Desensitization Of Nicotinic Receptors Research Articles

Alkaloid‐dependent fetal muscle‐type nicotinic acetylcholine receptor desensitization (1059.9)

The exposure of a developing embryo or fetus to teratogenic alkaloids from plants has the potential to cause developmental defects in humans and animals due to the inhibition of fetal movement. The mechanism behind the inhibition of fetal movement is postulated to be the desensitization of fetal muscle type nicotinic acetylcholine receptors (nAChR). In this study, we compared the endogenous ligand acetylcholine (ACh), to coniine from poison hemlock (Conium maculatum) and nicotine from tobacco (Nicotiana tabacum) for the ability to desensitize fetal muscle‐type nAChR expressed by TE‐671 cells. The experiments were conducted using a membrane potential sensing dye‐based system and a two‐step agonist addition protocol. Treatment of the TE‐671 cells with 10 μM ACh reduced the subsequent dye response of the cells to 100 μM ACh by 72%. The sequential changes in membrane potential to nicotine and coniine were only reduced at 1 mM which was most likely due blockade of the nAChR ion channel by the agonist. This research was supported by USDA/ARS.

Effects of the sazetidine-a family of compounds on the body temperature in wildtype, nicotinic receptor β2−/− and α7−/− mice

Nicotine elicits hypothermic responses in rodents. This effect appears to be related to nicotinic receptor desensitization because sazetidine-A, an α4β2 nicotinic receptor desensitizing agent, produces marked hypothermia and potentiates nicotine-induced hypothermia in mice. To determine the specificity of sazetidine-A induced hypothermia to β2 subunit-containing nicotinic receptors, we tested its efficacy in β2 knockout (β2−/−) mice. These effects were compared with wildtype (WT) and α7 knockout (α7−/−) mice. Confirming our earlier results, sazetidine-A elicited a pronounced and long-lasting hypothermia in WT mice. In comparison, sazetidine-A induced a much attenuated and shorter hypothermic response in β2−/− mice. This indicates that the greater proportion of sazetidine-A induced hypothermia is mediated via actions on β2-containing nicotinic receptors, while a smaller component of hypothermia induced by sazetidine-A is mediated by non-β2 receptors. Similar to WT mice, α7−/− mice showed the full extent of the sazetidine-A effect, suggesting that the hypothermia produced by sazetidine-A did not depend on actions on α7 nicotinic receptor subtype. Three other novel nicotinic receptor desensitizing agents derived from sazetidine-A, triazetidine-O, VMY-2-95 and YL-1-127 also produced hypothermia in WT and α7−/− mice. Furthermore, unlike sazetidine-A, triazetidine-O and YL-1-127 did not show any hint of a hypothermic effect in β2−/− mice. VMY-2-95 like sazetidine-A did show a residual hypothermic effect in the β2−/− mice. These studies show that the hypothermic effects of sazetidine-A and the related compound VMY-2-95 are mainly mediated by nicotinic receptors containing β2 subunit, but that a small component of the effect is apparently mediated by non-β2 containing receptors.

The dual effect of PNU-120596 on α7 nicotinic acetylcholine receptor channels

PNU-120596 (1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea), a Type-II positive allosteric modulator of α7 nicotinic acetylcholine receptors inhibits α7 desensitization and robustly prolongs openings of α7 channels. However, these effects may render α7 channels more accessible to positively charged molecules and thus, more susceptible to voltage-dependent open-channel-block-like inhibition. To test this hypothesis, choline chloride (i.e., choline), a selective endogenous α7 agonist, and bicuculline methochloride (i.e., bicuculline), a competitive α7 antagonist, were used as membrane voltage-sensitive probes in whole-cell voltage-clamp recordings from hippocampal CA1 interneurons in acute brain slices in the absence and presence of PNU-120596. PNU-120596 enhanced voltage-dependent inhibition of α7 responses by bicuculline and choline. In the presence of PNU-120596, α7 channels favored a burst-like kinetic modality in the presence, but not absence of bicuculline and bursts of α7 openings were voltage-dependent. These results suggest that PNU-120596 alters the pharmacology of α7 channels by making these channels more susceptible to voltage-dependent inhibitory interactions with positively charged drugs at concentrations that do not potently inhibit α7 channels without PNU-120596. This inhibition imitates α7 nicotinic receptor desensitization and compromises the potentiating anti-desensitization effects of PNU-120596 on α7 nicotinic receptors. This unexpected dual action of PNU-120596, and possibly other Type-II positive allosteric modulators of α7 nicotinic receptors, may lead to unanticipated α7 channel-drug interactions and misinterpretation of α7 single-channel data.

Enhancement of α9α10 Nicotinic Acetylcholine Receptor Desensitization by Nimodipine

Entry of calcium ions through nicotinic acetylcholine receptors hyperpolarizes outer hair cells (OHCs) by activating colocalized SK potassium channels. This process, termed efferent inhibition, plays a central role in the protective reflex by suppressing the amplification of incoming sound. OHC nicotinic receptors are composed of α9 and α10 subunits that are activated by acetylcholine (ACh) release from efferent neurons originating within the medial olivocochlear nucleus. Fast application of ACh to GH4C1 cells transiently transfected with plasmids encoding mouse α9 and α10 subunits, produced an inward current that displayed a concentration-dependent amplitude, with an EC50 of 41 ± 5.4 μM and Hill slope (n H) of 0.89 ± 0.1 (n=5). This current was antagonized by nicotine, displaying a KB of 9.0 ± 1.0 μM (n=8), consistent with the ACh-mediated current arising from activation of heteromeric α9α10 receptor channels. Inward ACh currents in the presence of the dihydropyridine antagonist nimodipine (10 μM) were reduced in amplitude and displayed faster decay kinetics. Exponential decay rates (τ) increased from 647.5 ± 66.0 ms (n=6) in control, to 160.6 ± 25.8 ms (n=6) in the presence of nimodipine (10 μM) (p 0.05), suggesting that the increased decay rate of ACh currents did not result from a decrease in agonist binding affinity. using a double-pulse protocol, ACh (300 μM)-mediated responses recovered from desensitization with an exponential time-course (τ = 3.81 ± 0.67 s, n=3). Recovery was slowed in the presence of nimodipine (τ = 8.88 ± 2.54 s; n=3), indicating that nimodipine stabilizes the desensitization state of the α9α10 receptor and suggests that these receptors can be modulated to fine-tune efferent inhibition.

Low dose nicotine and antagonism of β2 subunit containing nicotinic acetylcholine receptors have similar effects on affective behavior in mice.

Nicotine leads to both activation and desensitization (inactivation) of nicotinic acetylcholine receptors (nAChRs). This study tested the hypothesis that nicotine and a selective antagonist of β2*nAChRs would have similar effects on affective behavior. Adult C57BL/6J male mice were tested in a conditioned emotional response (CER) assay which evaluates the ability of an aversive stimulus to inhibit goal-directed behavior. Mice lever-pressed for a saccharin reinforcer according to a variable schedule of reinforcement during sessions in which two presentations of a compound light/tone conditioned stimulus (CS) co-terminated with a 0.1 or 0.3 mA, 0.5 s footshock unconditioned stimulus (US). During testing in the absence of the US, mice received doses of i.p. nicotine (0, 0.0032, 0.01, 0.032, 0.1 mg/kg) or a selective β2 subunit containing nAChR (β2*nAChR) antagonist dihydro-beta-erythroidine (0, 0.1, 0.3, 1.0, 3.0 mg/kg DHβE). There was a dose-dependent effect of nicotine revealing that only low doses (0.01, 0.032 mg/kg) increased CER suppression ratios (SR) in these mice. DHβE also dose-dependently increased SR at the 3 mg/kg dose. In ethological measures of fear−/anxiety-like behavior, these doses of nicotine and DHβE significantly reduced digging behavior in a marble burying task and 0.3 mg/kg DHβE promoted open-arm activity in the elevated plus maze. Doses of nicotine and DHβE that altered affective behavior had no effect on locomotor activity. Similar to previous reports with anxiolytic drugs, low dose nicotine and DHβE reversed SR in a CER assay, decreased digging in a marble burying assay and increased open arm activity in the elevated plus maze. This study provides evidence that inactivation of β2*nAChRs reduces fear-like and anxiety-like behavior in rodents and suggests that smokers may be motivated to smoke in part to desensitize their β2*nAChRs. These data further identify β2*nAChR antagonism as a potential therapeutic strategy for relief of negative affect and anxiety.

Effects of chronic sazetidine-A, a selective α4β2 neuronal nicotinic acetylcholine receptors desensitizing agent on pharmacologically-induced impaired attention in rats

Nicotine and nicotinic agonists have been shown to improve attentional function. Nicotinic receptors are easily desensitized, and all nicotinic agonists are also desensitizing agents. Although both receptor activation and desensitization are components of the mechanism that mediates the overall effects of nicotinic agonists, it is not clear how each of the two opposed actions contributes to attentional improvements. Sazetidine-A has high binding affinity at α4β2 nicotinic receptors and causes a relatively brief activation followed by a long-lasting desensitization of the receptors. Acute administration of sazetidine-A has been shown to significantly improve attention by reversing impairments caused by the muscarinic cholinergic antagonist scopolamine and the NMDA glutamate antagonist dizocilpine. In the current study, we tested the effects of chronic subcutaneous infusion of sazetidine-A (0, 2, or 6mg/kg/day) on attention in Sprague-Dawley rats. Furthermore, we investigated the effects of chronic sazetidine-A treatment on attentional impairment induced by an acute administration of 0.02mg/kg scopolamine. During the first week period, the 6-mg/kg/day sazetidine-A dose significantly reversed the attentional impairment induced by scopolamine. During weeks3 and 4, the scopolamine-induced impairment was no longer seen, but sazetidine-A (6mg/kg/day) significantly improved attentional performance on its own. Chronic sazetidine-A also reduced response latency and response omissions. This study demonstrated that similar to its acute effects, chronic infusions of sazetidine-A improve attentional performance. The results indicate that the desensitization of α4β2 nicotinic receptors with some activation of these receptors may play an important role in improving effects of sazetidine-A on attention.

P.1.g.015 Is neurogenesis in the dentate gyrus compromised in Parkinson's disease?

The major goal of the present study was to examine acute tolerance to nicotine-induced disruption of operant behavior following a single, noncontingent injection. Rats were trained to lever press for food reinforcement under a fixed ratio-30 schedule. Once trained, rats were injected with either saline or nicotine (0.8 mg/kg) in their home cages. After either a 90- or 180-min delay, each rat was injected with nicotine (0.4 mg/kg) and placed in the operant chamber for a 30-min behavioral evaluation session. This experiment was replicated with slight modifications 1 week later. The results of the present study suggest that 0.8 mg/kg of nicotine produces acute tolerance to the response rate decreasing effects of 0.4 mg/kg of nicotine. Because the tolerance-producing dose of nicotine was injected while rats were not in the test environment, they did not have an opportunity to practice the target behavior while under the influence of the drug. Hence, the acute tolerance observed in this study appears to be, at least partly, pharmacological (vs. behavioral) in nature, and may be related to a desensitization of central nicotinic acetylcholinergic receptors (nAChRs).

P.1.g.016 Modulation of nicotinic acetylcholinergic receptors differentially affects auditory event related potentials in awake rats

The major goal of the present study was to examine acute tolerance to nicotine-induced disruption of operant behavior following a single, noncontingent injection. Rats were trained to lever press for food reinforcement under a fixed ratio-30 schedule. Once trained, rats were injected with either saline or nicotine (0.8 mg/kg) in their home cages. After either a 90- or 180-min delay, each rat was injected with nicotine (0.4 mg/kg) and placed in the operant chamber for a 30-min behavioral evaluation session. This experiment was replicated with slight modifications 1 week later. The results of the present study suggest that 0.8 mg/kg of nicotine produces acute tolerance to the response rate decreasing effects of 0.4 mg/kg of nicotine. Because the tolerance-producing dose of nicotine was injected while rats were not in the test environment, they did not have an opportunity to practice the target behavior while under the influence of the drug. Hence, the acute tolerance observed in this study appears to be, at least partly, pharmacological (vs. behavioral) in nature, and may be related to a desensitization of central nicotinic acetylcholinergic receptors (nAChRs).

Analgesic effects mediated by neuronal nicotinic acetylcholine receptor agonists: Correlation with desensitization of α4β2* receptors

Nicotinic α4β2* agonists are known to be effective in a variety of preclinical pain models, but the underlying mechanisms of analgesic action are not well-understood. In the present study, we characterized activation and desensitization properties for a set of seventeen novel α4β2*-selective agonists that display druggable physical and pharmacokinetic attributes, and correlated the in vitro pharmacology results to efficacies observed in a mouse formalin model of analgesia. ABT-894 and Sazetidine-A, two compounds known to be effective in the formalin assay, were included for comparison. The set of compounds displayed a range of activities at human (α4β2)2β2 (HS-α4β2), (α4β2)2α5 (α4β2α5) and (α4β2)2α4 (LS-α4β2) receptors.We report the novel finding that desensitization of α4β2* receptors may drive part of the antinociceptive outcome. Our molecular modeling approaches revealed that when receptor desensitization rather than activation activitiesat α4β2* receptors are considered, there is a better correlation between analgesia scores and combined in vitro properties.Our results suggest that although all three α4β2 subtypes assessed are involved, it is desensitization of α4β2α5 receptors that plays a more prominent role in the antinociceptive action of nicotinic compounds. For modulation of Phase I responses, correlations are significantly improved from an r2 value of 0.53 to 0.67 and 0.66 when HS- and LS-α4β2 DC50 values are considered, respectively. More profoundly, considering the DC50 at α4β2α5 takes the r2 from 0.53 to 0.70. For Phase II analgesia scores, adding HS- or LS-α4β2 desensitization potencies did not improve the correlations significantly. Considering the α4β2α5 DC50 value significantly increased the r2 from 0.70 to 0.79 for Phase II, and strongly suggested a more prominent role for α4β2α5 nAChRs in the modulation of pain in the formalin assay.The present studies demonstrate that compounds which are more potent at desensitization of α4β2* receptors display better analgesia scores in the formalin test. Consideration of desensitization propertiesat α4β2* receptors, especially at α4β2α5, in multiple linear regression analyses significantly improves correlations with efficacies of analgesia. Thus, α4β2* nicotinic acetylcholine receptor desensitization may contribute to efficacy in the mediation of pain, and represent a mechanism for analgesic effects mediated by nicotinic agonists.

The AChE membrane-binding tail PRiMA is down-regulated in muscle and nerve of mice with muscular dystrophy by merosin deficiency

Since Duchenne muscular dystrophy was attributed to mutations in the dystrophin gene, more than 30 genes have been found to be causally related with muscular dystrophies, about half of them encoding proteins of the dystrophin–glycoprotein complex (DGC). Through laminin-2, the DGC bridges the muscle cytoskeleton and the extracellular matrix. Decreased levels of PRiMA-linked acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) have been observed in dystrophic muscle and nerve of dystrophin-deficient (mdx) and laminin-2 deficient (Lama2dy) mice. To help explain these observations, the relative content of AChE, BuChE and PRiMA mRNAs were compared in normal and Lama2dy mouse muscle and sciatic nerve. The 17-fold lower level of PRiMA mRNA in Lama2dy muscle explained the deficit in PRiMA-linked ChEs. This would increase acetylcholine availability and, eventually, the desensitization of nicotinic receptors. Abnormal development of the Schwann cells led to peripheral neuropathy in the Lama2dy mouse. Compared with normal nerve, dystrophic nerve displayed 4-fold less AChE-T mRNA, 3-fold more BuChE mRNA and 2.5-fold less PRiMA mRNA, which agreed with the lower AChE activity in dystrophic nerve, its increased BuChE activity and the specific drop in PRiMA-linked BuChE. The widely accepted role of glial cells as the source of BuChE, the observed dysmyelination of Lama2dy nerve and its increased BuChE activity support the idea that BuChE up-regulation is related with the aberrant differentiation of the Schwann cells.

Potential State-selective Hydrogen Bond Formation Can Modulate Activation and Desensitization of the α7 Nicotinic Acetylcholine Receptor

A series of arylidene anabaseines were synthesized to probe the functional impact of hydrogen bonding on human α7 nicotinic acetylcholine receptor (nAChR) activation and desensitization. The aryl groups were either hydrogen bond acceptors (furans), donors (pyrroles), or neither (thiophenes). These compounds were tested against a series of point mutants of the ligand-binding domain residue Gln-57, a residue hypothesized to be proximate to the aryl group of the bound agonist and a putative hydrogen bonding partner. Q57K, Q57D, Q57E, and Q57L were chosen to remove the dual hydrogen bonding donor/acceptor ability of Gln-57 and replace it with hydrogen bond donating, hydrogen bond accepting, or nonhydrogen bonding ability. Activation of the receptor was compromised with hydrogen bonding mismatches, for example, pairing a pyrrole with Q57K or Q57L, or a furan anabaseine with Q57D or Q57E. Ligand co-applications with the positive allosteric modulator PNU-120596 produced significantly enhanced currents whose degree of enhancement was greater for 2-furans or -pyrroles than for their 3-substituted isomers, whereas the nonhydrogen bonding thiophenes failed to show this correlation. Interestingly, the PNU-120596 agonist co-application data revealed that for wild-type α7 nAChR, the 3-furan desensitized state was relatively stabilized compared with that of 2-furan, a reversal of the relationship observed with respect to the barrier for entry into the desensitized state. These data highlight the importance of hydrogen bonding on the receptor-ligand state, and suggest that it may be possible to fine-tune features of agonists that mediate state selection in the nAChR.

Increased nicotinic receptor desensitization in hypoglossal motor neurons following chronic developmental nicotine exposure

Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on hypoglossal motor neurons (XII MNs) that innervate muscles of the tongue. Activation of XII MN nAChRs evokes depolarizing currents, which are important for regulating the size and stiffness of the upper airway. Although data show that chronic developmental nicotine exposure (DNE) blunts cholinergic neurotransmission in the XII motor nucleus, it is unclear how nAChRs are involved. Therefore, XII MN nAChR desensitization and recovery were examined in tissues from DNE or control pups using a medullary slice preparation and tight-seal whole cell patch-clamp recordings. nAChR-mediated inward currents were evoked by brief pressure pulses of nicotine or the α4β2 nAChR agonist RJR-2403. We found that, regardless of treatment, activatable nAChRs underwent desensitization, but, following DNE, nAChRs exhibited increased desensitization and delayed recovery. Similar results were produced using RJR-2403, showing that DNE influences primarily the α4β2 nAChR subtype. These results show that while some nAChRs preserve their responsiveness to acute nicotine following DNE, they more readily desensitize and recover more slowly from the desensitized state. These data provide new evidence that chronic DNE modulates XII MN nAChR function, and suggests an explanation for the association between DNE and the incidence of central and obstructive apneas.

Tramadol requirements may need to be increased for the perioperative management of pain in smokers

Evidence indicates that smokers have hyperalgesia perioperatively as characterized by a higher postoperative pain score as well as increased requirements of opioids during surgery and postoperative patient-controlled analgesia compared with non-smokers. The possible mechanism of hyperalgesia for smokers is related to nicotinic acetylcholine receptor (nAChR) desensitization as well as competitive occupancy for binding sites. For smokers, high doses of opioids are needed perioperatively whereas small doses of nicotine do not reduce postoperative opioid requirements. Nicotine replacement treatment has been proved to be effective only for non-smokers. The serotonergic system plays an important part in modulating anti-nociception, and decreasing the concentration of serotonin in vesicles in neurons of the brain and spinal cord is an effective method. Intraoperative application of tramadol could result in an analgesic effect via enhancement of descending inhibitory pain pathways. Therefore, increasing the amount of tramadol given intraoperatively and postoperatively may reduce overall opioid requirements, and decrease the pain score as well as morphine consumption postoperatively.

Desensitization of nicotinic acetylcholine receptors in central nervous system neurons of the stick insect ( Carausius morosus) by imidacloprid and sulfoximine insecticides

Imidacloprid, sulfoxaflor and two experimental sulfoximine insecticides caused generally depressive symptoms in stick insects, characterized by stillness and weakness, while also variably inducing postural changes such as persistent ovipositor opening, leg flexion or extension and abdomen bending that could indicate excitation of certain neural circuits. We examined the same compounds on nicotinic acetylcholine receptors in stick insect neurons, which have previously been shown to desensitize in the presence of ACh. Brief U-tube application of 10 −4 M solutions of insecticides for 1 s evoked currents that were much smaller than ACh-evoked currents, and depressed subsequent ACh-evoked currents for several minutes, indicating that the compounds are low-efficacy partial agonists that potently desensitize the receptors. Much lower concentrations of insecticides applied in the bath for longer periods did not activate currents, but inhibited ACh-evoked currents via desensitization of the receptors. Previously described fast- and slowly-desensitizing nACh currents, I ACh1 and I ACh2 respectively, were each found to consist of two components with differing sensitivities to the insecticides. Imidacloprid applied in the bath desensitized high-sensitivity components, I ACh1H and I ACh2H with IC 50s of 0.18 and 0.13 pM, respectively. It desensitized the low-sensitivity slowly desensitizing component, I ACh2L, with an IC 50 of 2.6 nM, while a component of the fast-desensitizing current, I ACh1L, was least sensitive, with an IC 50 of 81 nM I ACh1L appeared to be insensitive to the three sulfoximines tested, whereas all three sulfoximines potently desensitized I ACh1H and both slowly desensitizing components, with IC 50s between 2 and 7 nM. We conclude that selective desensitization of certain nAChR subtypes can account for the insecticidal actions of imidacloprid and sulfoximines in stick insects.

Oxime Reactivators and Their in Vivo and in Vitro Effects on Nicotinic Receptors

Current treatment of organophosphorus poisoning, resulting in overstimulation and desensitization of muscarinic and nicotinic receptors by acetylcholine (ACh), consists of the administration of atropine and oxime reactivators. However, no versatile oxime reactivator has been developed yet and some mortality still remains after application of standard atropine treatment, probably due to its lack of antinicotinic action. In our study, we focused on the interesting non-acetylcholinesterase property of oximes, i.e. antinicotinic effect of reactivators. Two standard reactivators (HI-6, obidoxime) and two new compounds (K027 and K203) were chosen for in vitro (patch clamp) and in vivo (nerve-evoked muscle contraction) testings. Both examinations showed antinicotinic effects of the reactivators. In vitro inhibition of acetylcholine-evoked currents by obidoxime, HI-6 and K203 was equivalent while K027 was less potent. Similar order of potency was observed by the in vivo examinations. We thus confirm previous in vitro results, which describe antinicotinic effects of oxime reactivators, and furthermore, we show in vivo antagonism of oxime reactivators exerted by the inhibition of ACh effect on the nicotinic receptor in the neuromuscular junction. Taking together, the effects of tested oxime reactivators indicate an antagonism on both embryonic and adult form of the muscle nicotinic receptors.

Desensitization of α7 Nicotinic Receptor Is Governed by Coupling Strength Relative to Gate Tightness

Binding of a neurotransmitter to its membrane receptor opens an integral ion conducting pore. However, prolonged exposure to the neurotransmitter drives the receptor to a refractory state termed desensitization, which plays an important role in shaping synaptic transmission. Despite intensive research in the past, the structural mechanism of desensitization is still elusive. Using mutagenesis and voltage clamp in an oocyte expression system, we provide several lines of evidence supporting a novel hypothesis that uncoupling between binding and gating machinery is the underlying mechanism for α7 nicotinic receptor (nAChR) desensitization. First, the decrease in gate tightness was highly correlated to the reduced desensitization. Second, nonfunctional mutants in three important coupling loops (loop 2, loop 7, and the M2-M3 linker) could be rescued by a gating mutant. Furthermore, the decrease in coupling strength in these rescued coupling loop mutants reversed the gating effect on desensitization. Finally, coupling between M1 and hinge region of the M2-M3 linker also influenced the receptor desensitization. Thus, the uncoupling between N-terminal domain and transmembrane domain, governed by the balance of coupling strength and gate tightness, underlies the mechanism of desensitization for the α7 nAChR.

On the Mechanism Underlying the Irreversible Desensitization of Human Ganglionic Nicotinic Receptors

alpha3-containing nicotinic acetylcholine receptors (AChRs) are expressed in autonomic ganglia and are thought to mediate fast synaptic transmission. These receptors have largely eluded rigorous kinetic characterization due to a combination of poor expression in heterologous systems and the total loss of receptor responsiveness during the course of excised-patch electrophysiology experiments. Recently, it has been demonstrated that a cysteine-to-alanine mutation at the −4’ position of the cytosolic M1-M2 linker can prevent this rundown in whole-cell patches of rodent superior cervical ganglion neurons (Campanucci et al., 2010). This work also suggested that this rundown, which plays a pivotal role in diabetic neuropathy, is due to oxidation of these cysteine residues. Here, we heterologously expressed human alpha3-subunit-containing AChRs in HEK-293 cells and found that the −4’ cysteine mutation cannot prevent the irreversible rundown of currents observed in outside-out patches of membrane. Furthermore, we found that the irreversible loss of receptor responsiveness does not occur in the whole-cell or cell-attached configurations, implying that patch excision leads to this phenomenon. We also investigated the effects of the reciprocal −4’ cysteine mutation in all five subunits of the human muscle AChRs, which do not natively have cysteines at this position. We found that introduction of the cysteines does not lead to an irreversible rundown, although it does slow down the rate of recovery from desensitization. Taken together, our results thus far suggest that the mechanism underlying the rundown of currents mediated by alpha3-containing AChRs in outside-out patches is fundamentally different from that mediated by cysteine oxidation in whole-cell recordings.

Neuronal networks of nicotine addiction

Nicotine is the main psychoactive substance present in tobacco, targeting neuronal nicotinic acetylcholine receptors. The main effects of nicotine associated with smoking are nicotinic receptor activation, desensitization, and upregulation, with the subsequent modulation of the mesocorticolimbic dopaminergic system. However, there is a lack of a comprehensive explanation of their roles that effectively makes clear how nicotine dependence might be established on those grounds. Receptor upregulation is an unusual effect for a drug of abuse, because theoretically this implies less need for drug consumption. Receptor upregulation and receptor desensitization are commonly viewed as opposite, homeostatic mechanisms. We here review the available information on smoking addiction, especially under a recently presented model of nicotine dependence. In this model both receptor upregulation and receptor desensitization are responsible for establishing a biochemical mechanism of nicotine dependence, which have an important role in starting and maintaining tobacco addiction.

Diversity and Variability of the Effects of Nicotine on Different Cortical Regions of the Brain. Therapeutic and Toxicological Implications

Nicotine/nicotine agonists or allosteric modulators of nicotine receptors have been suggested as the most important therapeutic agents in the prevention and clinical control of cognitive impairment which characterize neuropsychiatric and neurodegenerative disorders such as schizophrenia, attention deficit/hyperactivity disorder and Alzheimer's disease. Both clinical studies and animal experiments support the important role of the nicotinic systems in learning, different kind of memory and cognition. For development of nicotinic treatments we have a well characterized lead compound, nicotine. However, the neural nicotinic mechanisms underlying cognitive functions are not well known because the side effects of nicotine overdose have hindered the development of this therapeutical line. The new development of non-toxic, brain specific nicotine drugs need a full knowledge of these mechanism and a reevaluation of the nicotine effects. This review aims to analyze the different kind of effects of nicotine on the Central Nervous System (CNS), especially on the cortex and hippocampus. Nicotine effects are, theoretically and/or practically, of variable character depending on daily dose and time of treatment; on the subtype and density of the different nicotinic receptors existing in the distinct brain regions; on the processes of desensitization and tolerance of nicotinic receptors and on other neuronal factors. Nicotine produces the above mentioned activation of the cognitive functions acting directly or indirectly on cortical neurons. In some experiments, high doses of nicotine can impair memory. This substance induces increases in the glycolytic pathway and Krebs cycle of neurons, as well as brain blood flow. Nicotine also produces an increase in NGF immunoreactivity in frontoparietal cortex. All these neuronal changes may cause different positive effects such as neuroprotection, neuroplasticity and better performance of synaptic circuits. The benefit of other neuronal changes can be matter of discussion such as some modifications in synaptic transmission, the COX-2 increase in frontoparietal cortex and hippocampus or the changes in the antioxidant systems. Finally, other neuronal changes can be of negative effect such as the induction of apoptosis and oxidative stress (DNA damage, ROS and lipid peroxide increase). All these described effects explain both the beneficial and neurotoxic consequences of the activation of the nicotinic receptors. The diversity and variability of the nicotinic effects should take into account when nicotine agonists will be used as a possible cognitive treatment.

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

320 The nAChR-subunit topology is common for all other pentameric neurotransmitter receptor channels. These ionic channels constitute the Cys-loop receptor superfamily whose members share a conserved disulfide bridge in their extracellular ligand-binding domain. The binding pockets for ACh are formed by the interface between each α -subunit and the adjacent subunit. The α -subunit contributes to the ACh-binding pocket the so-called principal component, which consists of a series of loops carrying highly conserved ACh-binding residues, mainly aromatic but also a cysteine pair. The neighboring subunit contributes a series of complementary ACh-binding residues. The role of negatively charged amino acids in the F-loop of the β 4 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 α 3 β 4 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 EC50 of Epi and Nic were increased by a factor of 3 to 6 due to single mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC50 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 longterm desensitization induced by Epi.