Acetylcholine Sensitivity Research Articles

The Role of Myosins in Depression of Neuron Sensitivity to Acetylcholine in a Cellular Analog of Habituation in the Common Snail

The involvement of cytoskeletal motor proteins, i.e., myosins, in the molecular mechanism of depression of the acetylcholine sensitivity of defensive behavior command neurons was studied in the common snail. Thus, the effects of compounds impairing myosin functioning on ACh-evoked current depression curves in neurons were studied – the myosin light chain kinase blockers ML-7 and MLCK-IP-18, the non-muscle myosin II inhibitor blebbistatin, and the ROCK-I and ROCK-II kinase (which mainly activate non-muscle myosin II) inhibitor Y-27632. ML-7 and MLCK-IP-18 were found to weaken depression of the current, while blebbistatin and Y-27632 had no effect on depression. The experimental results and mathematical models of the effects of these blockers on the number of membrane-bound cholinoreceptors suggest the involvement of myosins (except non-muscle myosin II) in the endo- and exocytosis of cholinoreceptors and the resultant depression of the cholinosensitivity of the somatic membranes of neurons in a cellular analog of habituation.

Effects of ovarian hormone treatment on the gene expression of muscarinic acetylcholine receptors in the ovariectomized rat myometrium

We investigate the effects of ovarian hormone on the gene expression of muscarinic acetylcholine receptors (M1–M5) in the myometrium using real-time PCR and evaluate the relationships between their expression and that of ovarian hormone receptors (ERα, ERβ, and PgR). Wistar rats were sham operated (SO) or ovariectomized (OVX) and treated with vehicle, estradiol (E2), progesterone (P4), or both E2 and P4 for 2 days beginning on postoperative day 33.M1 and M4 mRNA expressions were not detected in the myometrium. M2 mRNA expression did not change significantly in the OVX and OVX+P4 groups compared to the SO group, but increased significantly in the OVX+E2 group and was normalized in the OVX+E2P4 group. M3 mRNA expression increased significantly in the OVX and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2 and OVX+E2P4 groups. M5 mRNA expression did not change significantly in all experimental groups. ERα mRNA expression increased significantly in the OVX, OVX+E2, and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2P4 group. The changes in ERβ mRNA expression were similar to those of M3 mRNA expression in all experimental groups. In contrast, the changes in PgR mRNA expression did not correspond with that of M2, M3, or M5 mRNA expression in any of the experimental groups.Additionally, we evaluated the relationship between the expression of muscarinic acetylcholine receptors and ovarian hormone receptors in estrus cycle. M2 mRNA expression increased significantly in diestus and metaestrus compared in proestrus and estrus. M3 mRNA expression increased significantly in only diestrus compared in the other stages. In contrast, M5 mRNA expression did not change in estrus cycle. The changes in ERα mRNA expression appeared to be similar to those of M2 in estrus cycle, but no significant difference was found. The changes in ERβ mRNA expression were similar to those of M3 mRNA expression. The change in PgR mRNA expression increased significantly in diestrus compared in metaestrus, but did not correspond with that of M2, M3, or M5 mRNA expression in estrus cycle. When acetylcholine sensitivity in the myometrium was compared between diestrus and estrus, the sensitivity is significantly lower in estrus than in diestrus.These results suggest that ovarian hormones influence the expression of M2 and M3 in the myometrium by regulating the expression of hormone receptors. E2 may upregulate M2 via ERα, but P4 may downregulate M2 by inhibiting ERα via PgR. E2 may downregulate M3 by inhibiting ERβ, but P4 may not regulate the expression of M3 and ERβ. M5 may be a constitutive muscarinic receptor in the myometrium because neither E2 nor P4 influence the expression of M5. The combination of E2 and P4 may contribute the reproduction by quieting down the acetylcholine-induced myometrial contraction.

Two Distinct Allosteric Binding Sites at α4β2 Nicotinic Acetylcholine Receptors Revealed by NS206 and NS9283 Give Unique Insights to Binding Activity-associated Linkage at Cys-loop Receptors

Positive allosteric modulators (PAMs) of α4β2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4β2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4β2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4β2 stoichiometries (2α:3β and 3α:2β). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (Emax(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2β receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2β receptors, which results in current levels exceeding Emax(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4β2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging.

Neonatal Lesions to the Catecholaminergic System Prevents Formation of the Cholinergic Innervation of the Rat Neocortex

The studies reported here addressed the effects of the catecholaminergic system on the functioning of the somatosensory and cholinergic systems during early ontogeny in rats. Systemic administration of the neurotoxin 6-hydroxydopamine (6-OHDA) to neonates was found to modify the sensitivity of somatosensory cortex neurons to peripheral stimulation (stimulation of the sciatic nerve). Animals with lesioned catecholaminergic systems showed decreases in the numbers of neocortical neurons responding to stimulation of the ascending cholinergic pathways. The acetylcholine sensitivity of these neurons did not change. It is suggested that damage to the function-regulating catecholaminergic mechanisms during early ontogeny prevents maturation of the cholinergic afferents of the neocortex.

Tibetan herbal formula Padma Digestin modulates gastrointestinal motility in vitro

To examine the effects of Padma Digestin on the smooth muscle motility of different gastrointestinal segments in vitro. The effects of the ethanolic extract of Padma Digestin (at 8.16 mg/mL or 81.6 mg/mL) on the contractility and susceptibility to acetylcholine (ACh) of muscle strips from the cardia, antrum, pylorus, duodenum, jejunum, ileum and colon of male Wistar rats were analyzed. Compared with the control treatment, the Padma Digestin extract had a procontractile effect on the antral smooth muscle strips. Padma Digestin decreased ACh sensitivity in cardia muscle strips and increased it in those from the antrum and pylorus. In the intestinal segments, spontaneous contractility was inhibited in both the duodenal and jejunal strips, whereas reactivity to ACh was inhibited in the jejunal strips only. In the colonic samples, Padma Digestin inhibited spontaneous and ACh-stimulated contractility at a low dose but seems to have increasing effects at a high dose. Padma Digestin extract has region-specific effects on the contractility and excitability of gastrointestinal smooth muscle. Our results support the traditional use of Padma Digestin for maldigestion and functional gastrointestinal disorders.

Physiological and comparative proteomic analyses in response to nitrogen application in an Amaryllidaceae plant, Lycoris aurea

Galanthamine is used as a strong reversible inhibitor of cholinesterase to increase acetylcholine sensitivity. Here, we sought to determine the effect of nitrogen (N) application on physiological and proteomic properties of Lycoris aurea (L. aurea) in order to explore the factors that influence galanthamine biosynthesis. A marked increase in plant growth was observed under absent nitrogen condition (0 mmol L−1 N), while a clear stunting of growth was seen under high nitrogen condition (3.57 mmol L−1 N). Levels of photosynthetic pigments also showed a preference for the absent nitrogen condition. The leaves and roots of absent nitrogen plants had significantly higher galanthamine content and lower contents of free amino acids and proline compared with those of control (0.75 mmol L−1 N). On the other hand, galanthamine content in L. aurea was significantly reduced with high nitrogen treatment. However, the contents of free amino acids and proline were increased with high nitrogen application, suggesting that these play important roles in the storage of nitrogen in L. aurea. Next, we carried out proteomic analysis of the leaves, bulbs and roots of L. aurea treated with nitrogen by two-dimensional gel electrophoresis. Using comparative proteomic strategies, we found 11 proteins that showed significant differences among the three groups. These proteins are involved in a variety of cellular processes including energy metabolism, scavenging oxygen free radicals, cytoskeleton stabilization, galanthamine biosynthesis and cellular homeostasis. Out of the 11 proteins, the level of phenylalanine ammonia-lyase was found to correlate with that of galanthamine biosynthesis.

Muscle-specific kinase myasthenia gravis IgG4 autoantibodies cause severe neuromuscular junction dysfunction in mice

Myasthenia gravis is a paralytic disorder with autoantibodies against acetylcholine receptors at the neuromuscular junction. A proportion of patients instead has antibodies against muscle-specific kinase, a protein essential for acetylcholine receptor clustering. These are generally of the immunoglobulin-G4 subclass and correlate with disease severity, suggesting specific myasthenogenic activity. However, immunoglobulin-G4 subclass antibodies are generally considered to be 'benign' and direct proof for their pathogenicity in muscle-specific kinase myasthenia gravis (or other immunoglobulin-G4-associated disorders) is lacking. Furthermore, the exact electrophysiological synaptic defects caused at neuromuscular junctions by human anti-muscle-specific kinase autoantibodies are hitherto unknown. We show that purified immunoglobulin-G4, but not immunoglobulin-G1-3, from patients with muscle-specific kinase myasthenia gravis binds to mouse neuromuscular junctions in vitro, and that injection into immunodeficient mice causes paralysis. Injected immunoglobulin-G4 caused reduced density and fragmented area of neuromuscular junction acetylcholine receptors. Detailed electrophysiological synaptic analyses revealed severe reduction of postsynaptic acetylcholine sensitivity, and exaggerated depression of presynaptic acetylcholine release during high-rate activity, together causing the (fatigable) muscle weakness. Intriguingly, compensatory transmitter release upregulation, which is the normal homeostatic response in acetylcholine receptor myasthenia gravis, was absent. This conveys extra vulnerability to neurotransmission at muscle-specific kinase myasthenia gravis neuromuscular junctions. Thus, we demonstrate that patient anti-muscle-specific kinase immunoglobulin-G4 is myasthenogenic, independent of additional immune system components, and have elucidated the underlying electrophysiological neuromuscular junction abnormalities.

Rapid Plasticity of Visual Responses in the Adult Lateral Geniculate Nucleus

Compared to the developing visual system, where neuronal plasticity has been well characterized at multiple levels, little is known about plasticity in the adult, particularly within subcortical structures. We made intraocular injections of 2-amino-4-phosphonobutyric acid (APB) in adult cats to block visual responses in On-center retinal ganglion cells and examined the consequences on visual responses in the lateral geniculate nucleus (LGN) of the thalamus. In contrast to current views of retinogeniculate organization, which hold that On-center LGN neurons should become silent with APB, we find that ∼50% of On-center neurons rapidly develop Off-center responses. The time course of these emergent responses and the actions of APB in the retina indicate the plasticity occurs within the LGN. These results suggest there is greater divergence of retinogeniculate connections than previously recognized and that functionally silent, nonspecific retinal inputs can serve as a substrate for rapid plasticity in the adult.

Modification of drug responses by hydrolytic enzymes.

Abstract α-Chymotrypsin and phospholipase enzymes have been used to alter the cell membranes of the smooth muscle of the guinea-pig taenia coli. The effects of these treatments on the electrical and mechanical responses of the muscle to acetylcholine have been studied. Prolonged treatment with both enzymes causes depolarization with a consequent loss of acetylcholine sensitivity. After controlled trypsinization a membrane response to acetylcholine was obtained in the absence of a mechanical response. The sensitivity of the taenia coli to acetylcholine was depressed by controlled phospholipolysis without affecting the membrane potential or contractile apparatus. The implications of these findings are discussed.

Human α3β4 Neuronal Nicotinic Receptors Show Different Stoichiometry if They Are Expressed in Xenopus Oocytes or Mammalian HEK293 Cells

BackgroundThe neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the α3 and β4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the α5 and/or β2 subunits. We compared the properties of human α3β4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and α∶β subunit ratio.Methodology/Principal FindingsTwo distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the α subunit, as reported for α4β2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9′ of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike α4β2 channels, for α3β4 receptors the putative two-α form is the predominant one in oocytes (at 1∶1 α∶β cRNA ratio). This two-α form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-α form is the predominant one in HEK cells transfected with a 1∶1 α∶β DNA ratio or in oocytes at 9∶1 α∶β RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-α form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-α form gave rise to short bursts (14 ms) of high conductance (39 pS).Conclusions/SignificanceLike other neuronal nicotinic receptors, the α3β4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected.

86Rb + efflux mediated by α4β2*-nicotinic acetylcholine receptors with high and low-sensitivity to stimulation by acetylcholine display similar agonist-induced desensitization

The nicotinic acetylcholine receptors (nAChR) assembled from α4 and β2 subunits are the most densely expressed subtype in the brain. Concentration–effect curves for agonist activation of α4β2*-nAChR are biphasic. This biphasic agonist sensitivity is ascribed to differences in subunit stoichiometry. The studies described here evaluated desensitization elicited by low concentrations of epibatidine, nicotine, cytisine or methylcarbachol of brain α4β2-nAChR function measured with acetylcholine-stimulated 86Rb + efflux from mouse thalamic synaptosomes. Each agonist elicited concentration-dependent desensitization. The agonists differed in potency. However, IC 50 values for each agonist for desensitization of 86Rb + efflux both with high (EC 50 ≈ 3 μM) and low (EC 50 ≈ 150 μM) acetylcholine sensitivity were not significantly different. Concentrations required to elicit desensitization were higher that their respective K D values for receptor binding. Even though the two components of α4β2*-nAChR-mediated 86Rb + efflux from mouse brain differ markedly in EC 50 values for agonist activation, they are equally sensitive to desensitization by exposure to low agonist concentrations. Mice were also chronically treated with nicotine by continuous infusion of 0, 0.5 or 4.0 mg/kg/h and desensitization induced by nicotine was evaluated. Consistent with previous results, chronic nicotine treatment increased the density of epibatidine binding sites. Acute exposure to nicotine also elicited concentration-dependent desensitization of both high-sensitivity and low-sensitivity acetylcholine-stimulated 86Rb + efflux from cortical and thalamic synaptosomes. Although chronic nicotine treatment reduced maximal 86Rb + efflux from thalamus, IC 50 values in both brain regions were unaffected by chronic nicotine treatment.

Deletion of protein tyrosine phosphatase 1B (PTP1B) prevents type 1 diabetes‐induced endothelial dysfunction.

PTP1B dephosphorylates receptors tyrosine kinase and acts as a brake on various signaling pathways. Deletion of PTP1B protects against metabolic dysfunction by increasing insulin sensitivity, but little is known about its role in the control of vascular function. We hypothesized that PTP1B deletion prevents vascular dysfunction in the absence of insulin. Insulin secretion was abolished in Balb/C and PTP1B‐deficient mice with streptozotocin (STZ) injection. 24 days after treatment, aortic reactivity was assessed via myography. STZ induced similar increases in glycemia in Balb/C and PTP1B KO mice (384±24 vs 444±21 mg/dL, Balb/C vs PTP1B). Phenylephrine (PE)‐induced constriction was increased by STZ treatment in Balb/C mice only (115±13 vs 136±6% of KCl, vehicle vs STZ). Endothelial function assessed via acetylcholine (ACh)‐mediated relaxation was not affected by PTP1B deficiency but was reduced by STZ in Balb/C mice (63±5 vs 43±5% of precontraction, vehicle vs STZ). In PTP1B KO mice, STZ treatment did not affect maximum relaxation (56±7 vs 58±7% of preconstriction, vehicle vs STZ) but increased ACh sensitivity (logEC50 7.4±0.3 vs 8.2±0.2, vehicle vs STZ). Western blot analysis revealed a trend towards increased baseline Akt activation without changes in eNOS expression or activation. These data indicate that PTP1B deletion protects against the harmful vascular effects of type I diabetes.

Motor Axonal Sprouting and Neuromuscular Junction Loss in an Animal Model of Charcot-Marie-Tooth Disease

Muscle weakness in Charcot-Marie-Tooth Type 1A disease (CMT1A) caused by mutations in peripheral myelin protein 22 (PMP22) has been attributed to an axonopathy that results in denervation and muscle atrophy. The underlying pathophysiological mechanisms involved are not understood. We investigated motor performance, neuromuscular junctions (NMJs), physiological parameters, and muscle morphometry of PMP22 transgenic mice. Neuromuscular junctions were progressively lost in hindlimb muscles of PMP22 transgenic mice, but their motor performance did not completely deteriorate during the observation period. There was considerable variability, including in laterality, in deficits among the animals. Cross-sectional areas and mean fiber size measurements indicated variable myofiber atrophy in hindlimb muscles. There was substantial concomitant axonal sprouting, and loss of neuromuscular junctions was inversely correlated with the accumulated length of axonal branches. Synaptic transmission studied in isolated nerve/muscle preparations indicated variable partial muscle denervation. Acetylcholine sensitivity was higher in the mutant muscles, and maximum tetanic force evoked by direct or indirect stimulation, specific force, and wet weights were markedly reduced in some mutant muscles. In summary, there is partial muscle denervation, and axons may retain some regenerative capacity but fail to reinnervate muscles in PMP22 transgenic mice.

Effect of the Heat-exposure on Peripheral Sudomotor Activity Including the Density of Active Sweat Glands and Single Sweat Gland Output

Tropical inhabitants are able to tolerate heat through permanent residence in hot and often humid tropical climates. The goal of this study was to clarify the peripheral mechanisms involved in thermal sweating pre and post exposure (heat-acclimatization over 10 days) by studying the sweating responses to acetylcholine (ACh), a primary neurotransmitter of sudomotor activity, in healthy subjects (n=12). Ten percent ACh was administered on the inner forearm skin for iontophoresis. Quantitative sudomotor axon reflex testing, after iontophoresis (2 mA for 5 min) with ACH, was performed to determine directly activated (DIR) and axon reflex-mediated (AXR) sweating during ACh iontophoresis. The sweat rate, activated sweat gland density, sweat gland output per single gland activated, as well as oral and skin temperature changes were measured. The post exposure activity had a short onset time (p<0.01), higher active sweat rate [(AXR (p<0.001) and DIR (p<0.001)], higher sweat output per gland (p<0.001) and higher transepidermal water loss (p<0.001) compared to the pre-exposure measurements. The activated sweat rate in the sudomotor activity increased the output for post-exposure compared to the pre-exposure measurements. The results suggested that post-exposure activity showed a higher active sweat gland output due to the combination of a higher AXR (DIR) sweat rate and a shorter onset time. Therefore, higher sudomotor responses to ACh receptors indicate accelerated sympathetic nerve responsiveness to ACh sensitivity by exposure to environmental conditions.

Reduced oxidant stress, increased NO‐dependent vasodilatation, and improved endothelial function with voluntary exercise in old mice: another excuse for long walks on the beach

The study by Durrant and coworkers (2009) in this issue of The Journal of Physiology investigates the effect of aerobic exercise on endothelium-dependent (vaso)dilatation (EDD) in B6D2F1 mice, a model of age-associated endothelial dysfunction (Lesniewski et al. 2009). The authors note that increasing age is a major risk factor for cardiovascular disease (Lakatta & Levy, 2003) that is likely to be mediated via increased oxidative stress and reduced NO levels (Cai & Harrison, 2000; Taddei et al. 2001). Current evidence indicates that habitual aerobic exercise improves EDD in older humans by reducing oxidative stress and increasing NO availability (Seals et al. 2008). The multiple beneficial effects of exercise that the authors report in the older mice are consistent with existing studies in humans, but provide more detailed insight into the potential mechanisms by which habitual aerobic exercise may improve EDD with age and potentially reduce age-associated risks of cardiovascular disease. The study evaluates multiple indices of EDD from the whole animal level to the vascular and enzymatic level in young and old mice with and without access to an exercise wheel for voluntary running. The authors report that voluntary exercise ameliorates the decreased whole body sensitivity to systemic infusion of acetylcholine in conscious older mice. The improvement in whole body acetylcholine sensitivity in older mice was accompanied by a restoration of both the maximum capacity for EDD and the NO-dependent component of EDD in the carotid artery, increased eNOS expression and activation (Ser1177 phosphorylation) in the aorta, and reduced nitrotyrosine levels in the aorta. The beneficial effects of voluntary exercise in the older mice are mediated via effects on both pro-oxidant and antioxidant mechanisms. For example, exercise not only increased total SOD activity, MnSOD activity, and Cu/Zn SOD + ecSOD activity in aortas of the older mice, but also ameliorated the increased expression of the p67 subunit of NADPH oxidase and the increased NADPH oxidase activity in aortas of the older mice. An important and striking observation in this study was that the beneficial effects of voluntary exercise in the older mice occurred even though their exercise levels were only about 10% of those in the younger mice. The importance of endothelial dysfunction and vascular oxidant stress in contributing to cardiovascular morbidity and mortality in humans was strongly emphasized in a recent review (Widlansky et al. 2003) citing 10 different studies showing that the presence of endothelial dysfunction is predictive of future adverse cardiovascular events (including death), and suggesting that evaluation of endothelial function in peripheral arteries may be beneficial in guiding the diagnosis and treatment of cardiovascular disease. In this regard, the results of the present study raise the possibility that voluntary exercise might not only reduce the risk of cardiovascular disease in older asymptomatic humans (see Lakatta and Levy, 2003), but also be beneficial in ameliorating more serious conditions such as peripheral artery disease, which increases with age and is associated with increased cardiovascular morbidity and mortality (McDermott et al. 2008; Sakamoto et al. 2009). The latter possibility is supported by studies showing that antioxidant treatment with glutathione infusion increases pain free walking distance and improves several indicators of microcirculatory and whole bed blood flow in humans with peripheral artery disease (Arosio et al. 2002) and that peripheral artery disease patients completing a supervised exercise training programme show reduced cardiovascular morbidity and mortality (Sakamoto et al. 2009).

Human Nocturnal Frontal Lobe Epilepsy: Pharmocogenomic Profiles of Pathogenic Nicotinic Acetylcholine Receptor β-Subunit Mutations outside the Ion Channel Pore

Certain mutations in specific parts of the neuronal nicotinic acetylcholine receptor (nAChR) subunit genes CHRNA4, CHRNB2, and probably CHRNA2, can cause autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). All but one of the known causative mutations are located in the second transmembrane region (TM2), which serves as the major ion poreforming domain of the receptor. Functional characterization of these ADNFLE mutations has shown that although each mutant exhibits specific properties, they all confer a gain of function with increased sensitivity to acetylcholine. In this work, we characterize the second and third ADNFLE-associated mutations that are external to TM2 but affect different amino acid residues within the third transmembrane region (TM3). The two new CHRNB2 mutations were identified in three families of Turkish Cypriot, Scottish, and English origin. These TM3 mutations elicit the same gain of function pathomechanism as observed for the TM2 mutations with enhanced acetylcholine sensitivity, despite their unusual localization within the gene. Electrophysiological experiments, including single channel measurements, revealed that incorporation of these new mutant subunits does not affect the conductance of the ionic pore but increases the probability of opening. Determination of the sensitivity to nicotine for nAChRs carrying mutations in TM2 and TM3 showed clear differences in the direction and the extent to which the window current for nicotine sensitivity was shifted for individual mutations, indicating differences in pharmacogenomic properties that are not readily correlated with increased ACh affinity.

Partial Deletion of the Nicotinic Cholinergic Receptor α4 or β2 Subunit Genes Changes the Acetylcholine Sensitivity of Receptor-Mediated86Rb+Efflux in Cortex and Thalamus and Alters Relative Expression of α4 and β2 Subunits

Alpha4 and beta2 nicotinic cholinergic receptor (nAChR) subunits can assemble in heterologous expression systems as pentameric receptors with different subunit stoichiometries that exhibit differential sensitivity to activation by acetylcholine, yielding biphasic concentration-effect curves. nAChR-mediated (86)Rb(+) efflux in mouse brain synaptosomes also displays biphasic acetylcholine (ACh) concentration-response curves. Both phases are mediated primarily by alpha4beta2(*)-nAChR, because deletion of either the alpha4 or beta2 subunit reduces response at least 90%. A relatively larger decrease in the component of (86)Rb(+) efflux with lower ACh sensitivity occurred with partial deletion of alpha4 (alpha4(+/-)), whereas a larger decrease in the component with higher ACh sensitivity was elicited by partial deletion of beta2 (beta2(+/-)). Immunoprecipitation with selective antibodies demonstrated that more than 70% of [(3)H]epibatidine binding sites in both regions contained only alpha4 and beta2 subunits. Subsequently, alpha4 and beta2 subunit content in the cortex and thalamus of alpha4 and beta2 wild types and heterozygotes was analyzed with Western blots. Partial deletion of alpha4 decreased and partial deletion of beta2 increased the relative proportion of the alpha4 subunit in assembled receptors. Although these methods do not allow exact identification of stoichiometry of the subtypes present in wild-type cortex and thalamus, they do demonstrate that cortical and thalamic nAChRs of the alpha4(+/-) and beta2(+/-) genotypes differ in relative expression of alpha4 and beta2 subunits a result that corresponds to the relative functional changes observed after partial gene deletion. These results strongly suggest that alpha4beta2-nAChR with different stoichiometry are expressed in native tissue.

Properties of mutated murine α4β2 nicotinic receptors linked to partial epilepsy

We characterized, by electrophysiological methods, two biophysical properties of murine recombinant alpha4beta2 nicotinic acetylcholine receptors (nAChR) bearing a mutation (alpha4:+L264alpha4:beta2 or alpha4:S252Falpha4:beta2) linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Sensitivity to acetylcholine (ACh) was increased by the S252F substitution expressed in heterozygosis (alpha4:S252Falpha4:beta2) but was markedly reduced when this mutation was expressed in homozygosis (S252Falpha4:beta2). ACh sensitivity was not altered by the +L264 insertion. Moreover, receptor desensitization was significantly increased by both mutations expressed in heterozygosis. These results are in general agreement to those of rat and human recombinant receptors bearing the same mutations, thus contributing to validate the use of knock-in mice harboring ADNFLE mutations as models to study this pathology.

Ricardo Miledi and the foundations of synaptic and extra‐synaptic neurotransmitter receptor physiology

Ricardo Miledi and the foundations of synaptic and extra‐synaptic neurotransmitter receptor physiology

Acute Late Sepsis Attenuates Effects of a Nondepolarizing Neuromuscular Blocker, Rocuronium, by Facilitation of Endplate Potential and Enhancement of Membrane Excitability In Vitro

Sepsis attenuates the muscle-relaxing effects of nondepolarizing neuromuscular blockers. The authors investigated the effects of acute late sepsis on neuromuscular transmission and neuromuscular actions of rocuronium to clarify the mechanisms by which sepsis attenuates the effects of nondepolarizing neuromuscular blockers. Sepsis was induced by cecal ligation and puncture operation. Endplate potentials, acetylcholine potentials, and electrotonic potentials were recorded from the motor endplates of isolated diaphragms from acute late septic and nonseptic rats. (1) Sepsis did not influence the effect of rocuronium to decrease endplate potential amplitude, which was increased by sepsis itself; (2) sepsis facilitated the effect of rocuronium to decrease quantal acetylcholine release, which was increased by sepsis itself; (3) sepsis did not influence the effect of rocuronium to decrease acetylcholine sensitivity, which was decreased by sepsis itself; (4) sepsis decreased critical depolarization, and rocuronium did not influence critical depolarization. These results indicate that acute late sepsis facilitates endplate potentials and enhances excitability of the muscle membrane, indicated by a decrease of critical depolarization. It is thought that these elicit the sepsis-induced attenuation of the muscle-relaxing effects of rocuronium.