Nicotinic cholinergic mechanisms causing elevated dopamine release and abnormal locomotor behavior.

Firing rates of dopamine (DA) neurons in substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) control DA release in target structures such as striatum and prefrontal cortex. DA neuron firing in the soma and release probability at axon terminals are tightly regulated by cholinergic transmission and nicotinic acetylcholine receptors (nAChRs). To understand the role of α6* nAChRs in DA transmission, we studied several strains of mice expressing differing levels of mutant, hypersensitive (leucine 9' to serine [L9'S]) α6 subunits. α6 L9'S mice harboring six or more copies of the hypersensitive α6 gene exhibited spontaneous home-cage hyperactivity and novelty-induced locomotor activity, whereas mice with an equal number of WT and L9'S α6 genes had locomotor activity resembling that of control mice. α6-dependent, nicotine-stimulated locomotor activation was also more robust in high-copy α6 L9'S mice versus low-copy mice. In wheel-running experiments, results were also bi-modal; high-copy α6 L9'S animals exhibited blunted total wheel rotations during each day of a 9-day experiment, but low-copy α6 L9'S mice ran normally on the wheel. Reduced wheel running in hyperactive strains of α6 L9'S mice was attributable to a reduction in both overall running time and velocity. ACh and nicotine-stimulated DA release from striatal synaptosomes in α6 L9'S mice was well-correlated with behavioral phenotypes, supporting the hypothesis that augmented DA release mediates the altered behavior of α6 L9'S mice. This study highlights the precise control that the nicotinic cholinergic system exerts on DA transmission and provides further insights into the mechanisms and consequences of enhanced DA release.

Altered activity-rest patterns in mice with a human autosomal-dominant nocturnal frontal lobe epilepsy mutation in the ß2 nicotinic receptor.

High-affinity nicotinic receptors containing ß2 subunits (ß2*) are widely expressed in the brain, modulating many neuronal processes and contributing to neuropathologies such as Alzheimer's disease, Parkinson's disease and epilepsy. Mutations in both the α4 and ß2 subunits are associated with a rare partial epilepsy, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In this study, we introduced one such human missense mutation into the mouse genome to generate a knock-in strain carrying a valine-to-leucine mutation ß2V287L. ß2(V287L) mice were viable and born at an expected Mendelian ratio. Surprisingly, mice did not show an overt seizure phenotype; however, homozygous mice did show significant alterations in their activity-rest patterns. This was manifest as an increase in activity during the light cycle suggestive of disturbances in the normal sleep patterns of mice; a parallel phenotype to that found in human ADNFLE patients. Consistent with the role of nicotinic receptors in reward pathways, we found that ß2(V287L) mice did not develop a normal proclivity to voluntary wheel running, a model for natural reward. Anxiety-related behaviors were also affected by the V287L mutation. Mutant mice spent more time in the open arms on the elevated plus maze suggesting that they had reduced levels of anxiety. Together, these findings emphasize several important roles of ß2* nicotinic receptors in complex biological processes including the activity-rest cycle, natural reward and anxiety.

Pharmacological differences between immunoisolated native brain and heterologously expressed rat alpha4beta2 nicotinic receptors.

Native brain and heterologously expressed rat alpha4beta2 nicotinic receptors (in Xenopus oocytes and CV-1 cells) were immunoisolated with the anti-alpha4 antibody mAb 299 and their pharmacological properties were compared using [3H](+/-)epibatidine, the novel N-alkylnicotinium analog N-n-octylnicotinium iodide (NONI), and the ganglionic antagonist trimethaphan (TRM). The equilibrium dissociation constant (K(d)) for [3H](+/-)epibatidine binding to the native and heterologously expressed receptors ranged from 13 to 21 pM. The Hill coefficients for [3H](+/-)epibatidine binding to the native and expressed receptors ranged from 0.8 to 1.1 and were consistent with a single high-affinity site. NONI inhibited 30 pM [3H](+/-)epibatidine binding to the native and expressed receptors with similar potency (IC(50) values of 6-7 microM). However, [3H](+/-)epibatidine dissociated 2-3 times more slowly from the native, than from the expressed receptors and TRM inhibited 30 pM [3H](+/-)epibatidine binding to the native receptors (IC(50) value of 330 microM) less potently than it did to the receptors expressed in oocytes (IC(50) value of 16 microM) or CV-1 cells (IC(50) value of 55 microM). The differences between the native and expressed [3H](+/-)epibatidine dissociation rate constants and IC(50) values for TRM were significant for both host cell types, although the values for the CV-1-expressed receptors were closer to the native ones than were those for the oocyte-expressed receptors. Thus, the epibatidine and trimethaphan binding sites in native and expressed alpha4beta2 receptors appear to have significantly different structural or chemical properties.

Psychosocial correlates of suicidal intent among patients with schizophrenia.

Suicidal intent is common among patients with schizophrenia, resulting in extreme personal distress, increased hospitalizations and healthcare expenditures, and early mortality. However, the psychosocial correlates of current suicidality among schizophrenic patients are not fully understood. This study attempted to identify psychosocial correlates of current suicidal intent in a sample of 267 patients with schizophrenia. Multiple regression analyses were used to assess the relationship between suicidality and current symptomatology, psychosocial problems, global functioning, and demographic characteristics. Results showed that depressive symptomatology as a single variable accounted for approximately 50% of the variance in ratings of suicidal intent. In addition, female gender, younger age, recent traumatic stress, and less severe psychotic symptoms each significantly predicted current suicidality independent of depressive symptoms. The combination of depression among younger female schizophrenic patients whose psychotic symptoms have abated and who are experiencing current traumatic stress may indicate extreme suicidal intent.

The transporter-like protein inebriated mediates hyperosmotic stimuli through intracellular signaling.

We cloned the inebriated homologue MasIne from Manduca sexta and expressed it in Xenopus laevis oocytes. MasIne is homologous to neurotransmitter transporters but no transport was observed with a number of putative substrates. Oocytes expressing MasIne respond to hyperosmotic stimulation by releasing intracellular Ca(2+), as revealed by activation of the endogenous Ca(2+)-activated Cl(-) current. This Ca(2+) release requires the N-terminal 108 amino acid residues of MasIne and occurs via the inositol trisphosphate pathway. Fusion of the N terminus to the rat gamma-aminobutyric acid transporter (rGAT1) also renders rGAT1 responsive to hyperosmotic stimulation. Immunohistochemical analyses show that MasIne and Drosophila Ine have similar tissue distribution patterns, suggesting functional identity. Inebriated is expressed in tissues and cells actively involved in K(+) transport, which suggests that it may have a role in ion transport, particularly of K(+). We propose that stimulation of MasIne releases intracellular Ca(2+) in native tissues, activating Ca(2+)-dependent K(+) channels, and leading to K(+) transport.

UNCL, the mammalian homologue of UNC-50, is an inner nuclear membrane RNA-binding protein.

We isolated a mammalian homologue of the C. elegans gene unc-50 that we have named UNCL. The 777 kb rat UNCL cDNA encodes a 259 amino acid protein that is expressed in a wide variety of tissues with highest mRNA levels in brain, kidney and testis. Hydropathy plot analysis and in vitro translation experiments with microsomal membranes indicate that UNCL is a transmembrane protein. Hemagglutinin tagged UNCL was stably transfected into SaOS-2 osteosarcoma cells and exhibited a nuclear rim staining pattern which was retained following extraction with 1% Triton X-100, suggesting that UNCL localizes to the inner nuclear membrane. UNCL-HA was extractable in 350 mM NaCl, suggesting that UNCL is not associated with the nuclear matrix. Homopolymer RNA-binding assays performed on in vitro translated UNCL protein and 'structural modeling by homology' suggest that UNCL binds RNA via an amino-terminal RNA Recognition-like Motif. Since unc-50 is required for expression of assembled muscle-type nicotinic receptors in the nematode we investigated whether UNCL had a similar function for mammalian nicotinic receptors. When UNCL was co-expressed with neural nicotinic receptors in Xenopus oocytes or COS cells it increased expression of functional cell surface receptors up to 1. 6-fold. We conclude that UNCL is a novel inner nuclear membrane protein that associates with RNA and is involved in the cell-surface expression of neuronal nicotinic receptors. UNCL plays a broader role because UNCL homologues are present in two yeast and a plant species, none of which express nicotinic receptors and it is also found in tissues that lack nicotinic receptors.

The subunit dominates the relaxation kinetics of heteromeric neuronal nicotinic receptors.

The ACh-induced voltage-jump relaxation currents of the nicotinic receptors formed by pair-wise expression of the rat alpha2, alpha3, or alpha4 subunits with the beta2 or beta4 subunit in Xenopus oocytes were fitted best by the sum of two exponentials and a constant between -60 and -150 mV. As the ACh concentration approached zero, the relaxation time constants approached limiting values that should equal the single-channel burst duration at low ACh concentrations and the synaptic current decay time constants. beta4 co-expression prolonged the zero ACh concentration limits for the relaxation time constants. The fast beta4 zero ACh concentration limits ranged from 40 to 121 ms between -60 and -150 mV, and the slow beta4 zero ACh concentration limits ranged from 274 to 1039 ms. In contrast, the fast beta2 limits were 4-6 ms over the same voltage range and the slow beta2 limits were 30-53 ms. Expression with the beta4 subunit increased the voltage sensitivity of the alpha2, alpha3 and slow alpha4 relaxation time constants but not that of the fast alpha4 relaxation time constant. Reducing the temperature from 22 C to 8-9 C increased the alpha4beta2 and alpha3beta4 relaxation time constants 2.3- to 6.6-fold and reduced the fractional amplitude of the fast relaxation component. It also increased the voltage dependence of the fast alpha3beta4 relaxation time constant and decreased that of the slow time constant. The Q10 for alpha4beta2 and alpha3beta4 relaxation time constants ranged from 1.9 to 3.9 between 10 and 20 C. The beta subunit appears to have a dominant influence on the voltage-jump relaxation kinetics of heteromeric neuronal nicotinic receptors.

Pharmacological similarities between native brain and heterologously expressed alpha4beta2 nicotinic receptors.

1 We studied the pharmacological properties of native rat brain and heterologously expressed rat alpha4beta2 nicotinic receptors immunoprecipitated onto a fixed substrate with the anti-alpha4 antibody mAb 299. 2 Immunodepletion with the anti-beta2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained beta2. 3 The association and dissociation rate constants for 30 pM +/-[3H]-epibatidine binding to alpha4beta2 receptors expressed in oocytes were 0.02+/-0.01 and 0.03+/-0.01 min-1 (+/-standard error, degrees of freedom=7 - 8) at 20 - 23 degrees C. 4 The Hill coefficients for +/-[3H]epibatidine binding to the native brain, alpha4beta2 receptors expressed in oocytes, and alpha4beta2 receptors expressed in CV-1 cells (using recombinant adenovirus) were 0.69 - 0.70 suggesting a heterogeneous receptor population. Fits of the +/-[3H]-epibatidine concentration-binding data to a two-site model gave KD s of 8 - 30 and 560 - 1,200 pM. The high-affinity sites comprised 73 - 74% of the native brain and oocyte alpha4beta2 receptor population, 85% of the CV-1 alpha4beta2 receptor population. 5 The expression of rat alpha4beta2 receptors in CV-1 cells using vaccinia viral infection-transfection resulted in a more homogeneous receptor population (Hill coefficient of 1. 0+/-0.2). Fits of the +/-[3H]-epibatidine binding data to a single-site model gave a KD of 40+/-3 pM. 6 DHbetaE (IC50=260-470 nM) and the novel nicotine analogue NDNI (IC50=7-10 microM) inhibited 30 pM+/-[3H]-epibatidine binding to the native brain and heterologously expressed alpha4beta2 receptors equally well. 7 The results show that alpha4beta2-containing nicotinic receptors in the rat brain and heterologously expressed rat alpha4beta2 receptors have similar affinities for +/-[3H]-epibatidine, DHbetaE, and NDNI.

Two mutations linked to nocturnal frontal lobe epilepsy cause use-dependent potentiation of the nicotinic ACh response.

1. We constructed rat homologues (S252F and +L264) of two human alpha4 nicotinic mutations - alpha4(S248F) and alpha4(777ins3) - that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co-expressed them with wild-type rat beta2 subunits in Xenopus oocytes. 2. The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use-dependent potentiation of the response during a train of brief 100 nM ACh pulses. Second, they delayed the rise times of the 5-15 nM (+L264) and 30 nM (S252F) ACh responses. Third, they reduced extracellular Ca2+-induced increases in the 30 microM ACh response. 3. Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage-jump relaxations, enhanced steady-state desensitization and reduced the single-channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single-channel conductance. Neither mutation affected the number of surface receptors measured by antibody binding but the S252F mutation reduced the maximum ACh response. 4. The ACh concentration dependence of use-dependent potentiation and the delay in the rising phase of the mutant ACh response suggest that these effects are caused by a slow unblocking of the closed mutant receptors. Use-dependent potentiation of the mutant response during a series of high-frequency cholinergic inputs to the presynaptic terminal could trigger ADNFLE seizures by suddenly increasing nicotinic-mediated transmitter release.

The alpha4 subunit of rat alpha4beta2 nicotinic receptors is phosphorylated in vivo.

The intracellular domains of the alpha4 and beta2 neuronal nicotinic subunits between transmembrane segments 3 and 4 contain a number of predicted phosphorylation sites but there is no direct evidence that any of these sites are actually phosphorylated in vivo. We expressed rat alpha4beta2 nicotinic receptors in Xenopus oocytes, labeled them by an overnight incubation in [32P]orthophosphate, and analyzed the immunoprecipitated receptors by autoradiography and Western blotting. Our results show that the oocytes contained three kinds of alpha4 subunits with apparent weights of 69, 79, and 89 kDa. The 89 kDa alpha4 subunit was the most heavily phosphorylated.

Two domains of the beta subunit of neuronal nicotinic acetylcholine receptors contribute to the affinity of substance P.

Substance P is known to noncompetitively inhibit activation of muscle and neuronal nicotinic acetylcholine receptors. Neuronal nicotinic receptors formed from different combinations of alpha and beta subunits exhibited differential sensitivity to substance P, with those containing beta-4 subunits having a 25-fold higher affinity than those having beta-2 subunits. To identify the regions and/or amino acid residues of the beta subunit responsible for this difference, chimeric beta subunits were coexpressed with alpha-3 in Xenopus oocytes and the IC50 values for substance P were determined. Amino acid residues between 105 and 109 (beta4 numbering), in the middle of the N-terminal domain, and between 214 and 301, between the extracellular side of M1 and the intracellular side of M3, were identified as major contributors to the apparent affinity of substance P. The affinity of acetylcholine was only affected by residue changes between 105 and 109. Site-directed mutagenesis revealed two amino acids that are important determinants of the affinity of substance P, beta4(V108)/beta2(F106), which is in the middle of the first extracellular domain, and beta4(F255)/beta2(V253), which is within the putative channel lining transmembrane domain M2. However, other residues within these domains must be making subtle but significant contributions, since simultaneous mutation of both these amino acids did not cause complete interconversion of the beta subunit-dependent differences in the receptor affinity for substance P.

Environmental factors and clients' self-disclosure in counseling.

Self-disclosure by clients is regarded as a fundamental component of counseling, with increased self-disclosure being related to positive outcome of treatment; however, scant attention has focused on environmental characteristics that may facilitate self-disclosure among clients. An evaluation of prior research in this area leads to suggestions for studies on environmental factors and self-disclosure.

Does insight affect long-term impatient treatment outcome in chronic schizophrenia?

The purpose of this study was to investigate whether the degree of insight into illness is associated with long-term inpatient treatment outcome in chronic schizophrenia. Inpatients diagnosed with schizophrenia were evaluated using the Global Assessment of Functioning Scale (GAF) and the Functional Skills Rating Form (FSRF) at baseline and at follow-up evaluation 1 year after treatment. The Scale to Assess Unawareness of Mental Disorder (SUMD) was used to evaluate insight into illness at follow-up evaluation. Pearson correlation coefficients were used to evaluate the relationship between insight and outcome variables, and multivariate analyses of variance (MANOVAs) were used to assess differences in treatment outcome between patients with good versus poor insight. The results suggest that a patient's insight is significantly related to global and specific measures of functional outcome. Moreover, patients with good insight showed better improvement after long-term inpatient treatment. These findings both support and expand on previous research indicating that increased insight into illness is associated with better treatment compliance and outcome. We propose that further research is necessary to specify the etiology of insight and to develop new interventions focused on increasing insight into illness.

Voltage-jump relaxation kinetics for wild-type and chimeric beta subunits of neuronal nicotinic receptors.

We have studied the voltage-jump relaxation currents for a series of neuronal nicotinic acetylcholine receptors resulting from the coexpression of wild-type and chimeric beta 4/beta 2 subunits with alpha 3 subunits in Xenopus oocytes. With acetylcholine as the agonist, the wild-type alpha 3 beta 4 receptors displayed five- to eightfold slower voltage-jump relaxations than did the wild-type alpha 3 beta 2 receptors. In both cases, the relaxations could best be described by two exponential components of approximately equal amplitudes over a wide range of [ACh]'s. Relaxation rate constants increased with [ACh] and saturated at 20- to 30-fold lower concentrations for the alpha 3 beta 2 receptor than for the alpha 3 beta 4 receptor, as observed previously for the peak steady state conductance. Furthermore, the chimeric beta 4/beta 2 subunits showed a transition in the concentration dependence of the rate constants in the region between residues 94 and 109, analogous to our previous observation with steady state conductances. However, our experiments with a series of beta-subunit chimeras did not localize residues that govern the absolute value of the kinetic parameters. Hill coefficients for the relaxations also differed from those previously measured for steady state responses. The data reinforce previous conclusions that the region between residues 94 and 109 on the beta subunit plays a role in binding agonist but also show that other regions of the receptor control gating kinetics subsequent to the binding step.

Regions of beta 2 and beta 4 responsible for differences between the steady state dose-response relationships of the alpha 3 beta 2 and alpha 3 beta 4 neuronal nicotinic receptors.

We constructed chimeras of the rat beta 2 and beta 4 neuronal nicotinic subunits to locate the regions that contribute to differences between the acetylcholine (ACh) dose-response relationships of the alpha 3 beta 2 and alpha 3 beta 4 receptors. Expressed in Xenopus oocytes, the alpha 3 beta 2 receptor displays an EC50 for ACh approximately 20-fold less than the EC50 of the alpha 3 beta 4 receptor. The apparent Hill slope (n(app)) of alpha 3 beta 2 is near one whereas the alpha 3 beta 4 receptor displays an n(app) near two. Substitutions within the first 120 residues convert the EC50 for ACh from one wild-type value to the other. Exchanging just beta 2:104-120 for the corresponding region of beta 4 shifts the EC50 of ACh dose-response relationship in the expected direction but does not completely convert the EC50 of the dose-response relationship from one wild-type value to the other. However, substitutions in the beta 2:104-120 region do account for the relative sensitivity of the alpha 3 beta 2 receptor to cytisine, tetramethylammonium, and ACh. The expression of beta 4-like (strong) cooperativity requires an extensive region of beta 4 (beta 4:1-301). Relatively short beta 2 substitutions (beta 2:104-120) can reduce cooperativity to beta 2-like values. The results suggest that amino acids within the first 120 residues of beta 2 and the corresponding region of beta 4 contribute to an agonist binding site that bridges the alpha and beta subunits in neuronal nicotinic receptors.

Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates.

The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 microM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subconductance state, measuring approximately 60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 microM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at Vm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.

Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations.

We measured the permeability ratios (PX/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xenopus oocytes for alkali metal and organic cations using shifts in the bi-ionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced PCs/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na+ and glycine methyl ester/Na+ permeability ratios. Two subunit alterations also affected selectivity: omission of the delta subunit reduced PCs/PNa by 16%, and substitution of Xenopus delta for mouse delta increased Pguanidinium/PNa more than twofold and reduced PCs/PNa by 34% and PLi/PNa by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 A; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/PNa by 38% and Pbutylammonium/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222.

Regions of beta 4.beta 2 subunit chimeras that contribute to the agonist selectivity of neuronal nicotinic receptors.

Fifteen chimeric nicotinic receptor beta subunits were constructed consisting of N-terminal neuronal beta 4 sequences and C-terminal beta 2 sequences. Responses to cytisine, nicotine, or tetramethylammonium were compared to acetylcholine responses for these subunits expressed in Xenopus oocytes with alpha 3 subunits. The results show that (i) two residues in the extracellular domain of chimeric beta 4.beta 2 subunits (108 beta 2F/beta 4V, 110 beta 2S/beta 4T) account for much of the relative cytisine sensitivity; and (ii) four extracellular residues of chimeric beta 4.beta 2 subunits (112 beta 2A/beta 4V, 113 beta 2V/beta 4I and 115 beta 2S/beta 4R, 116 beta 2Y/beta 4S) account for most of the relative tetramethylammonium sensitivity. The data did not permit localization of nicotine sensitivity to any particular region.

Pharmacological and kinetic properties of alpha 4 beta 2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

1. Co-injection of RNA synthesized from cloned neuronal acetylcholine receptor (nAChR) subunits (alpha 4 and beta 2) in Xenopus oocytes produced functional receptors. In macroscopic voltage-clamp experiments, the agonist-induced current exhibited a strong inward rectification. 2. Voltage jumps from +50 mV to more negative potentials produced relaxations of the agonist-induced current with a single exponential time course. The relaxation rate constant was only weakly voltage dependent. 3. At the single-channel level, three conductances were recorded of 12, 22 and 34 pS. Their burst durations were similar and varied only weakly with voltage (e-fold for 120 to 370 mV), consistent with the poorly voltage-dependent relaxation rate constants. However, the burst durations were less than 10 ms, or less than 1/5 the value expected from voltage-jump relaxations. 4. Hexamethonium (Hex, 0.5 to 8 microM) inhibited the agonist-induced current and produced voltage-jump relaxations characterized by a rapid conductance increase and a slower conductance decrease. Analysis of these relaxations suggested that the Hex-receptor interaction is open-channel blockade characterized by a forward binding rate of 1 x 10(7) M-1 s-1 and a dissociation rate constant of about 25 s-1. 5. For the relaxations produced by QX222, the slowest phase was a conductance increase, suggesting that the dissociation rate constant for QX222 is 10-30-fold greater than for Hex. 6. Hex but not QX222 produced an additional use-dependent blockade that was manifest during repetitive hyperpolarizing pulses. 7. With mouse muscle ACh receptors expressed in oocytes, the blockade by Hex did not depend strongly on voltage. Neither Hex nor QX222 produced appreciable use-dependent block on muscle ACh receptors. 8. Of the four conditions studied (neuronal and muscle receptors, Hex and QX222), only the blockade of the neuronal AChR by Hex is characterized by a residence time longer than the normal open time. 9. It is concluded that the modest differences in primary amino acid sequence between muscle and neuronal receptors lead to profound changes in their interactions with channels blockers.

Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region.

Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.