Vitamin E regulates acetylcholine receptor function of molluscan neurons.

1. Intracellular recordings were made from identified LP11, RBc4, D1 and E4 neurons in perioesophageal ganglionic ring with buccal ganglia of the mollusc Helix pomatia. 2. The modulations of acetylcholine (ACh)-induced current by vitamin E in these neurons were investigated using two-microelectrode intracellular recording and voltage-clamp techniques. 3. ACh receptors function on LP11 and RBc4 neurons was strongly regulated by intracellular calcium ions. For these ACh receptors application of 10(-6) to 10(-4) M vitamin E and calcium influx both induced an enhancement of the ACh-induced chloride current. Application of 10(-5) to 5.10(-5) M arachidonic acid on the same identified LP11 and RBc4 neurons was shown to evoke a decrease of the ACh-induced chloride current. 4. The elevation of calcium levels into D1 and E4 neurons induced a faint decrease of ACh-induced chloride current, but vitamin E and arachidonic acid were ineffective. 5. The calmodulin inhibitor, chloropromazine (6.10(-5) M), strongly inhibited the enhancing effect of calcium influx on ACh-induced chloride current in LP11 and RBc4 neurons, but it had a weak influence on the effect of vitamin E. 6. The effect of vitamin E on surface distribution of functional ACh receptors in LP11 and RBc4 neurons was found. 7. Application of 10(-4) to 10(-6) M vitamin E (DL-alpha-tocopherol) triggered mechanisms, which after a 5 to 45-min period lead to appearance of functional ACh receptors on the parts of neuronal soma, which were further from the axon. 8. Arachidonic acid (vitamin F) evoked a disappearance of functional ACh receptors, which were activated by vitamin E.     

Modulatory effects of oxytocin on functional properties of three types of cholinoreceptors in molluskan neurons

It was found that applying 10^–8 M oxytocin (OT) affects the functional properties of three types of cholinoreceptors under conditions of voltage clamping at the membrane of identified ganglia neurons inHelix pomatia. This neuropeptide depressed acetycholine-(ACh-)induced sodium-potassium-calcium current in neuron RB3 without altering reversal potential of ACh-induced current. Two (sub-) types of cholinoreceptors were distinguished on the basis of findings on OT effects on ACh-induced chloride currents; ACh-induced chloride current was reduced by the action of OT on the cholinoreceptors of one of these (neuron F4) and increased in the case of neurons D5 and F86. The effects of applying OT and serotonin were reversible but not cumulative. Injection of OT exerted an action on ACh-induced chloride current independent of that of OT application. Involvement of cyclic adenosine monophosphate in OT-induced bimodal modulation of functional properties of three types of cholinoreceptors was demonstrated.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziology, Vol. 22, No. 1, pp. 87–93, January–February, 1990.     

Vitamin e regulates acetylcholine receptor function of molluscan neurons

1. Intracellular recorclings were made from identified LP11, RBc4, D1 and E4 neurons in perioesophageal ganglionic ring with buccal ganglia of the mollusc Helix pomatia.2. The modulations of acetylcholine (ACh)-induced current by vitamin E in these neurons were investigated using two-microelectrode intracellular recorcling and voltage-clamp techniques.3. ACh receptors function on LP11 and RBc4 neurons was strongly regulated by intracellular calcium ions. For these ACh receptors application of 10⁻⁶ to 10⁻⁴ M vitamin E and calcium influx both induced an enhancement of the ACh-induced chloride current. Application of 10⁻⁵ to 5.10⁻⁵M arachidonic acid on the same identified LP11 and RBc4 neurons was shown to evoke a decrease of the ACh-induced chloride current.4. The elevation of calcium levels into D1 and E4 neurons induced a faint decrease of ACh-induced chloride current, but vitamin E and arachidonic acid were ineffective.5. The calmodulin inhibitor, chloropromazine (6.10^−-5M), strongly inhibited the enhancing effect of calcium influx on ACh-induced chloride current in LP11 and RBc4 neurons, but it had a weak influence on the effect of vitamin E.6. The effect of vitamin E on surface distribution of functional ACh receptors in LP11 and RBc4 neurons was found.7. Application of 10⁻⁴ to 10⁻⁶ M vitamin E (DL-α-tocopherol) triggered mechanisms, which after a 5 to 45-min period lead to appearance of functional ACh receptors on the parts of neuronal soma, which were further from the axon.8. Arachidonic acid (vitamin F) evoked a disappearance of functional ACh receptors, which were activated by vitamin E.     

Modulatory effect of oxytocin and arginine-vasopressin on functional properties of three types of acetylcholine receptors in molluscan neurons.

It was found that 10(-7)-10(-8) mol/l oxytocin (OT) or arginine-vasopressin (AVP) applications produced effects on functional properties of three types of acetylcholine (ACh) receptors on various neurons identified in the ganglia of Helix pomatia under voltage clamp conditions. OT and AVP depressed ACh-induced sodium-potassium-calcium current in neuron RBc3 without shift of reversal potential. Our data show that there are two types (subtypes) of ACh receptors which are connected with chloride current in neurons of Helix pomatia. OT decreased ACh-induced chloride current in neuron D4 and enhanced ACh-induced chloride current in neuron D5. These effects of OT were mimicked by the intracellular injection of cyclic AMP or application of isobutylmethylxanthine and an active cyclic AMP analog. AVP as a rule mimicked the effects of OT on functional properties of ACh receptors, but in neuron F8 effects of OT and AVP were independent. The present results suggest that cyclic AMP may be the second messenger mediating the OT- and AVP-induced modulations of functional properties of three types of ACh-receptors.     

Acetylcholine,GABA and glutamate induce ionic currents in cultured antennal lobe neurons of the honeybee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

The honeybee, Apis mellifera, is a valuable model system for the study of olfactory coding and its learning and memory capabilities. In order to understand the synaptic organisation of olfactory information processing, the transmitter receptors of the antennal lobe need to be characterized. Using whole-cell patch-clamp recordings, we analysed the ligand-gated ionic currents of antennal lobe neurons in primary cell culture. Pressure applications of acetylcholine (ACh), γ-amino butyric acid (GABA) or glutamate induced rapidly activating ionic currents. The ACh-induced current flows through a cation-selective ionotropic receptor with a nicotinic profile. The ACh-induced current is partially blocked by α-bungarotoxin. Epibatidine and imidacloprid are partial agonists. Our data indicate the existence of an ionotropic GABA receptor which is permeable to chloride ions and sensitive to picrotoxin (PTX) and the insecticide fipronil. We also identified the existence of a chloride current activated by pressure applications of glutamate. The glutamate-induced current is sensitive to PTX. Thus, within the honeybee antennal lobe, an excitatory cholinergic transmitter system and two inhibitory networks that use GABA or glutamate as their neurotransmitter were identified.     

Reduction of the acetylcholine-induced K+ current in identifiedAplysia neurons by human interleukin-1 and interleukin-2

1. Effects of bath-applied recombinant human interleukin-1 (rhIL-1) and interleukin-2 (rhIL-2) on the acetylcholine (ACh)-induced K+ current recorded from identified neurons (R9 and R10) of Aplysia kurodai were investigated with voltage-clamp and pressure ejection techniques. 2. Bath-applied rhIL-1 and rhIL-2 (10-40 U/ml) reduced the ACh-induced current in the neurons without affecting the resting membrane conductance and holding current. 3. The suppressing effects of these cytokines on the current were completely reversible. 4. Heat-inactivated rhIL-1 and rhIL-2 were without effect. 5. These results suggest that the immunomodulators, IL-1 and IL-2, can modulate the ACh-induced response in the nervous system.     

Muscarinic inhibition of nicotinic transmission in rat sympathetic neurons and adrenal chromaffin cells

Little is known about the interactions between nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine-induced nAChR currents in sympathetic superior cervical ganglion neurons and adrenal chromaffin cells. The muscarine-induced inhibition (MiI) substantially reduced ACh-induced membrane currents through nAChRs and quantal neurotransmitter release. The MiI was time- and temperature-dependent. The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2), suggested, instead of a direct open-channel blockade, an intracellular metabotropic process. The effects of GTP-γ-S, GDP-β-S and pertussis toxin suggested that MiI was mediated by G-protein signalling. Inhibitors of protein kinase C (bisindolymaleimide–Bis), protein kinase A (H89) and PIP2 depletion attenuated the MiI, indicating that a second messenger pathway is involved in this process. Taken together, these data suggest that mAChRs negatively modulated nAChRs via a G-protein-mediated second messenger pathway. The time dependence suggests that MiI may provide a novel mechanism for post-synaptic adaptation in all cells/neurons and synapses expressing both types of AChRs.     

Synaptic contact between embryonic neurons and acetylcholine receptor-fibroblast

1. Mouse fibroblast cell lines were established that stably express Torpedo californica acetylcholine receptors (AChR) on their cell surface in quantities sufficient for biochemical and pharmacological analyses, as well as electrophysiological analysis at the single channel level. 2. Surface-expressed AChRs were shown to be assembled into proper alpha 2 beta gamma delta pentamers. 3. The distribution of surface-AChRs was uniform and identical in every cell. 4. We were able to successfully coculture AChR-fibroblasts with 1-day old Xenopus laevis embryonal neurons and maintain expression of cell surface AChRs. 5. Using the voltage-clamp technique, miniature end-plate currents were recorded from AChR-fibroblasts which were contacted by neurons. The current amplitudes of these AChRs were approximately 10-fold smaller than those observed in Xenopus myocytes, and the rise-times were slower.     

Single amino acid substitutions in alpha-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor

The alpha-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) antagonists, are emerging as important probes of the role played by different nAChR subtypes in cell function and communication. In this study, the native alpha-conotoxins PnIA and PnIB were found to cause concentration-dependent inhibition of the ACh-induced current in all rat parasympathetic neurons examined, with IC(50) values of 14 and 33 nM, and a maximal reduction in current amplitude of 87% and 71%, respectively. The modified alpha-conotoxin [N11S]PnIA reduced the ACh-induced current with an IC(50) value of 375 nM and a maximally effective concentration caused 91% block. [A10L]PnIA was the most potent inhibitor, reducing the ACh-induced current in approximately 80% of neurons, with an IC(50) value of 1.4 nM and 46% maximal block of the total current. The residual current was not inhibited further by alpha-bungarotoxin, but was further reduced by the alpha-conotoxins PnIA or PnIB, and by mecamylamine. (1)H NMR studies indicate that PnIA, PnIB, and the analogues, [A10L]PnIA and [N11S]PnIA, have identical backbone structures. We propose that positions 10 and 11 of PnIA and PnIB influence potency and determine selectivity among alpha7 and other nAChR subtypes, including alpha3beta2 and alpha3beta4. Four distinct components of the nicotinic ACh-induced current in mammalian parasympathetic neurons have been dissected with these conopeptides.     

A study of the cholinolytic actions of strychnine using the technique of concentration jump relaxation analysis

The blocking actions of strychnine on excitatory acetylcholine (ACh) responses in isolated, voltage clamped Aplysia neuronal cell bodies has been studied using a rapid drug application technique. Rapid microperfusion of strychnine (10-50 microM) produced a reduction of the steady-state ACh-induced inward current in Aplysia neurons which decayed exponentially with a highly dose-dependent time constant. At the cessation of strychnine perfusion the ACh-induced current recovered to its original value with an exponential time course which was not sensitive to the dose of strychnine previously applied. The calculated association (k1) and dissociation (k-1) constants for a pseudo-first-order reaction between strychnine and its binding site were k1 = 1.2 X 10(4) M-1. sec-1 and k-1 = 0.12 sec-1 (KD = 1 X 10(-5) M-1). These results demonstrate that concentration jump relaxation experiments can be performed on isolated neurons for the study of voltage-independent antagonists by the use of rapid microperfusion systems and provide the first direct estimates to date of the rate constants of the cholinolytic effect of strychnine.     

Effects of preganglionic denervation and postganglionic axotomy on acetylcholine receptors in the chick ciliary ganglion

The regulation of nicotinic acetylcholine receptors (AChRs) in chick ciliary ganglia was examined by using a radiolabeled anti-AChR mAb to quantitate the amount of receptor in ganglion detergent extracts after preganglionic denervation or postganglionic axotomy. Surgical transection of the preganglionic input to the ciliary ganglion in newly hatched chicks caused a threefold reduction in the total number of AChRs within 10 d compared with that present in unoperated contralateral control ganglia. Surgical transection of both the choroid and ciliary nerves emerging from the ciliary ganglion in newly hatched chicks to establish postganglionic axotomy led to a nearly 10-fold reduction in AChRs within 5 d compared with unoperated contralateral ganglia. The declines were specific since they could not be accounted for by changes in ganglionic protein or by decreases in neuronal survival or size. Light microscopy revealed no gross morphological differences between neurons in operated and control ganglia. A second membrane component of cholinergic relevance on chick ciliary ganglion neurons is the alpha-bungarotoxin (alpha-Bgt)-binding component. The alpha-Bgt-binding component also declined in number after either postganglionic axotomy or preganglionic denervation, but appeared to do so with a more rapid time course than did ganglionic AChRs. The results imply that cell-cell interactions in vivo specifically regulate both the number of AChRs and the number of alpha-Bgt-binding components in the ganglion. Regulation of these neuronal cholinergic membrane components clearly differs from that previously described for muscle AChRs.     

Nicotinic acetylcholine receptors in health and disease

Nicotinic acetylcholine receptors (AChRs) are a family of acetylcholine-gated cation channels that form the predominant excitatory neurotransmitter receptors on muscles and nerves in the peripheral nervous system. AChRs are also expressed on neurons in lower amounts throughout the central nervous system. AChRs are even being reported on unexpected cell types such as keratinocytes. Structures of these AChRs are being determined with increasing precision, but functions of some orphan subunits are just beginning to be established. Functional roles for postsynaptic AChRs in muscle are well known, but in neurons the post-, peri-, extra-, and presynaptic roles of AChRs are just being revealed. Pathogenic roles of AChRs are being discovered in many diseases involving mechanisms ranging from mutations, to autoimmune responses, to the unknown; involving cell types ranging from muscles, to neurons, to keratinocytes; and involving signs and symptoms ranging from muscle weakness to epilepsy, to neurodegenerative disease, to psychiatric disease, to nicotine addiction. Awareness of AChR involvement in some of these diseases has provoked new interests in development of therapeutic agonists for specific AChR subtypes and the use of expressed cloned AChR subunits as possible immunotherapeutic agents. Highlights of recent developments in these areas will be briefly reviewed.     

Nitric oxide donors inhibit the acetylcholine-induced Cl− current in identified Onchidium neurons

The present study was undertaken to assess the effects of sodium nitroprusside (SNP) and diethylamine NO(C₂H₅)₂N[N(O)NO]⁻Na⁺ (DEA/NO), NO donors, on an acetylcholine (ACh)-induced Cl⁻ current in identified Onchidium neurons using voltage-clamp and pressure ejection techniques. Bath-applied SNP (10 μM) and DEA/NO (5–10 μM) reduced the ACh-induced Cl⁻ current in the neurons without affecting the resting membrane conductance and holding current. The suppressing effects of NO donors were concentration-dependent and completely reversible. Pretreatment with 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (1 μM), a specific inhibitor of NO-stimulated guanylate cyclase, and hemoglobin (50 μM), a nitric oxide scavenger, decreased the SNP-induced inhibition of the ACh-induced current. Intracellular injection of guanosine 3′,5′-cyclic monophosphate (cGMP) or bath-application of 3-isobutyl-1-methylxanthine (50 μM), a non-specific phosphodiesterase inhibitor, inhibited the ACh-induced current, mimicking the effect of NO donors. These results suggest that SNP and DEA/NO inhibit the ACh-induced Cl⁻ current and that this effect is mediated by an increase in intracellular cGMP. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 388–394, 1998     

乙酰胆碱对大鼠丘脑束旁核和中脑网状结构痛反应神经元电活动的影响

在54只大鼠上,用两支微电极同时记录神经元放电的方法,研究了脑室注射乙酰胆碱(ACh)对丘脑束旁核(Pf)和中脑网状结构(RF)痛反应神经元电活动的影响。结果表明,当脑内ACh含量增加时,Pf和RF中两个痛兴奋神经元(PEN)的电活动同时减弱,两个痛抑制神经元(PIN)的电活动同时加强,Pf中一个PEN电活动减弱的同时RF中一个PIN电活动加强,或者相反。阿托品可以阻断ACh的上述作用。这提示,ACh对不同中枢痛反应神经元的电活动的影响是通过M胆碱能受体而实现的。     

Kinetic analysis of acetylcholine-induced chloride current in isolated snail neurons

1. Kinetics of activation and desensitization phases of the acetylcholine (ACh)-induced chloride current (ICI) were studied using isolated single neurons of Japanese land snail and the "concentration clamp" technique. 2. The dose-response curve for the peak ICI gave a dissociation constant of 7.1 x 10(-6) M and a Hill coefficient of 1.8. 3. The current-voltage relationship was linear in the voltage range examined (-60 to +10 mV) and the reversal potential (EACh) was -7.2 +/- 1.5 mV (N = 10). The value was close to the calculated equilibrium potential for chloride ions (ECI). 4. Both activation and desensitization phases of the ACh-induced ICI consisted of a single exponential at concentrations less than 3 x 10(-6) M and a double exponential at higher concentrations. The time constants of both phases decreased with increasing ACh concentrations but showed no potential dependency. 5. The recovery from desensitization of the ICI induced by 5 x 10(-6) M ACh proceeded double exponentially, with time constants of 11 and 114 sec at a holding potential of -30 mV. 6. Noise analysis was performed on a steady-state current induced by 3 x 10(-7) to 2 x 10(-6) M ACh. The mean open time was about 60 msec at 10(-6) M ACh and the single-channel conductance was 14 PS. 7. These results suggest that the ACh receptor-Cl channel complex in snail neurons has two binding sites with the dissociation constant of 7.1 x 10(-6) M and is rapidly activated and desensitized to a steady level in the presence of the agonist.     

Nicotine decreases agrin signaling and acetylcholine receptor clustering in C2C12 myotube culture

The clustering of acetylcholine receptors (AChRs) in skeletal muscle fibers is a critical event in neuromuscular synaptogenesis. AChRs in concert with other molecules form postsynaptic scaffolds in response to agrin released from motor neurons as motor neurons near skeletal muscle fibers in development. Agrin drives an intracellular signaling pathway that precedes AChR clustering and includes the tyrosine phosphorylation of AChRs. In C2C12 myotube culture, agrin application stimulates the agrin signaling pathway and AChR clustering. Previous studies have determined that the frequency of spontaneous AChR clustering is decreased and AChRs are partially inactivated when bound by the acetylcholine agonist nicotine. We hypothesized that nicotine interferes with AChR clustering and consequent postsynaptic scaffold formation. In the present study, C2C12 myoblasts were cultured with growth medium to stimulate proliferation and then differentiation medium to stimulate fusion into myotubes. They were bathed in a physiologically relevant concentration of nicotine and then subject to agrin treatment after myotube formation. Our results demonstrate that nicotine decreases agrin-induced tyrosine phosphorylation of AChRs and decreases the frequency of spontaneous as well as agrin-induced AChR clustering. We conclude that nicotine interferes with postsynaptic scaffold formation by preventing the tyrosine phosphorylation of AChRs, an agrin signaling event that precedes AChR clustering.     

A fungal metabolite asperparaline a strongly and selectively blocks insect nicotinic acetylcholine receptors: the first report on the mode of action

Asperparalines produced by Aspergillus japonicus JV-23 induce paralysis in silkworm (Bombyx mori) larvae, but the target underlying insect toxicity remains unknown. In the present study, we have investigated the actions of asperparaline A on ligand-gated ion channels expressed in cultured larval brain neurons of the silkworm using patch-clamp electrophysiology. Bath-application of asperparaline A (10 μM) had no effect on the membrane current, but when delivered for 1 min prior to co-application with 10 μM acetylcholine (ACh), it blocked completely the ACh-induced current that was sensitive to mecamylamine, a nicotinic acetylcholine receptor (nAChR)-selective antaogonist. In contrast, 10 μM asperparaline A was ineffective on the γ-aminobutyric acid- and L-glutamate-induced responses of the Bombyx larval neurons. The fungal alkaloid showed no-use dependency in blocking the ACh-induced response with distinct affinity for the peak and slowly-desensitizing current amplitudes of the response to 10 μM ACh in terms of IC(50) values of 20.2 and 39.6 nM, respectively. Asperparaline A (100 nM) reduced the maximum neuron response to ACh with a minimal shift in EC(50), suggesting that the alkaloid is non-competitive with ACh. In contrast to showing marked blocking action on the insect nAChRs, it exhibited only a weak blocking action on chicken α3β4, α4β2 and α7 nAChRs expressed in Xenopus laevis oocytes, suggesting a high selectivity for insect over certain vertebrate nAChRs.     

Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions.

To test the hypothesis that agrin mediates motor neuron-induced aggregation of acetylcholine receptors (AChRs) in skeletal muscle fibers and to determine whether the agrin active in this process is released by motor neurons, we raised polyclonal antibodies to purified ray agrin that blocked its receptor aggregating activity. When the antibodies were applied to chick motor neuron--chick myotube cocultures, they inhibited the formation of AChR aggregates at and near neuromuscular contacts, demonstrating that agrin plays a role in the induction of the aggregates. Rat motor neurons, like chick motor neurons, induce AChR aggregates on chick myotubes. This effect was not inhibited by our antibodies, indicating that, although the antibodies inhibited the activity of chick agrin, they did not have a similar effect on rat agrin. We conclude that agrin released by rat motor neurons induced the chick myotubes to aggregate AChRs.     

Further study of the correlation between na-pump activity and membrane chemosensitivity

Using internally dialyzed neurons of Helix, we have examined the effects of sodium-pump activity and intracellular ATP concentration on transmembrane currents induced by acetylcholine (ACh) and gamma-aminobutyric acid (GABA). We also report on the effects of pump activity and levels of intracellular ATP on binding by Helix ganglia of 3H-alpha-bungarotoxin (3H-alpha-BT) and 3H-GABA. Both ouabain-containing and potassium-free solutions depressed the neurotransmitter-induced transmembrane current of one type of dialyzed neurons. An increase in the intracellular ATP concentration led to a depression of ACh-induced currents and to the disappearance of the blocking effect of ouabain on these currents. Intracellular ADP had a similar but smaller effect on transmitter-induced currents, and intracellular AMP was ineffective. The depressing effect of internal ATP on ACh-induced currents was absent in the presence of an inhibitor of membrane phosphorylation (dinitrophenol). The binding of tritium-labeled alpha-BT and GABA to the membranes was depressed by both ouabain-containing and K-free solutions and also by compounds (theophylline and NaF) which increase the levels of intracellular ATP. The results suggest that the Na pump modulates the affinity of ACh and GABA membrane receptors by the regulation of the phosphorylated state of membrane receptors.     

Functional properties of human nicotinic AChRs expressed by IMR-32 neuroblastoma cells resemble those of alpha3beta4 AChRs expressed in permanently transfected HEK cells.

We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human alpha3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express alpha3, alpha5, alpha7, beta2, and beta4 AChR subunits. From these subunits, several types of alpha3 AChRs as well as homomeric alpha7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect alpha3beta4 AChRs. alpha7 AChR function was not detected, yet we estimate that there are 70% as many surface alpha7 AChRs in IMR-32 when compared with alpha3 AChRs. Agonist potencies (EC(50) values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16+/-1 microM) > nicotine (Nic; 48 +/- 7 microM) > or = cytisine (Cyt; 57 +/- 3 microM) = acetylcholine (ACh; 59 +/- 6 microM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s(-1) (300 microM ACh; -60 mV). Assays that used mAbs confirmed the predominance of alpha3- and beta4-containing AChRs in IMR-32 cells. Although 18% of total alpha3 AChRs contained beta2 subunits, no beta2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface alpha3beta2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells. Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC(50) values) for this line was DMPP (14 +/- 1 microM) = Cyt (18 +/- 1 microM) > Nic (56 +/- 15 microM > ACh (79 +/- 8 microM). The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1). These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to alpha3beta4 AChRs.