation, then long-term depression resulted. Outside of these time frames, the mismatch of nAChR activity and stimulation led to short-term potentiation. The results indicate that nAChRs may have various influences over excitatory events in the hippocampus. Ongoing nAChR activity likely modulates the impact of glutamate transmission and alters the probabilities for various forms of synaptic plasticity. The fine network of cholinergic fibers running through the hippocampus forms synaptic contacts onto pyramidal cells, granule cells, and interneurons, ensuring continual modulatory influence by nicotinic mechanisms throughout the hippocampal complex. Disruption of events such as those described here may contribute to the deficits associated with the decline of nicotinic cholinergic functions during degenerative diseases such as Alzheimer's dementia.

Loss of functional neuronal nicotinic receptors in dorsal root ganglion neurons in a rat model of neuropathic pain

Recent evidence has suggested that the anti-allodynic effect of neuronal acetylcholine receptor (nAChR) agonists may have a peripheral component [L.E. Rueter, K.L. Kohlhaas, P. Curzon, C.S. Surowy, M.D. Meyer, Peripheral and central sites of action for A-85380 in the spinal nerve ligation model of neuropathic pain, Pain 103 (2003) 269–276]. In further studies of the peripheral anti-allodynic mechanisms of nAChR agonists, we investigated the function of nAChRs in acutely isolated dorsal root ganglion (DRG) neurons from allodynic [L5–L6 spinal nerve ligation (SNL)] and naïve adult rats. Following determination of cell diameter and membrane capacitance, responses to rapid applications of nAChR agonists were recorded under whole cell patch clamp. nAChR inward currents were observed in ∼60% of naïve neurons, across small, medium, and large diameter cells. Evoked nAChR currents could be clustered into three broad classes: fast transient, biphasic, and slow desensitizing currents, consistent with multiple subtypes of nAChR expressed in DRG [J.R. Genzen, W. Van Cleve, D.S. McGehee, Dorsal root ganglion neurons express multiple nicotinic acetylcholine receptor subtypes, J. Neurophysiol. 86 (2001) 1773–1782]. In contrast, in neurons from allodynic animals, the occurrence and amplitude of responses to nAChR agonists were significantly reduced. Reduced responsiveness to nAChR agonists covered the range of DRG neuron sizes. The decrease in the responsiveness to nAChR agonists was not seen in neighboring uninjured L4 neurons. The significant decrease in the number of cells with nAChR agonist responses, compounded with the significant decrease in response amplitude, indicates that there is a marked down regulation of functional nAChRs in DRG somata associated with SNL.

Volatile organic compounds inhibit human and rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes

The relative sensitivity of rats and humans to volatile organic compounds (VOCs) such as toluene (TOL) and perchloroethylene (PERC) is unknown and adds to uncertainty in assessing risks for human exposures to VOCs. Recent studies have suggested that ion channels, including nicotinic acetylcholine receptors (nAChRs), are targets of TOL effects. However, studies comparing TOL effects on human and rat ligand-gated ion channels have not been conducted. To examine potential toxicodynamic differences between these species, the sensitivity of human and rat nAChRs to TOL was assessed. Since PERC has similar effects, in vivo, to TOL, effects of PERC on nAChR function were also examined. Two-electrode voltage-clamp techniques were utilized to measure acetylcholine-induced currents in neuronal nAChRs (α4β2, α3β2, and α7) expressed in Xenopus oocytes. PERC (0.065 mM) inhibited α7 nAChR currents by 60.1 ± 4.0% (human, n = 7) and 40 ± 3.5% (rat, n = 5), and inhibited α4β2 nAChR currents by 42.0 ± 5.2% (human, n = 6) and 52.2 ± 5.5% (rat, n = 8). Likewise, α3β2 nAChRs were significantly inhibited by 62.2 ± 3.8% (human, n = 7) and 62.4 ± 4.3% (rat, n = 8) in the presence of 0.065 mM PERC. TOL also inhibited both rat and human α7, α4β2, and α3β2 nAChRs. Statistical analysis indicated that although there was not a species (human vs. rat) difference with PERC (0.0015–0.065 mM) or TOL (0.03–0.9 mM) inhibition of α7, α4β2, or α3β2 nAChRs, all receptor types were more sensitive to PERC than TOL. These results demonstrate that human and rat nACh receptors represent a sensitive target for VOCs. This toxicodynamic information will help decrease the uncertainty associated with animal to human extrapolations in the risk assessment of VOCs.

The allosteric potentiation of nicotinic acetylcholine receptors by galantamine is transduced into cellular responses in neurons: Ca2+ signals and neurotransmitter release.

Neuronal nicotinic acetylcholine receptors (nAChR) modulate a variety of cellular responses, including Ca2+ signals and neurotransmitter release, which can influence neuronal processes such as synaptic efficacy and neuroprotection. In addition to receptor activation through the agonist binding site, an allosteric modulation of nAChR has also been described for a novel class of allosteric ligands. Of these, the acetylcholinesterase inhibitor and Alzheimer drug galantamine represents the prototypical allosteric ligand, based on its potentiation of nAChR-evoked single-channel and whole-cell currents. The aim of this study was to establish whether the allosteric potentiation of nAChR currents is transduced in downstream cellular responses to nAChR activation, namely increases in intracellular Ca2+ and [3H]noradrenaline release. In SH-SY5Y cells, galantamine potentiated nicotine-evoked increases in intracellular Ca2+ and [3H]noradrenaline release with a bell-shaped concentration-response profile; maximum enhancement of nicotine-evoked responses occurred at 1 muM galantamine. This potentiation was blocked by mecamylamine, whereas galantamine had no effect on these measures in the absence of nicotine. Galantamine did not compete for radioligand binding to the agonist binding sites of several nAChR subtypes, consistent with an allosteric mode of action. Unlike galantamine, the acetylcholinesterase inhibitors rivastigmine and donepezil did not potentiate nAChR-mediated responses, whereas donepezil was a reasonably potent inhibitor of nicotine- and KCl-evoked increases in Ca2+. nAChR-mediated [3H]noradrenaline release from hippocampal slices was also potentiated by galantamine, with an additional component attributable to acetylcholinesterase inhibition and subsequent increase in acetylcholine. These results indicate that the allosteric regulation of nAChR results in the potentiation of receptor-dependent cellular processes relevant to many of the physiological consequences of nAChR activation.

Differential Desensitization and Distribution of Nicotinic Acetylcholine Receptor Subtypes in Midbrain Dopamine Areas

Although many psychopharmacological factors contribute to nicotine addiction, midbrain dopaminergic systems have received much attention because of their roles in reinforcement and associative learning. It is generally thought that the mesocorticolimbic dopaminergic system is important for the acquisition of behaviors that are reinforced by the salient drives of the environment or by the inappropriate stimuli of addictive drugs. Nicotine, as obtained from tobacco, can activate nicotinic acetylcholine receptors (nAChRs) and excite midbrain neurons of the mesocorticolimbic system. Using midbrain slices from rats, wild-type mice, and genetically engineered mice, we have found differences in the nAChR currents from the ventral tegmental area (VTA) and the substantia nigra compacta (SNc). Nicotinic AChRs containing the alpha7 subunit (alpha7* nAChRs) have a low expression density. Electrophysiological analysis of nAChR currents, autoradiography of [125I]-alpha-bungarotoxin binding, and in situ hybridization revealed that alpha7* nAChRs are more highly expressed in the VTA than the SNc. In contrast, beta2* nAChRs are move evenly distributed at a higher density in both the VTA and SNc. At the concentration of nicotine obtained by tobacco smokers, the slow components of current (mainly mediated by beta2* nAChRs) become essentially desensitized. However, the minority alpha7* component of the current in the VTA/SNc is not significantly desensitized by nicotine in the range < or =100 nm. These results suggest that nicotine, as obtained from tobacco, can have multiple effects on the midbrain areas by differentially influencing dopamine neurons of the VTA and SNc and differentially desensitizing alpha7* and non-alpha7 nAChRs.

Memantine inhibits efferent cholinergic transmission in the cochlea by blocking nicotinic acetylcholine receptors of outer hair cells.

Memantine is a blocker of Ca(2+)-permeable glutamate and nicotinic acetylcholine receptors (nAChR). We investigated the action of memantine on cholinergic synaptic transmission at cochlear outer hair cells (OHCs). At this inhibitory synapse, hyperpolarization of the postsynaptic cell results from opening of SK-type Ca(2+)-activated K(+) channels via a highly Ca(2+)-permeable nAChR containing the alpha 9 subunit. We show that inhibitory postsynaptic currents recorded from OHCs were reversibly blocked by memantine with an IC(50) value of 16 microM. RT-PCR revealed that a newly cloned nAChR subunit, alpha 10, is expressed in OHCs. In contrast to homomeric expression, coexpression of alpha 9 and alpha 10 subunits in Xenopus laevis oocytes resulted in robust acetylcholine-induced currents, indicating that the OHC nAChR may be an alpha 9/alpha 10 heteromer. Accordingly, nAChR currents evoked by application of the ligand to OHCs and currents through alpha 9/alpha 10 were blocked by memantine with a similar IC(50) value of about 1 microM. Memantine block of alpha 9/alpha 10 was moderately voltage dependent. The lower efficacy of memantine for inhibition of inhibitory postsynaptic currents (IPSCs) most probably results from a blocking rate that is slow with respect to the short open time of the receptor channels during an IPSC. Thus, synaptic transmission in OHCs is inhibited by memantine block of Ca(2+) influx through nAChRs. Importantly, prolonged receptor activation and consequently massive Ca(2+) influx, as might occur under pathological conditions, is blocked at low micromolar concentrations, whereas the fast IPSCs initiated by short receptor activation are only blocked at concentrations above 10 microM.

Weakening of ion-channel interactions of Na+ and Li+ in acetylcholine-receptor channels of frog skeletal muscle with an increase in agonist concentration.

The possibility that increases in agonist concentration beyond threshold levels may force changes in the character of high-conductance open states of skeletal muscle nicotinic acetylcholine receptor channels (nAChR) was examined by seeing whether differences in several critical ionic properties of nAChR currents could be detected with changes in agonist level. Single- and bi-ionic whole-cell currents of Na+ and Li+ in voltage-clamped frog (Rana pipiens) muscle fibers were measured during local superfusion of endplates with carbamylcholine (carb) at concentrations of 54 microm (low-carb) and 270 microM (high-carb). Three ionic properties that would be affected by changes in the open-state configuration of channel subunits were tested. First, ion-saturation characteristics. Peak Na+ and Li+ currents in low-carb trials showed sublinear dependence on ion concentrations from 0 to 60 mM with Km values of 78 (Na+) and 49 (Li+) mM and a power function slope of 0. 75 on double-log plot. In contrast, the concentration dependence of Na+ and Li+ currents in high-carb tests was linear through the origin with a power function slope of 1.02. Second, Na+/Li+ selectivity. The ratio of peak Na+ and Li+ currents in low-carb tests varied from 1.86 to 2.28 for ion concentrations of from 20 to 60 mM [mean = 2.02 +/- 0.06 (SEM)] whereas the ratio for high-carb trials ranged from only 1.29 to 1.52 [mean = 1.42 +/- 0.40 (SEM)]. Third, competitive interactions of Na+ and Li+ currents. Equimolar mixtures of Na+ and Li+ in low-carb tests produced bi-ionic inward currents which were never larger than the single-ion Na+ current alone, but bi-ionic currents at the high-carb level were always greater than the single-ion Na+ current, approximating the sum of the single-ion Na+ and Li+ currents in most cases. The results are consistent with a decrease in ion-channel binding at the high-carb level and support the possibility of agonist-induced changes in the high-conductance open-state configuration of nAChR subunits which result in a weakening of constraints on cation movements through the channel.

Two ion currents activated by acetylcholine in the ARC muscle of Aplysia.

1. This work continues our examination of the electrophysiology and contractions of single fibers dissociated from a widely studied molluscan muscle, the accessory radula closer (ARC) muscle of Aplysia californica, aimed at understanding its excitation-contraction mechanisms and their modulation. 2. Extensive previous work has characterized a number of basal ion currents present in the fibers and effects of transmitters and peptide cotransmitters that modulate ARC-muscle contractions in vivo. Here we use current clamp, voltage clamp, and contraction measurements to examine the actions of acetylcholine (ACh), the transmitter that induces the contractions. 3. As in the whole ARC muscle, ACh depolarizes unclamped fibers maximally to about -25 mV where, no matter how much ACh is applied, the depolarization saturates. 4. The underlying ACh-activated current is in fact the sum of two quite distinct components, IACh,cat and IACh,Cl. 5. IACh,cat is itself a mixed current carried by cations (physiologically mainly by Na+, but to a significant degree also by Ca2+), reverses near +20 mV, rectifies inwardly, exhibits prominent voltage-dependent kinetics of activation with hyperpolarization, and is selectively blocked by hexamethonium. 6. In contrast, IACh,Cl is carried by Cl-, reverses near -60 mV, exhibits little rectification or voltage-dependent kinetics, is activated selectively by suberyldicholine, and is blocked by alpha-bungarotoxin. 7. Both currents activate fast when ACh is applied, desensitize relatively slowly in its presence, then deactivate fast. Both currents are activated at similar ACh concentrations, half-maximally at approximately 10 microM. Both currents also are activated by carbachol and propionylcholine and blocked by d-tubocurarine, bicuculline and paraoxon. Picrotoxin and atropine block IACh,cat better, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene 9-carboxylic acid IACh,Cl better. 8. The two currents are virtually identical to ACh-activated cationic (Na) and Cl currents that are ubiquitous in molluscan neurons. As has been proposed for the neuronal currents, IACh,cat resembles vertebrate neuronal nicotinic ACh-receptor (nAChR) currents, whereas IACh,Cl resembles vertebrate skeletal muscle nAChR currents. 9. Functionally, we believe that IACh,cat serves primarily to depolarize the ARC muscle to open voltage-activated L-type Ca channels, allow Ca2+ influx, and initiate contraction. Physiologically significant Ca2+ may also enter through the ACh,cat channels themselves. 10. By superimposing on IACh,cat, IACh,Cl brings the reversal potential of the combined current to around -25 mV and thereby sets a relatively negative upper limit to the ACh-induced depolarization. We propose that this is its physiological role. By limiting the depolarization, IACh,Cl limits the degree of activation of the Ca current and Ca2+ influx, and so prevents excessive contraction. More importantly, it moderates the voltage during contraction to a range where small voltage changes can finely grade contraction amplitude in this nonspiking muscle. 11. Indeed, in contraction experiments on the single fibers, there is an inverse correlation between the IACh,Cl/IACh,cat ratio and the magnitude of the ACh-induced depolarization and contraction. Furthermore, increased pharmacological activation of IACh,Cl depresses, and block of IACh,Cl enhances, both the depolarization and contraction. 12. Obligatory simultaneous coactivation of IACh,cat and IACh,Cl in the ARC muscle may be part of a peripheral control mechanism that automatically keeps the size of its contractions within behaviorally optimal limits.

Interaction of Na+ and Mg2+ ions in acetylcholine receptor channels of frog skeletal muscle changes in character with an increase in agonist concentration.

Measurements of agonist-induced single-channel currents of nicotinic acetylcholine receptor channels (nAChR) have shown that addition of divalent metal cations (Ca2+, Mg2+, Ba2+) in millimolar amounts causes a decrease of 40% or more in currents carried by monovalent cations (Na+, Cs+). Conventional voltage-clamp measurements were made at frog (Rana pipiens) sartorius endplates to determine if this occlusive interaction between mono- and divalent cations is preserved in macroscopic currents produced by higher concentrations of nAChR agonists, as would be expected on the basis of simple summation of unitary currents. Single ion nAChR currents of Na+ (INa+ ) and Mg2+(IMg2+ ) and the composite current when both cations were present (INa+, Mg2+ ) were measured in high-K+, low ionic strength bathing media during local superfusion with various concentrations of carbamylcholine. It was found that, at lower test carbamylcholine levels of 27 and 54 microM, peak values of INa+, Mg2+ were 40% and 53% of the respective INa+ , in agreement with the effects observed from single-channel measurements. At all higher carbamylcholine levels, however, peak INa+, Mg2+ exceeded INa+ and increased instead with the sum of INa+ and IMg2+ as if there was an independent movement of these ions through the channels rather than the occlusive interaction found at lower carbamylcholine levels. This suggests that with exposure to increasing agonist concentrations above those commonly used in single-channel measurements there are changes in the open state of nAChR affecting cation selectivity possibly in the narrow pore region of the channels.

Acetylcholine receptor desensitization induced by nicotine in rat medial habenula neurons.

1. The activation and desensitization properties of nicotinic acetylcholine receptor (nAChR) channels were examined in acutely isolated medial habenula (MHb) neurons using whole cell patch-clamp recordings. nAChR-mediated currents were evoked by applying known concentrations of nicotinic agonists using rapid solution exchange techniques. 2. At a membrane potential of -60 mV, nAChR currents were observed above a concentration of approximately 100 mM nicotine. The peak current amplitude at low doses of agonist was proportional to the square of the concentration of nicotine, indicating that at least two molecules of agonist were required for channel opening. The concentration of nicotine required for half-maximal nAChR activation was estimated as 77 microM from a complete concentration-response curve. 3. During the continuous activation (2-5 s) of nAChRs by high concentrations of nicotine (300 microM), the current desensitized rapidly and extensively. The desensitization phase was described by the sum of two exponentials, with time constants of 210 and 1,435 ms. The fast component comprised 74% of the desensitizing phase of the current. Recovery from desensitization induced by 2- s applications of 300 microM nicotine was also fast and could be reasonably well described by a single exponential with a time constant of approximately 800 ms. Both the time courses of desensitization and recovery from desensitization were slightly slower at positive membrane potentials. 4. Incubation of neurons with low concentrations of nicotine (100 nM-10 microM) caused a slowly developing but pronounced desensitization of the nAChRs. In these cases desensitization was assessed from the reduction in the amplitude of the peak nicotinic current induced by repetitively applied pulses of a higher test concentration of agonist. A 5-min continuous exposure to 1 microM nicotine reduced the amplitude of the acetylcholine (30 microM, 1 s) test response to < 30% of its control value. As with higher concentrations of nicotine, the onset of the desensitization induced by 1 microM nicotine was biexponential, with fast and slow time constants of 15 s and 1.74 min, respectively. Recovery from the desensitization induced by these longer applications of nicotine was much slower than that observed with the brief pulses of high concentrations of nicotine. The concentration required for half-maximal desensitization after a 5-min incubation was approximately 300 nM. 5. Peak nAChR currents were approximately 85% smaller at +40 mV compared with -40 mV. The receptors that do not open at positive potentials desensitize almost as well as they would at negative potentials after channel opening.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxantel-activated single channel currents in the muscle membrane ofAscaris suum

The patch clamp technique was used to investigate the action of the anthelmintic drug, oxantel, on nicotinic acetylcholine receptor (nAChR) currents recorded from vesicles of the somatic muscle cells of the nematode parasite Ascaris suum. The amplitudes of the currents were analysed at different membrane potentials to determine the single channel conductance. Also the open and closed durations were measured to determine the kinetic properties of the activated channel. Oxantel activated single nAChR currents throughout a concentration range 10-100 microM, these currents were not observed with oxantel-free pipette solutions. The mean open time of the activated channels at a membrane potential of -75 mV and a concentration of 10 microM was 1.34 ms. At higher concentrations the open times were shorter and voltage sensitive, decreasing in duration on hyperpolarization, thus suggesting open channel block. The kinetics were analysed using a simple channel block model. The forward block rate, K + B, increased with increasing oxantel concentration but showed little increase as the membrane was hyperpolarized. K + B was 2.41 x 10(7) M-1 s-1 at -50 mV and 2.64 x 10(7) M-1 s-1 at -100 mV. The unblocking rate constant, K-B, did exhibit voltage sensitivity being 443.6 s-1 at -50 mV and 86.8 s-1 at -100 mV. Thus the blocking dissociation constant KB (= K-B/K + B) was 18.5 microM at -50 mV and 3.3 microM at -100 mV. The simple channel block scheme was found to be insufficient to explain fully the observations made; reasons for this are discussed.

Inhibition of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells by Y3-type neuropeptide Y receptors via the adenylate cyclase/protein kinase A system.

The effect of neuropeptide Y [NPY(1-36)] and related peptides on the voltage-dependent currents and the nicotinic acetylcholine receptor (nAChR) currents (IACh) of bovine adrenal chromaffin cells was investigated using the whole-cell patch clamp technique. Catecholamine release from single chromaffin cells was measured by means of fast cyclic voltammetry. The potency order of these peptides in inhibiting IACh evoked by nicotine was NPY(1-36), NPY (16-36) > peptide YY(PYY) > [Leu31, Pro34]NPY. NPY(16-36) produced a similar degree of inhibition, irrespective of whether nicotine or an equipotent concentration of acetylcholine was used to evoke IACh. NPY(16-36) failed to alter voltage-dependent inward or outward currents. Intracellular cAMP, and extracellular dibutyryl-cAMP, produced a slowly developing increase in IACh. Intracellular cAMP, extracellular 8-Br-cAMP or dibutyryl-cAMP, and an inhibitor of cyclic nucleotide phosphodiesterases 3-isobutyl-1-methyl-xanthine (IBMX), decreased the inhibitory effect of NPY(16-36) on IACh. Although the intracellular application of the cAMP-dependent protein kinase A inhibitor [PKI(14-24)amide] alone did not alter IACh, it potentiated the effect of NPY(16-36) in interaction experiments. While the NPY(16-36)-induced inhibition of IACh was reversed on washout of the peptide, the slightly shorter C-terminal fragment NPY(18-36) caused a long-lasting depression of both IACh and catecholamine secretion evoked by nicotine. This depression was smaller in the presence of extracellular 8-Br-cAMP than in its absence. NPY(18-36) did not alter the secretory activity induced by a high concentration of potassium. It appears that, by activating Y3-receptors, NPY inhibits nAChR-current and the resulting secretion of catecholamines from bovine chromaffin cells. This process may involve a G protein-mediated decrease in intracellular cAMP with a subsequent decrease in the degree of phosphorylation of the nAChR-channel.

Alpha-Bungarotoxin-sensitive hippocampal nicotinic receptor channel has a high calcium permeability

The hippocampal nicotinic acetylcholine receptor (nAChR) is a newly identified ligand-gated ion channel that is blocked by the snake toxin alpha-bungarotoxin (alpha-BGT) and that probably contains the alpha 7 nAChR subunit in its structure. Here its ion selectivity was characterized and compared with that of the N-methyl-D-aspartate (NMDA) receptor channel. The reversal potentials (VR) of acetylcholine- and NMDA-activated whole-cell currents were determined under various ionic conditions. Using ion activities and a Goldman-Hodgkin-Katz equation for VR shifts in the presence of Ca2+, permeability ratios were calculated. For the alpha-BGT-sensitive nAChR, PNa/PCs was close to 1 and Cl- did not contribute to the currents. Changing the [Ca2+]0 from 1 to 10 mM, the VRs of the nAChR and NMDA currents were shifted by +5.6 +/- 0.4 and +8.3 +/- 0.4 mV, respectively, and the nAChR current decay was accelerated. These shifts yielded PCa/PCss of 6.1 +/- 0.5 for the nAChR channel and 10.3 +/- 0.7 for the NMDA channel. Thus, the neuronal alpha-BGT-sensitive nAChR is a cation channel considerably selective to Ca2+ and may mediate a fast rise in intracellular Ca2+ that would increase in magnitude with membrane hyperpolarization.

Clinical concentrations of edrophonium enhance desensitization of the nicotinic acetylcholine receptor.

The principal acetylcholinesterase inhibitors used in clinical practice, edrophonium, neostigmine, and pyridostigmine, differ in their abilities to reverse profound neuromuscular block. This difference may reflect differential inhibition of the nicotinic acetylcholine receptor (nAChR) itself. To investigate this possibility, we studied the effects of these drugs on the function of nAChR (alpha 2 beta gamma delta subtype expressed in Xenopus laevis oocytes) using a whole-cell voltage clamp technique. All three drugs produced concentration-dependent inhibition of nAChR currents induced by the nicotinic agonist dimethylphenyl piperazinium iodide (DMPP). However, only with edrophonium did the effective inhibitory concentration overlap with the clinical range, producing 47% inhibition of nAChR current at the peak serum concentration (60 microM) obtained from a 1 mg/kg dose. The inhibition by edrophonium was voltage-dependent, being more potent at hyperpolarized membrane potentials [IC50(-60 mV) = 82.1 +/- 5.0 microM; IC50(-90 mV) = 50.8 +/- 2.7 microM; IC50(-120 mV) = 41.1 +/- 1.3 microM] and implying some degree of channel block within the ion-conducting pore. Edrophonium also enhanced desensitization of the nAChR within the clinically observed range. Edrophonium desensitization of the nAChR was further increased by simultaneous exposure to other drugs known to promote desensitization of the receptor. These two mechanisms, channel block and enhanced desensitization, may provide molecular explanations for the lesser capacity of edrophonium to promote complete reversal of profound neuromuscular block.

Plasma from patients with seronegative myasthenia gravis inhibit nAChR responses in the TE671/RD cell line.

Myasthenia gravis (MG) is an autoimmune disorder in which anti-acetylcholine receptor (AChR) antibodies cause muscle weakness. In 10-15% of MG patients anti-AChR antibodies are undetectable (seronegative MG, SMG), though clinical and experimental evidence points to causative circulating factors. Using whole-cell patch-clamp techniques, we investigated the effects of heat-inactivated plasma from SMG patients (n = 7) on voltage-gated sodium [INa(V)] and ACh-induced nicotinic AChR (nAChR) currents in the human rhabdomyosarcoma cell line TE671/RD, comparing the results to those obtained with plasma from healthy individuals (HC, n = 6), patients with Guillain-Barré syndrome (GBS, n = 3) or those with other neurological diseases (OND, n = 3). None of the plasma samples inhibited INa(V). nAChR currents were rapidly (< 1 min) and significantly (P < 0.01) reduced by a 1:10 dilution of plasma from SMG patients compared with plasma from healthy controls and were not restored by washing. The inhibition appeared in some cases to be calcium dependent since for one of three plasmas it was prevented by 10 mM EGTA in the patch pipette. Currents were also reduced by two of three plasmas obtained from GBS patients at 1:3 dilution, but not by the three plasmas from patients with ONDs. The rapid action of plasma from SMG patients argues against an antibody-induced reduction in nAChR numbers; its calcium dependence in one case suggests action by a second messenger that might involve nAChR phosphorylation.

The expression of nicotinic acetylcholine receptors by PC12 cells treated with NGF

The expression of neuronal nicotinic ACh receptors (nAChRs) and the subunits that compose these receptors by PC12 cells exposed to NGF has been studied. The analysis of total RNA reveals that the neuronal nAChR subunits alpha 3, alpha S, beta 2, beta 3, and beta 4, but not alpha 2 and alpha 4, are expressed in our PC12 cells. Within 48 hr of adding NGF to cultures, the RNA corresponding to alpha 3, alpha 5, beta 3, and beta 4 is decreased, but the beta 2 RNA increases for up to 6 d after NGF treatment. To determine the influence of NGF treatment on subunit protein expression, subunit-specific antisera were prepared. Immunocytochemistry detected antigen for alpha 3, alpha 5, beta 2, beta 3, and beta 4 (but not alpha 2 and alpha 4) in both NGF-treated and nontreated PC12 cells. The expression of nAChR subunit proteins, as measured by direct binding of antibodies to PC12 cells, does not change subsequent to 6 d of treatment with NGF. Whole-cell recording of PC12 cells shows that both the individual cell current density and response to the agonist cytisine were not altered after 5-7 d in NGF. However, the number of cells exhibiting detectable ACh-induced currents doubled. These results indicate that NGF increases the number of PC12 cells expressing ACh-sensitive nAChR currents but the activation is not the result of altering the amounts of individual nAChR subunit proteins. These data, taken together with the decrease in most nAChR subunit RNAs (except beta 2), suggest that NGF regulation of nAChRs may be through a posttranscriptional mechanism.

Neuronal nicotinic acetylcholine receptor currents in phaeochromocytoma (PC12) cells: dual mechanisms of rectification.

1. Neuronal nicotinic acetylcholine receptor (nAChR) currents in PC12 cells were studied using single-channel and whole-cell gigaohm seal voltage-clamp techniques. Nicotinic AChR agonists were applied using external pipettes. 2. The single-channel conductance of neuronal nAChRs in outside-out patches under Mg(2+)-free ionic conditions was 48 pS. In the absence of internal Mg2+ single-channel currents (outside-out patches) did not rectify. 3. Whole-cell nAChR currents recorded with normal internal solution (lacking Mg2+ chelators as described below) were strongly inwardly rectifying. Current vs. voltage relations showed a sharp inflexion near 0 mV, and outward current was never observed. 4. Extensive dialysis with internal solution containing EDTA and Na2-ATP, chelators of intracellular Mg2+ (Mgi2+), relieved rectification of instantaneous nAChR currents during voltage jumps from negative potentials. The instantaneous I-V relation became linear with voltage, in agreement with the expectation from single-channel measurements made under similar ionic conditions. 5. Agonist-induced currents recorded under Mgi(2+)-free conditions relaxed towards zero current during depolarizing voltage steps. Rectification of ACh-induced currents at the steady state could be described by a Boltzmann relation, with one-half of channels available at +3.4 mV and an effective gating charge of -0.97. Channel availability was approximately 95% at the resting potential. Opening and closing relaxations under Mgi(2+)-free conditions could be fitted by single exponential functions whose time constants were weakly voltage dependent. 6. A model of rectification was constructed incorporating two voltage-dependent processes: block by Mgi2+ and intrinsic channel gating. The I-V relations predicted by this model for both normal and Mgi(2+)-free conditions were in good agreement with the experimental data. We suggest that rectification of nAChR currents in these cells is due to concurrent activity of these two voltage-dependent processes. The relative contributions of these two mechanisms are frequency dependent, with block by Mgi2+ dominant for fast events and intrinsic channel gating more important at the steady state.