Use-Dependent Suppression of the Nicotinic Acetylcholine Receptor Response by the Proadrenomedullin N-Terminal 20-Amino Acid Peptide in Rat Locus Coeruleus Neurons

Abstract: The effects of the proadrenomedullin N-terminal 20-amino acid peptide (PAMP) on the nicotinic acetylcholine (ACh) receptor (nAChR)-mediated inward current were investigated in neurons acutely dissociated from the rat locus coeruleus using whole-cell recording under voltage clamp. Nicotine and cytidine mimicked the ACh response, whereas the maximal response to dimethyl-phenylpiperazinium was lower in amplitude compared with that to ACh. Nicotine-induced current ( I _nic) was suppressed more effectively by mecamylamine than by hexamethonium. In addition, neither atropine nor α-bungarotoxin affected the I _nic. PAMP reversibly and noncompetitively suppressed the peak amplitude of 10⁻⁴ M I _nic. PAMP concentrations for the threshold, half-maximal inhibition, and maximal inhibition of 10⁻⁴ M I _nic were 10⁻⁸, 2.6 × 10⁻⁷, and 10⁻⁵ M , respectively. The peak amplitudes of 10⁻⁴ M I _nic elicited at 2-min intervals showed a gradual decline in the presence of 10⁻⁷ M PAMP. This decline in the I _nic was independent of the period of PAMP pretreatment. The suppression of I _nic by PAMP did not show any voltage dependency at a holding potential ( V _H) of <0 mV, although the inhibitory effect was masked by the marked inward rectification of I _nic at a V _H of 0 mV. These results suggest that PAMP could thus be a unique endogenous peptide that antagonizes the nAChR in the CNS.     

Mechanism Underlying the Effect of Mecamylamine on Nicotinic Acetylcholine Receptors of Rat Sympathetic Ganglion Neurons

On neurons of the superior cervical ganglion of 3-week-old rats, we studied the mechanism underlying the blocking effect of mecamylamine on transmembrane currents evoked by iontophoretic application of acetylcholine (ACh currents); these currents were recorded with the use of a patch-clamp technique in the whole-cell configuration. The IC₅₀ of the above agent equaled (2.7 ± 0.3) · 10^-10 M. The blocking effect of mecamylamine on ACh current did not depend on the membrane potential and decreased with rise in the concentration of the drug. Thus, a competitive blocking mechanism mostly underlies the above phenomenon.     

Ion Channel Topography of the Neuronal Nicotinic Acetylcholine Receptor: Pharmacochemical Approaches

Forty-three bisammonium ganglionic blockers were synthesized to study the structure of the ion channel of nicotinic acetylcholine receptor. The conformational parameters of these blockers were studied, and their effects toward the ganglionic transmission in situ on the sympathetic feline superior cervical ganglia and in vitro on the parasympathetic guinea-pig small intestine ganglia were determined. A model of the binding site for the bisammonium ganglionic blockers in the neuronal ion channel was proposed.     

昆虫离子通道及其抗药性研究进展

昆虫细胞膜离子通道是多种杀虫剂的作用靶标,通道功能特性的变异等与害虫抗药性密切相关.电压钳及膜片钳等电生理技术在离子通道功能研究中具有独特优势,在杀虫剂作用机理及害虫抗性机理研究中越来越受到重视.昆虫细胞膜离子通道主要包括配体门控通道和电压门控通道两大类.配体门控通道主要包括乙酰胆碱受体、GABA和谷氨酸受体通道等.电压门控通道主要有钠、钾和钙通道等,其中钠通道研究成果较多,与害虫抗性关系密切.由于钙离子的重要生理功能,随着研究深入,钙通道将成为研究重点.     

The Nicotinic Acetylcholine Receptor: Structure and Autoimmune Pathology

Abstract

The nicotinic acetylcholine receptors (AChR) are presently the best-characterized neurotransmitter receptors. They are pentamers of homologous or identical subunits, symmetrically arranged to form a transmembrane cation channel. The AChR subunits form a family of homologous proteins, derived from a common ancestor. An autoimmune response to muscle AChR causes the disease myasthenia gravis. This review summarizes recent developments in the understanding of the AChR structure and its molecular recognition by the immune system in myasthenia.     

Subtype Identification in Acutely Dissociated Rat Nodose Ganglion Neurons Based on Morphologic Parameters

Nodose ganglia are composed of A-, Ah- and C-type neurons. Despite their important roles in regulating visceral afferent function, including cardiovascular, pulmonary, and gastrointestinal homeostasis, information about subtype-specific expression, molecular identity, and function of individual ion transporting proteins is scarce. Although experiments utilizing the sliced ganglion preparation have provided valuable insights into the electrophysiological properties of nodose ganglion neuron subtypes, detailed characterization of their electrical phenotypes will require measurements in isolated cells. One major unresolved problem, however, is the difficulty to unambiguously identify the subtype of isolated nodose ganglion neurons without current-clamp recording, because the magnitude of conduction velocity in the corresponding afferent fiber, a reliable marker to discriminate subtypes in situ, can no longer be determined. Here, we present data supporting the notion that application of an algorithm regarding to microscopic structural characteristics, such as neuron shape evaluated by the ratio between shortest and longest axis, neuron surface characteristics, like membrane roughness, and axon attachment, enables specific and sensitive subtype identification of acutely dissociated rat nodose ganglion neurons, by which the accuracy of identification is further validated by electrophysiological markers and overall positive predictive rates is 89.26% (90.04%, 76.47%, and 98.21% for A-, Ah, and C-type, respectively). This approach should aid in gaining insight into the molecular correlates underlying phenotypic heterogeneity of nodose ganglia. Additionally, several critical points that help for neuron identification and afferent conduction calibration are also discussed.     

Properties of Proton-Gated Ion Channels in Sensory Neurons of Rats

In experiments on the somata of sensory neurons isolated from the spinal and trigeminal ganglia of rats, we characterized three subclasses of proton-gated currents differing from each other in their kinetics of desensitization and characteristics of stationary desensitization (but not in the characteristics of stationary activation). A voltage clamp technique in the whole cell configuration and intracellular perfusion were used. Expression of the channels providing currents of each subclass depended on the soma diameter but not on anatomical localization of the neuron. Proton-gated channels of type I were characterized by mono- or biexponential kinetics of current desensitization with the duration of complete decay within a 1 to 15 sec range; the mean pH₅₀ of the curve of stationary desensitization was 7.21 ± 0.02. Channels of type II possessed mostly monoexponential desensitization kinetics with the duration of decay within a 1 to 3 sec range; their pH₅₀ of the stationary desensitization curve was 7.11 ± 0.02. Channels of type III showed mostly biexponential desensitization kinetics; the complete current decay lasted about 5 sec, while the mean pH₅₀ was about 6.78 ± 0.02. Channels of type I were typical of small neurons (soma diameter 10-20 m), while those of types II and III were found mostly in large cells (35-60 m).     

大鼠急性分离下丘脑神经元的自发性放电特性

目的和方法：采用电流钳技术,观察SD大鼠急性分离下丘脑神经元的自发性放电。结果：放电形式包括不放电、连续的单个放电和簇状放电等3种,但放电过程中出现一些阈下电位,这些闯下电位会明显影响神经元的放电过程,使放电表现为规则或不规则,规则放电通常由缓慢的去极化电位触发,而不规则放电常出现较多的阈下电位。结论：急性分离神经元具有与脑片和培养神经元不完全相同的放电特性,对此展开研究,有利于揭示下丘脑参与机体稳态的调控机制。     

Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates

We identify two heteroallelic mutations in the acetylcholine receptor δ-subunit from a patient with severe myasthenic symptoms since birth: a novel δD140N mutation in the signature Cys-loop and a mutation in intron 7 of the δ-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that δD140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the α-, β-, and ϵ-subunits also suppresses apparent channel gating, but the suppression is much greater in the α-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that αAsp-138 is energetically coupled to αArg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved αAsp-138 and αArg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.     

Ion Conduction through C-Type Inactivated Shaker Channels

C-type inactivation of Shaker potassium channels involves entry into a state (or states) in which the inactivated channels appear nonconducting in physiological solutions. However, when Shaker channels, from which fast N-type inactivation has been removed by NH₂-terminal deletions, are expressed in Xenopus oocytes and evaluated in inside-out patches, complete removal of K⁺ ions from the internal solution exposes conduction of Na⁺ and Li⁺ in C-type inactivated conformational states. The present paper uses this observation to investigate the properties of ion conduction through C-type inactivated channel states, and demonstrates that both activation and deactivation can occur in C-type states, although with slower than normal kinetics. Channels in the C-type states appear “inactivated” (i.e., nonconducting) in physiological solutions due to the summation of two separate effects: first, internal K⁺ ions prevent Na⁺ ions from permeating through the channel; second, C-type inactivation greatly reduces the permeability of K⁺ relative to the permeability of Na⁺, thus altering the ion selectivity of the channel.     

Linear Free-Energy Relationships and the Dynamics of Gating in the Acetylcholine Receptor Channel

The muscle acetylcholine receptor channel (AChR) is a large (M_r 290K) transmembrane protein that mediates synaptic transmission. Theactivation of this ion channel can be understood in the framework of athermodynamic cycle with spontaneous gating (i.e., the closed open reaction) and ligand-binding events as the elementary steps. Becauseagonists bind more tightly to the open than to the closed state, gating ofliganded receptors is more favorable than that of unliganded receptors.Accordingly, channel opening must involve two major conformationalchanges: the ACh-binding sites switch from a low-affinity to a high-affinityform, and the pore (located 45 Å away from the binding sites)switches from an ion-impermeable to an ion-permeable conformation. Togain insight into the reaction mechanism of fully-liganded gating, wecharacterized the corresponding transition state in the context of the `linearfree-energy relationships' of physical organic chemistry (-valueanalysis). Gating of fully-liganded AChRs was studied by recordingsingle-channel currents using the patch-clamp technique. Perturbations tothe wild-type receptor were either series of different mutations at individualpositions or series of different agonists. Based on the obtained `snapshot'of the gating reaction at the transition state, and aware of the lack ofinformation about the rest of the energy profile, the most parsimoniousmechanism seems to be one where opening proceeds asynchronously, withthe low-to-high affinity change at the binding sites preceding the completeopening of the distant pore.     

新霉素抑制PC12细胞烟碱受体介导的反应

新霉素是一种氨基甙类抗生素,在细胞水平可以抑制磷脂酶C介质的信号转导系统,本研究采用全细胞膜片钳技术,以大鼠肾上腺嗜铬细胞瘤细胞（PC12)为标本,观察了新霉素参考书国酰胆碱诱发电流（IACh)的影响,药理学鉴定表明,PC12细胞上的IACh是通过ACh激动烟碱受体引起的,钳制电压为－80mV时,ACh(30umol/L)诱发一内向电流;细胞外同时给予新霉素（0．01－1mmol/L)和ACh(30μmol/L)可显著抑制IACh峰值,此抑制作用迅速,可逆,呈浓度依赖性,用新霉素预处理细胞3－8min不影响其对IACh的抑制作用,用外源性蛋白激酶C（PKC）激剂激活PKC,同样可抑制IACh,而细胞内透析PKC抑制剂（PKCI19-31,0.1-5μmol/L）不影响新霉素对IACh的抑制作用,以上结果提示,新霉对PC12细胞的IACh的有抑制作用,这是一种与磷脂酶C阻断无关的药理学效应。     

Ion channels are linked to differentiation in keratinocytes

Summary In vivo and in vitro, keratinocyte differentiation is linked with increased extracellular Ca²⁺. In order to correlate ion channels with cell differentiation and investigate keratinocyte membrane responses to Ca²⁺, keratinocyte single channel currents were studied using the patch-clamp technique. The most frequently observed channel was a 14 pS nonspecific cation channel. This channel was permeable to Ca²⁺ and activated by physiological concentrations of Ca²⁺. We also found a 35 pS Cl^– channel whose open probability increased with depolarization. Finally, a 70 pS K⁺ channel was seen only in cell-attached or nystatin-permeabilized patches. We correlated channel types with staining for involucrin, an early marker of keratinocyte differentiation. While the nonspecific cation channel and Cl^– channel were seen in both involucrin positive and involucrin negative cells, all channels in which the K⁺ channel activity was present were involucrin positive. Membrane currents through these channels may be one pathway by which signals for keratinocyte proliferation or differentiation are sent.This work was supported in part by a National Institutes of Health grant K08 AR01853-03 and a National Science Foundation grant DCB-9009915 (to T.M.M.); National Institutes of Health Research Career Development Award K04 ARO 1803 and AR 39031 (to R.R.I.) and a National Institutes of Health grant GM-44840 (to P.A.P.).     

IP3-Gated Channels and their Occurrence Relative to CNG Channels in the Soma and Dendritic Knob of Rat Olfactory Receptor Neurons

Olfactory receptor neurons respond to odorants with G protein-mediated increases in the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) and/or inositol-1,4,5-trisphosphate (IP3). This study provides evidence that both second messengers can directly activate distinct ion channels in excised inside-out patches from the dendritic knob and soma membrane of rat olfactory receptor neurons (ORNs). The IP3-gated channels in the dendritic knob and soma membranes could be classified into two types, with conductances of 40 +/- 7 pS (n = 5) and 14 +/- 3 pS (n = 4), with the former having longer open dwell times. Estimated values of the densities of both channels from the same inside-out membrane patches were very much smaller for IP3-gated than for CNG channels. For example, in the dendritic knob membrane there were about 1000 CNG channels x microm(-2) compared to about 85 IP3-gated channels x microm(-2). Furthermore, only about 36% of the dendritic knob patches responded to IP3, whereas 83% of the same patches responded to cAMP. In the soma, both channel densities were lower, with the CNG channel density again being larger ( approximately 57 channels x microm(-2)) than that of the IP3-gated channels ( approximately 13 channels x microm(-2)), with again a much smaller fraction of patches responding to IP3 than to cAMP. These results were consistent with other evidence suggesting that the cAMP-pathway dominates the IP3 pathway in mammalian olfactory transduction.     

Cloning of a Putative Neuronal Nicotinic Acetylcholine Receptor Subunit

A cDNA clone was isolated from a rat superior cervical ganglion cDNA library with an oligonucleotide that hybridized to muscle-like nicotinic acetylcholine receptor (nAChR) subunit cDNA. The deduced amino acid sequence possesses characteristics expected of a nAChR subunit that does not bind acetylcholine, in addition to distinctive features such as unique cysteine residues and N-linked glycosylation sites.     

Gating of Single N-type Calcium Channels Recorded from Bullfrog Sympathetic Neurons

For many neurons, N-type calcium channels provide the primary pathway for calcium influx during an action potential. We investigated the gating properties of single N-type calcium channels using the cell-attached patch technique. With 100 mM Ba²⁺ in the pipet, mean N-channel open probability (P _o, measured over 100 ms) increased with depolarization, but the range at a single voltage was large (e.g., P _o at +40 mV ranged from 0.1 to 0.8). The open dwell time histograms were generally well fit by a single exponential with mean open time (τ_o) increasing from 0.7 ms at +10 mV to 3.1 ms at +40 mV. Shut time histograms were well fit by two exponentials. The brief shut time component (τ_sh1 = 0.3 ms) did not vary with the test potential, while the longer shut time component (τ_sh2) decreased with voltage from 18.9 ms at +10 mV to 2.3 ms at +40 mV. Although N-channel P _o during individual sweeps at +40 mV was often high (∼0.8), mean P _o was reduced by null sweeps, low P _o gating, inactivation, and slow activation. The variability in mean P _o across patches resulted from differences in the frequency these different gating processes were expressed by the channels. Runs analysis showed that null sweeps tended to be clustered in most patches, but that inactivating and slowly activating sweeps were generally distributed randomly. Low P _o gating (P _o = 0.2, τ_o = 1 ms at +40 mV) could be sustained for ∼1 min in some patches. The clustering of null sweeps and sweeps with low P _o gating is consistent with the idea that they result from different modes of N-channel gating. While P _o of the main N-channel gating state is high, the net P _o is reduced to a maximum value of close to 0.5 by other gating processes.     

Subunit Composition of Nicotinic Acetylcholine Receptors in Neurons of the Rat Intracardiac Ganglia

The effect of the antibodies against 3, 4, 5, and 7 subunits of neuronal nicotinic acetylcholine receptors (nAChR) on ACh-induced shifts of the membrane potential (ACh potentials) in neurons of the intracardiac ganglia of the rat left atrium was studied using intracellular electrodes. It was found that (i) antibodies against each of the above subunits caused only a partial block of ACh potential; (ii) the blocking effect of each particular antibody could be observed only in some of the examined neurons; (iii) in all neurons under study the block caused by a single antibody was only partial; (iv) the blocking effects of two or more antibodies on the ACh potential recorded from the same neuron were additive. It was concluded that (1) each of the above nAChR subunits is present only in a part of the intracardiac neurons, and (2) each intracardiac neuron has nAChR composed of more than one subunit.     

Affinity to the Nicotinic Acetylcholine Receptor and Insecticidal Activity of Chiral Imidacloprid Derivatives with a Methylated Imidazolidine Ring

Four imidacloprid derivatives with an asymmetrically methylated imidazolidine ring were synthesized. Their affinity to the nicotinic acetylcholine receptor of housefly Musca domestica and insecticidal activity against the housefly were measured. The compound with a 5R-methylated imidazolidine ring demonstrated intrinsic activity comparable to that of the unsubstituted compound. Most of the compounds were synergized by oxygenase inhibitors.     

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

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