Desensitization Of Nicotinic Receptors Research Articles

Activation and Desensitization of Nicotinic α7-type Acetylcholine Receptors by Benzylidene Anabaseines and Nicotine

Nicotinic receptor activation is inextricably linked to desensitization. This duality affects our ability to develop useful therapeutics targeting nicotinic acetylcholine receptor (nAChR). Nicotine and some alpha7-selective experimental partial agonists produce a transient activation of alpha7 receptors followed by a period of prolonged residual inhibition or desensitization (RID). The object of the present study was to determine whether RID was primarily due to prolonged desensitization or due to channel block. To make this determination, we used agents that varied significantly in their production of RID and two alpha7-selective positive allosteric modulators (PAMs): 5-hydroxyindole (5HI), a type 1 PAM that does not prevent desensitization; and 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596), a type 2 PAM that reactivates desensitized receptors. The RID-producing compounds nicotine and 3-(2,4-dimethoxybenzylidene)anabaseine (diMeOBA) could obscure the potentiating effects of 5HI. However, through the use of nicotine, diMeOBA, and the RID-negative compound 3-(2,4-dihydroxybenzylidene)anabaseine (diOHBA) in combination with PNU-120596, we confirmed that diMeOBA produces short-lived channel block of alpha7 but that RID is because of the induction of a desensitized state that is stable in the absence of PNU-120596 and activated in the presence of PNU-120596. In contrast, diOHBA produced channel block but only readily reversible desensitization, whereas nicotine produced desensitization that could be converted into activation by PNU-120596 but no demonstrable channel block. Steady-state currents through receptors that would otherwise be desensitized could also be produced by the application of PNU-120596 in the presence of a physiologically relevant concentration of choline (60 microM), which may be significant for the therapeutic development of type 2 PAMs.

Desensitization of Nicotinic Acetylcholine Receptors as a Strategy for Drug Development

The specific pharmacological response evoked by a nicotinic acetylcholine receptor (nAChR) agonist is governed by the anatomical distribution and expression of each receptor subtype and by the stoichiometry of subunits comprising each subtype. Contributing to this complexity is the ability of agonists that bind to the orthosteric site of the receptor to alter the affinity state of the receptor and induce desensitization and the observation that, at low doses, some nAChR antagonists evoke agonist-like nicotinic responses. Brain concentrations of nicotine rarely increase to the low-mid micromolar concentrations that have been reported to evoke direct agonist-like responses, such as calcium influx or neurotransmitter release. Low microgram per kilogram doses of nicotine administered to humans or to nonhuman primates to improve cognition and working memory probably result only in low nanomolar brain concentrations--more in line with the ability of nicotine to induce receptor desensitization. Here we review data illustrating that nicotine, its major metabolite cotinine, and two novel analogs of choline, JWB1-84-1 [2-(4-(pyridin-3-ylmethyl)piperazin-1-yl)ethanol] and JAY2-22-33, JWB1-84-1 [2-(methyl(pyridine-3-ylmethyl)amino)-ethanol], improve working memory in macaques. The effectiveness of these four compounds in the task was linearly related to their effectiveness in producing desensitization of the pressor response to ganglionic stimulation evoked by a nAChR agonist in rats. Only nicotine evoked an agonist-like action (increased resting blood pressure). Therefore, it is possible to develop new chemical entities that have the ability to desensitize nAChRs without an antecedent agonist action. Because these "silent desensitizers" are probably acting allosterically, an additional degree of subtype specificity could be attained.

P.4.10 Nicotinic acetylcholine receptor (nAChR) desensitization and its relevance to physiological and behavioral functions

P.4.10 Nicotinic acetylcholine receptor (nAChR) desensitization and its relevance to physiological and behavioral functions

Presynaptic opioid and nicotinic receptor modulation of dopamine overflow in the nucleus accumbens.

Behaviorally relevant stimuli prompt midbrain dopamine (DA) neurons to switch from tonic to burst firing patterns. Similar shifts to burst activity are thought to contribute to the addictive effects of opiates and nicotine. The nucleus accumbens DA overflow produced by these drugs is a key element in their pathological effects. Using electrochemical techniques in brain slices, we explored the effects of opioids on single-spike and burst stimuli-evoked DA overflow in the dorsal and ventral striatum. In specific subregions of the nucleus accumbens, mu-opioids inhibit DA overflow elicited with single-spike stimuli while leaving that produced by burst stimuli unaffected. This is similar to published effects of nicotinic receptor blockade or desensitization, and is mediated by opioid receptor-induced inhibition of cholinergic interneurons. Whereas delta-opioids have similar effects, kappa-opioids inhibit evoked DA overflow throughout the striatum in a manner that is not overcome with high-frequency stimuli. These observations reveal remarkable mechanistic overlap between the effects of nicotine and opiates within the dopamine reward pathway.

Role of negatively charged amino acids in β4 F-loop in activation and desensitization of α3β4 rat neuronal nicotinic receptors

The role of negatively charged amino acids in the F-loop of the β4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the α3β4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at − 40 mV produced inward currents which displayed biphasic desensitization. The EC 50 of Epi and Nic were increased by a factor of 3–6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC 50 increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.

Cellular nicotinic receptor desensitization correlates with nicotine-induced acute behavioral tolerance in rats

Individuals vary in their susceptibility to nicotine addiction. However, there is little evidence that behavioral sensitivity to nicotine is dependent upon the functional state of nicotinic cholinergic receptors (nAChRs). This study aims to determine the relationship between in vivo behavioral desensitization and in vitro desensitization of nAChR function. Male Sprague-Dawley rats trained to discriminate nicotine were tested for development of acute behavioral tolerance. The rats were injected with nicotine (0.4 mg/kg free base, s.c.), tested for nicotine discrimination for 2 min, then injected with the same dose of nicotine 90, 180, and 270 min after the first injection and tested for nicotine discrimination after each injection. Susceptibility of nAChRs of individual rats to desensitization was assessed by use of the (86)Rb(+) efflux assay using synaptosomes prepared from the "thalamus," which included the hypothalamus and midbrain as well as the thalamic nuclei. To desensitize nAChRs, synaptsosomes were superfused with low concentrations of nicotine (5, 10, 20, and 30 nM) before stimulation of (86)Rb(+) efflux with nicotine (10 muM). The slopes of the behavioral desensitization were plotted as a function of the decline of nicotine-stimulated (86)Rb(+) efflux after in vitro desensitization. A significant correlation was observed between the in vitro desensitization of thalamic (86)Rb(+) efflux and the extent of behavioral desensitization of individual rats. These findings are consistent with the idea that production of acute behavioral tolerance by nicotine is related to its ability to induce nAChR desensitization at the cellular level.

Disconnection between activation and desensitization of autonomic nicotinic receptors by nicotine and cotinine

Cotinine is the major metabolite of nicotine in humans, and the substance greatly outlasts the presence of nicotine in the body. Recently, cotinine has been shown to exert pharmacological properties of its own that include potential cognition enhancement, anti-psychotic activity, and cytoprotection. Since the metabolite is generally less potent than nicotine in vivo, we considered whether part of cotinine's efficacy could be related to a reduced ability to desensitize nicotinic receptors as compared with nicotine. Rats freely moving in their home cages were instrumented to allow ongoing measurement of mean arterial blood pressure (MAP). The ganglionic stimulant dimethylphenylpiperazinium (DMPP) maximally increased MAP by 25 mmHg. Slow (20 min) i.v. infusion of nicotine (0.25–1 μmol) produced no change in resting MAP, but the pressor response to subsequent injection of DMPP was significantly attenuated in a dose-dependent manner by up to 51%. Pre-infusion of equivalent doses of cotinine produced the same maximal degree of inhibition of the response to DMPP. Discrete i.v. injections of nicotine also produced a dose dependent increase in MAP of up to 43 mmHg after the highest tolerated dose. In contrast, injection of cotinine produced no significant change in MAP up to 13 times the highest dose of nicotine. These results illustrate the disconnection between nicotinic receptor activation and receptor desensitization, and they suggest that cotinine's pharmacological actions are either mediated through partial desensitization, or through non-ganglionic subtypes of nicotinic receptors.

Regulation of Nicotinic Acetylcholine Receptor Desensitization by Ca2+

The relationship between the concentration of intracellular Ca2+ ([Ca2+](i)) and recovery from desensitization of nicotinic acetylcholine receptors (nAChRs) in rat medial habenula (MHb) neurons was investigated using the whole cell patch-clamp techniques in combination with microfluorescent [Ca2+](i) measurements. Recovery from desensitization was assessed with a paired-pulse agonist application protocol. Application of 100 microM nicotine (5 s) caused pronounced desensitization of nAChRs, after which recovery proceeded with two components. The relative weight of the two phases of recovery was sensitive to the nature of the intracellular Ca2+ chelator, with a greater fraction of channels recovering during the fast phase in the presence of BAPTA than EGTA. Recovery was affected by differential Ca2+ buffering only when Ca2+ was present in the extracellular solution, implying that Ca2+ influx through nAChRs was responsible for slowing the recovery. Simultaneous [Ca2+](i) measurements showed that recovery from desensitization was inversely correlated with the instantaneous [Ca2+](i), further supporting the suggestion that elevation of [Ca2+](i) limits the return of nAChRs to the resting state. In a separate set of experiments, activation of voltage-gated Ca2+ channels during the recovery phase produced a sufficiently large increase in [Ca2+](i) to reduce recovery from desensitization even in the absence of Ca2+ influx through nAChRs. Overall, it is suggested that Ca2+ entry through both nAChRs and voltage-gated Ca2+ channels exerts a negative feedback on nAChR activity through stabilization of desensitized states. The interaction of these two Ca2+ sources could form the basis of a coincidence detector under specific circumstances.

Nicotine reverses consolidated long-term potentiation in the hippocampal CA1 region

Long-term potentiation (LTP) has a memory-like consolidation period during which it becomes progressively stabilized. However, it is unknown how the consolidation is achieved. The present study demonstrates that nicotine reverses stabilized LTP in the hippocampal CA1 region, providing the first evidence that consolidated LTP can be reversed. The nicotine-induced reversal appeared to work by reversing cellular processes involved in stabilizing LTP, as LTP was readily induced again after reversal. The effect of nicotine was mediated, in large part, via desensitization of α7 nicotinic acetylcholine receptors (nAChRs), as an α7 nAChR-selective antagonist mimicked the nicotine effect. A non-selective N-methyl- d-aspartate receptor (NMDAR) antagonist completely abolished the nicotine-induced reversal, whereas an NR2B-containing NMDAR-selective antagonist had no effect. Furthermore, both the protein phosphatase 1/protein phosphatase 2A inhibitor okadaic acid and the protein phosphatase 2B (calcineurin) inhibitor cyclosporin A blocked the nicotine-induced reversal. Taken together, our results suggest that the reversal of stabilized LTP depends on the activation of NR2A-containing NMDARs and dephosphorylation. Thus, the consolidation of LTP appears to be the interruption of signaling leading to NR2A-containing NMDAR-dependent activation of protein phosphatases, which can be circumvented by nicotine-induced signaling. LTP induced in chronic nicotine-treated hippocampi contained a component that is immune to reversal, and thus acute nicotine was no longer effective to reverse consolidated LTP. These results demonstrate the differential effects of acute and chronic nicotine exposure on the cellular processes that are potentially involved in learning and memory.

Nicotinic acetylcholine receptor (nAChR) desensitization and its relevance to physiological and behavioral functions

Nicotinic acetylcholine receptor (nAChR) desensitization and its relevance to physiological and behavioral functions

Desensitization of neuronal nicotinic receptors of human neuroblastoma SH‐SY5Y cells during short or long exposure to nicotine

Neuronal nicotinic ACh receptors (nAChRs) readily desensitize in the presence of an agonist. However, when the agonist is applied for minutes, hours or days, it is unclear how extensive desensitization is, how long it persists after agonist removal and whether nAChRs consequently change their pharmacological properties. These issues were explored with electrophysiological studies of native receptors of voltage-clamped human neuroblastoma SH-SY5Y cells. Puffer pulses of nicotine (1 mM)-evoked inward currents partly antagonized by methyllycaconitine (MLA; 10 nM) or alpha-conotoxin MII (MII; 10 nM), suggesting contribution by alpha7 and alpha3 subunit containing receptors, respectively. Nicotine-evoked currents desensitized with 150 ms time constant and fully recovered after a few s washout. Although the current induced by 10 min application of nicotine (10 microM) decayed to baseline indicating complete desensitization, puffer applications of maximally effective doses of nicotine still generated small responses (22% of control). Similar responses to puffer-applied nicotine were observed when nicotine was chronically incubated for 8 or 48 h. On nicotine washout, cells recovered their response amplitude within 5 min and then increased it (about 50% of untreated controls) after 30 min without altering response kinetics or sensitivity to MLA and MII. The present results suggest that native nAChRs of SH-SY5Y cells preserved a degree of responsiveness during chronic application of nicotine, and that they rapidly recovered on washout to generate larger responses without changes in kinetics or pharmacology. These data indicate strong compensatory mechanisms to retain nicotinic receptor function during long-term exposure to nicotine.

Activation and desensitization of neuronal nicotinic receptors modulate glutamatergic transmission on neonatal rat hypoglossal motoneurons

In the neonate the muscles of the tongue, which are exclusively innervated by the XII cranial nerves originating from the brainstem nucleus hypoglossus, must contract rhythmically in coincidence with breathing, suckling and swallowing. These motor commands are generated by hypoglossal motoneurons excited by glutamatergic inputs. Because in forebrain areas the efficiency of glutamatergic transmission is modulated by neuronal nicotinic receptors (nAChRs), the role and identity of nAChRs within the nucleus hypoglossus of the neonatal rat were explored using an in vitro brainstem slice preparation. This area expressed immunoreactivity for alpha4, alpha7 and beta2 nAChR subunits. Whole-cell patch-clamp recording from hypoglossal motoneurons showed lack of spontaneous cholinergic events mediated by nAChRs even in the presence of a cholinesterase inhibitor. However, pharmacological antagonism of alpha7- or beta2-containing receptors depressed glutamatergic currents arising either spontaneously or by electrical stimulation of the reticular formation. Hypoglossal motoneurons expressed functional nAChRs with characteristics of alpha4beta2 and alpha7 receptor subunits. Such receptors underwent fast desensitization (time constant of 200 ms) with full recovery within 1 min. Low (0.5 microm) concentration of nicotine first facilitated glutamatergic transmission on motoneurons and later depressed it through receptor desensitization. When 0.1 microm nicotine was used, only depression of synaptic transmission occurred, in keeping with the suggestion that nAChRs can be desensitized without prior activation. These results highlight the role of tonic nAChR activity in shaping excitatory inputs to hypoglossal motoneurons, and suggest that nAChR desensitization by ambient nicotine could contribute to disorders of tongue muscle movements.

Evidence of cellular nicotinic receptor desensitization in rats exhibiting nicotine-induced acute tolerance

Individuals vary in their susceptibility to nicotine addiction. However, there is little evidence that behavioral sensitivity to nicotine is dependent upon the functional state of nicotinic cholinergic receptors (nAChRs). To determine the relationship between in vivo pharmacological desensitization (in other words, acute tolerance) and brain regional nAChR function. Male Sprague-Dawley rats, trained to discriminate nicotine (0.4 mg/kg free base) from saline in a two-lever drug discrimination task, were tested for the development of acute tolerance. Rats were injected with 0.4 mg/kg nicotine, tested for nicotine discrimination for 2 min, then injected with the same dose of nicotine 90 min, 180 min, and 270 min after the first injection and tested for nicotine discrimination after each injection. These subjects were separated into two groups, desensitizers (DZ) and nondesensitizers (NDZ), based upon performance in the repetitive dosing drug discrimination paradigm. The sensitivity of nAChRs in specific brain regions of these two groups was assessed by the use of an 86Rb+ efflux assay using synaptosomes prepared from the frontal cortex, hippocampus, striatum, and "thalamus," which included the midbrain and hypothalamus as well as the thalamus. The nicotine-induced increase in 86Rb+ efflux was significantly greater in NDZ as compared to DZ in the "thalamus." There was no statistically significant difference in the effects of nicotine in the frontal cortex, hippocampus, and striatum of these two groups. A significant correlation was observed between thalamic 86Rb+ efflux and the rate of behavioral desensitization of individual rats. These findings are consistent with the concept that the production of acute tolerance by nicotine in vivo correlates directly with its ability to induce nAChR desensitization at the cellular level.

The effect of simulated microgravity on hybridoma cells

The effect of clinostat-simulated microgravity on SP-2/0 and 1D6 hybridoma cells was studied. Clinorotation during 4–5 days at 1.5 rounds per minute decreased dramatically their proliferating capacity: the rotated cells divided less than once while control cells performed 4–5 divisions. They decreased the non-specific adhesion to tissue culture plastic, but increased the number of cell-to-cell contacts. Such phenomenological changes were accompanied with the alterations in pericellular glycosaminoglycans: decreased accumulation of hyaluronic acid and increased accumulation of chondroitin/dermatan-sulfate, as well as with the increase of cytoplasmic Ca 2 + concentration. Clinorotation resulted in hybridoma nicotinic receptor desensitization but not down-regulation. In contrast, both the quantity and quality (molecular isoforms, affinity and specificity) of the antibody produced by 1D6 hybridoma cells were not altered by clinorotation. It is concluded that simulated microgravity affected the proliferating and adhesive, but not biosynthetic properties of hybridoma cells.

The smoking gun: many conditions associated with tobacco exposure may be attributable to paradoxical compensatory autonomic responses to nicotine

Tobacco exposure is implicated in many illnesses such as cardiovascular disease and cancer, but the mechanisms underlying these associations are poorly understood. The mechanisms by which tobacco induces pro-sympathetic and pro-inflammatory changes also remain elusive. Some studies have attributed these changes to the direct effects of nicotine, but such findings run counter to the pro-vagal, anti-inflammatory nature of the nicotinic pathway. We hypothesize that the illnesses associated with smoking may be partly attributable to autonomic dysfunction, sympathetic bias, and T helper (Th)2 inflammation induced by a paradoxical compensatory response to intermittent nicotinic exposure. The confusion of interpreting the adrenergia and inflammation associated with nicotine as a primary response instead of a secondary compensation may be explained by the unusually rapid absorption, action, and serum elimination of nicotine. Given the fast action and clearance of nicotine, even heavy smokers spend large portions of the day and the entire night in nicotine withdrawal, at which time rebound sympathetic bias may manifest as a result of desensitization of nicotinic receptors. This may help reconcile why the features observed in smokers such as tachycardia, hypertension, inflammation, insomnia, and anxiety, which are perhaps mistakenly attributed to the direct action of nicotine, are identical to those seen during acute nicotine withdrawal after smoking cessation. On the other hand, delayed responses to cessation of smoking such as weight gain and increased heart rate variability are compatible with reduced sympathovagal ratio and resensitization of nicotinic receptors. Sympathetic bias and the associated Th2 inflammation underlie many systemic diseases. Tobacco-related cancers may be partly attributable to immunomodulatory properties of chronic nicotine exposure by dampening Th1 immunity and enabling tumoral evasion of immune surveillance. Other conditions associated with tobacco exposure may also operate through similar autonomic and immune dysfunctions. Therapeutic implications are discussed.

Desensitization of α7 nicotinic receptors potentiated the inhibitory effect on M-current induced by stimulation of muscarinic receptors in rat superior cervical ganglion neurons

Whole-cell patch-clamp recording from rat superior cervical ganglion neurons in culture was used to investigate the modulatory effect of desensitized alpha7-nAChRs on mAChRs. An inward alpha-bungarotoxin and methyllycaconitine sensitive current was elicited by rapid application of choline, which consisted of a fast and a slow desensitizing component. The amplitude of choline-evoked currents recorded 0.5, 1, 2, and 3 min after the prolonged application of choline (10 mM, 30 s) decreased to 25.3 +/- 9.2%, 45.9 +/- 11.8%, 66.3 +/- 14.5%, and 73.9 +/- 13.3% of their baseline levels, respectively. The amplitudes of M-currents, recorded at the same time intervals after the similar prolonged stimulation with choline, were decreased to 52.7 +/- 17.4%, 63.9 +/- 4.2%, 70.9 +/- 2.8%, and 72.9 +/- 17.3% of initial values respectively by focal application of pilocarpine (1 mM, 5 s) onto the soma of neurons. By contrast, before the desensitization of alpha7-nAChRs, M-currents were only decreased to 79.8 +/- 13.7% of baseline levels by pilocarpine (1 mM, 5 s). Whereas the desensitization of alpha7-nAChRs had no direct effects on M-currents, and the facilitated effects on muscarinic agonists on the M-currents induced by desensitized alpha7-nAChRs, were removed in the presence of alpha-bungarotoxin and methyllycaconitine. These results indicated that desensitization of alpha7-nAChRs could potentiate the inhibitory effect on M-current by stimulation of mAChRs with their agonist.

Dynamics of fast synaptic excitation during trains of stimulation in myenteric neurons of guinea-pig ileum

Fast excitatory postsynaptic potentials (fEPSPs) occur in bursts in the myenteric plexus during evoked motor reflexes in the guinea-pig ileum in vitro. This study used electrophysiological methods to study fEPSPs during stimulus trains to mimic bursts of synaptic activity in vitro. The amplitude of fEPSPs or fast excitatory postsynaptic currents (EPSCs) declined (rundown) during stimulus trains at frequencies of 0.5, 5, 10 and 20 Hz. At 0.5 Hz, fEPSP or fEPSC amplitude declined by 50% after the first stimulus but remained constant for the remainder of the train. At 5, 10 and 20 Hz, synaptic responses ran down completely with time constants of 0.35, 0.21 and 0.11 s, respectively. Recovery from rundown occurred with a time constant of 7 s. Mecamylamine, a nicotinic cholinergic receptor antagonist, or PPADS, a P2X receptor antagonist, reduced fEPSP amplitude, but they had no effect on rundown. Responses caused by trains of ionophoretically applied ATP or ACh (to mimic fEPSPs) did not rundown. Blockade of presynaptic inhibitory muscarinic, adenosine A 1, opioid, α 2-adrenergic and 5-HT 1A receptors or pertussis toxin (PTX) treatment did not alter rundown. Antidromic action potentials followed a 10-Hz stimulus train. Iberiotoxin (100 nM), a blocker of large conductance calcium activated K + (BK) channels, did not alter rundown. These data suggest that synaptic rundown is not due to: (a) action potential failure; (b) nicotinic or P2X receptor desensitization; (c) presynaptic inhibition mediated by pertussis-toxin sensitive G-proteins, or (d) BK channel activation. Synaptic rundown is likely due to depletion of a readily releasable pool (RRP) of neurotransmitter.

Nicotine-induced Up-regulation and Desensitization of α4β2 Neuronal Nicotinic Receptors Depend on Subunit Ratio

Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different alpha4beta2 subunit ratios (1alpha:4beta, 2alpha:3beta, and 4alpha:1beta) expressed in Xenopus oocytes. The presence of alpha4 subunit in the oocyte plasmatic membrane increased linearly with the amount of alpha4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the alpha4 subunit. The 2alpha4:3beta2 subunit ratio displayed the highest ACh sensitivity. Nicotine dose-response curves for the 1alpha4:4beta2 and 2alpha4:3beta2 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 microm. A biphasic curve for 4alpha4:1beta2 was obtained at nicotine concentrations higher than 300 microm. The 1alpha4:4beta2 subunit ratio exhibited the lowest ACh- and nicotine-induced macroscopic current, whereas 4alpha4:1beta2 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of beta2:alpha4 subunits increased. All three alpha4beta2 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2alpha4: 3beta2 subunit ratio. Our data suggest that the subunit ratio of alpha4beta2 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate alpha4beta2 receptor activation and desensitization.

PKC-independent inhibition of neuronal nicotinic acetylcholine receptors by diacylglycerol

Diacylglycerol modulates cell functions primarily through activation of protein kinase C (PKC). In a previous study, however, we found that a diacylglycerol analogue, 1-oleoyl-2-acetylglycerol (OAG), accelerated desensitization of neuronal nicotinic acetylcholine receptors (nAchRs) independently of PKC activation in PC12 cells. In the present study, we investigated whether other analogues and endogenous diacylglycerol exert similar effects on neuronal nAchRs and characterized the modulation by diacylglycerol. We measured the nicotine-induced whole-cell current in the absence and presence of diacylglycerol analogues in PC12 cells. We also investigated the effects of a blockade of metabolic pathways of diacylglycerol by inhibiting diacylglycerol lipase and kinase. We found that all four diacylglycerol analogues studied promoted desensitization and depressed the nondesensitized component of the nicotine-induced current. These effects seemed independent of PKC activation because they were not antagonized by the PKC inhibitors staurosporine or bisindolylmaleimide I; one analogue that lacks the PKC-stimulating action was also effective. The effects of diacylglycerol analogues were not antagonized by high doses of nicotine and were independent of the membrane potential. Similar modulatory effects were observed by treatment with RHC80267, a blocker of diacylglycerol lipase, and R59949, an inhibitor of diacylglycerol kinase, in the presence of staurosporine. These results suggest that diacylglycerol, both exogenously applied and endogenously produced, modulates neuronal nAchRs independently of PKC activation in PC12 cells; further, these effects seemed consistent with a noncompetitive and voltage-independent block. They raised the possibility that PKC-independent inhibition of neuronal nAchRs by diacylglycerol may be a novel modulatory process.

Activation and desensitization of heteromeric neuronal nicotinic receptors: implications for non-synaptic transmission

Consideration of the activation and desensitization properties of neuronal nicotinic acetylcholine receptors (nAChRs) predicts that there should be a range of concentrations over which low ambient levels of agonist can continuously open nAChR channels. These findings support the idea that postsynaptic nAChRs may participate in unconventional cellular signaling mediated by the release of acetylcholine from diffusely distributed non-synaptic cholinergic varicosities.