Does Sotalol Still Have a Role in the Management of Arrhythmias?

Despite proven effectiveness in treating tachyarrhythmias, sotalol is proarrhythmic and can cause torsades de pointes. Given the emergence of studies that show no benefit from rhythm control strategies in managing atrial fibrillation, as well as the introduction of nonpharmacological approaches to treating arrhythmias, we felt it necessary to ascertain if there was any role for sotalol given its side effects. Review of the literature regarding sotalol use in the prevention and treatment of supraventricular and ventricular tachyarrhythmias seems to show that more effective and safer agents and nonpharmacological alternatives are currently available. However, sotalol still seems to be useful in preventing supraventricular tachyarrhythmias postcardiac surgery and in reverting hemodynamically stable sustained ventricular tachycardias in the setting of coronary artery disease. Its role in the prevention of tachyarrhythmias in the setting of arrhythmogenic right ventricular cardiomyopathy requires further investigation.

Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning.

Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency.

Integrating gross pathology into teaching of undergraduate medical science students using human cadavers.

Human cadavers offer a great opportunity for histopathology students for the learning and teaching of tissue pathology. In this study, we aimed to implement an integrated learning approach by using cadavers to enhance students' knowledge and to develop their skills in gross tissue identification, handling and dissection techniques. A total of 35 students enrolled in the undergraduate medical science program participated in this study. A 3-hour laboratory session was conducted that included an active exploration of cadaveric specimens to identify normal and pathological tissues as well as tissue dissection. The majority of the students strongly agreed that the integration of normal and morbid anatomy improved their understanding of tissue pathology. All the students either agreed or strongly agreed that this laboratory session was useful to improve their tissue dissection and instrument handling skills. Furthermore, students from both cohorts rated the session as very relevant to their learning and recommended that this approach be added to the existing histopathology curriculum. To conclude, an integrated cadaver-based practical session can be used effectively to enhance the learning experience of histopathology science students, as well as improving their manual skills of tissue treatment, instrument handling and dissection.

Fine-tuning the standard setting of objective structured practical examinations in clinical anatomy.

The objective structured practical examination (OSPE) is a reliable assessment of practical skills in anatomy teaching. It is often administered as low-stake assessments to track progress at multiple time points in anatomy curricula. Standard-setting OSPEs to derive a pass mark and to ensure assessment quality and rigor is a complex task. This study compared standard-setting outcomes of clinical anatomy OSPEs determined by traditional criterion-referenced (Ebel) and norm-referenced ("mean minus standard deviation") methods in comparison to hybrid methods which apply both criterion-referenced and norm-referenced approaches in setting examination standards. The hybrid approaches utilized included the "Cohen method" and an adaptation of the "Taylor's method," which is an improvement on the Cohen method. These diverse standard-setting methods were applied retrospectively to 16 anatomy OSPEs conducted over 4 years for first- and second-year medical students in a graduate Doctor of Medicine Program at Griffith Medical School, Australia; and the pass marks, failure rates, and variances of failure rates were compared. The application of the adaptation of Taylor's method to standard set OSPEs produced pass marks and failure rates comparable to the Ebel method, whereas the variability of failure rates was higher with the Ebel method than with the Cohen and Taylor's methods. This underscores this study's adaptation of Taylor's method as a suitable alternative to the widely accepted but resource intensive, panel-based criterion-referenced standard-setting methods such as the Ebel method, where panelists with relevant expertise are unavailable, particularly for the multiple low-stakes OSPEs in an anatomy curriculum.

Artificial intelligence software in pulmonary nodule assessment.

BACKGROUND: This study tests the impact of the addition of autonomous computed tomography (CT) interpreting software to radiologist assessment of pulmonary nodules. METHODS: Computed tomography scans for nodule assessment were identified retrospectively. Lung cancer risk factors, initial radiologist (RAD) report, Philips Lung Nodule software report (computer-aided nodule (CAD)) and radiologist report following the review of CT images and CAD (RAD + CAD) were collected. Follow-up recommendations based on current guidelines were derived from each report. RESULTS: In all, 100 patients were studied. Median maximal diameter of the largest nodule reported by RAD and RAD + CAD were similar at 10.0 and 9.0 mm, respectively (p = 0.06) but were reported as larger by CAD at 11.8 mm (p < 0.001). Follow-up recommendations derived from RAD + CAD were less intensive in 23 (23%) and more intensive in 34 (34%) than that of RAD. DISCUSSION: This study suggests that autonomous software use can alter radiologist assessment of pulmonary nodules such that suggested follow-up is altered.

Structural and functional characterization of a novel homodimeric three-finger neurotoxin from the venom of Ophiophagus hannah (king cobra).

Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (alphabetagammadelta) and neuronal (alpha(7), alpha(3)beta(2), and alpha(4)beta(2)) nicotinic acetylcholine receptors (nAChRs) with highest affinity for alpha(7)-nAChRs. The high resolution (1.5 A) crystal structure revealed haditoxin to be a homodimer, like kappa-neurotoxins, which target neuronal alpha(3)beta(2)- and alpha(4)beta(2)-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain alpha-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain alpha-neurotoxins and kappa-neurotoxins notwithstanding, haditoxin exhibited unique blockade of alpha(7)-nAChRs (IC(50) 180 nm), which is recognized by neither short-chain alpha-neurotoxins nor kappa-neurotoxins. This is the first report of a dimeric short-chain alpha-neurotoxin interacting with neuronal alpha(7)-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.

Irditoxin, a novel covalently linked heterodimeric three-finger toxin with high taxon-specific neurotoxicity.

A novel heterodimeric three-finger neurotoxin, irditoxin, was isolated from venom of the brown treesnake Boiga irregularis (Colubridae). Irditoxin subunit amino acid sequences were determined by Edman degradation and cDNA sequencing. The crystal structure revealed two subunits with a three-finger protein fold, typical for "nonconventional" toxins such as denmotoxin, bucandin, and candoxin. This is the first colubrid three-finger toxin dimer, covalently connected via an interchain disulfide bond. Irditoxin showed taxon-specific lethality toward birds and lizards and was nontoxic toward mice. It produced a potent neuromuscular blockade at the avian neuromuscular junction (IC(50)=10 nM), comparable to alpha-bungarotoxin, but was three orders of magnitude less effective at the mammalian neuromuscular junction. Covalently linked heterodimeric three-finger toxins found in colubrid venoms constitute a new class of venom peptides, which may be a useful source of new neurobiology probes and therapeutic leads.

Snake three-finger α-neurotoxins and nicotinic acetylcholine receptors: molecules, mechanisms and medicine.

Snake venom three-finger α-neurotoxins (α-3FNTx) act on postsynaptic nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction (NMJ) to produce skeletal muscle paralysis. The discovery of the archetypal α-bungarotoxin (α-BgTx), almost six decades ago, exponentially expanded our knowledge of membrane receptors and ion channels. This included the localisation, isolation and characterization of the first receptor (nAChR); and by extension, the pathophysiology and pharmacology of neuromuscular transmission and associated pathologies such as myasthenia gravis, as well as our understanding of the role of α-3FNTxs in snakebite envenomation leading to novel concepts of targeted treatment. Subsequent studies on a variety of animal venoms have yielded a plethora of novel toxins that have revolutionized molecular biomedicine and advanced drug discovery from bench to bedside. This review provides an overview of nAChRs and their subtypes, classification of α-3FNTxs and the challenges of typifying an increasing arsenal of structurally and functionally unique toxins, and the three-finger protein (3FP) fold in the context of the uPAR/Ly6/CD59/snake toxin superfamily. The pharmacology of snake α-3FNTxs including their mechanisms of neuromuscular blockade, variations in reversibility of nAChR interactions, specificity for nAChR subtypes or for distinct ligand-binding interfaces within a subtype and the role of α-3FNTxs in neurotoxic envenomation are also detailed. Lastly, a reconciliation of structure-function relationships between α-3FNTx and nAChRs, derived from historical mutational and biochemical studies and emerging atomic level structures of nAChR models in complex with α-3FNTxs is discussed.

Fulditoxin, representing a new class of dimeric snake toxins, defines novel pharmacology at nicotinic ACh receptors.

BACKGROUND AND PURPOSE: Animal toxins have contributed significantly to our understanding of the neurobiology of receptors and ion channels. We studied the venom of the coral snake Micrurus fulvius fulvius and identified and characterized the structure and pharmacology of a new homodimeric neurotoxin, fulditoxin, that exhibited novel pharmacology at nicotinic ACh receptors (nAChRs). EXPERIMENTAL APPROACH: Fulditoxin was isolated by chromatography, chemically synthesized, its structure determined by X-ray crystallography, and its pharmacological actions on nAChRs characterized by organ bath assays and two-electrode voltage clamp electrophysiology. KEY RESULTS: Fulditoxin's distinct 1.95-Å quaternary structure revealed two short-chain three-finger α-neurotoxins (α-3FNTxs) non-covalently bound by hydrophobic interactions and an ability to bind metal and form tetrameric complexes, not reported previously for three-finger proteins. Although fulditoxin lacked all conserved amino acids canonically important for inhibiting nAChRs, it produced postsynaptic neuromuscular blockade of chick muscle at nanomolar concentrations, comparable to the prototypical α-bungarotoxin. This neuromuscular blockade was completely reversible, which is unusual for snake α-3FNTxs. Fulditoxin, therefore, interacts with nAChRs by utilizing a different pharmacophore. Unlike short-chain α-3FNTxs that bind only to muscle nAChRs, fulditoxin utilizes dimerization to expand its pharmacological targets to include human neuronal α4ß2, α7, and α3ß2 nAChRs which it blocked with IC50 values of 1.8, 7, and 12 µM respectively. CONCLUSIONS AND IMPLICATIONS: Based on its distinct quaternary structure and unusual pharmacology, we named this new class of dimeric Micrurus neurotoxins represented by fulditoxin as Σ-neurotoxins, which offers greater insight into understanding the interactions between nAChRs and peptide antagonists.

Synthesis of trifluoromethylaryl diazirine and benzophenone derivatives of etomidate that are potent general anesthetics and effective photolabels for probing sites on ligand-gated ion channels.

To locate the binding sites of general anesthetics on ligand-gated ion channels, two derivatives of the intravenous general anesthetic etomidate (2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate), in which the 2-ethyl group has been replaced by photoactivable groups based on either aryl diazirine or benzophenone chemistry, have been synthesized and characterized pharmacologically. TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate) and BzBzl-etomidate (4-benzoylbenzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate are both potent general anesthetics with half-effective anesthetic concentrations of 700 and 220 nM, respectively. Both agents resembled etomidate in enhancing currents elicited by low concentrations of GABA on heterologously expressed GABAA receptors and in shifting the GABA concentration-response curve to lower concentrations. They also allosterically enhanced the binding of flunitrazepam to mammalian brain GABAA receptors. Both agents were also effective and selective photolabels, photoincorporating into some, but not all, subunits of the Torpedo nicotinic acetylcholine receptor to a degree that was allosterically regulated by an agonist or a noncompetitive inhibitor. Thus, they have the necessary pharmacological and photochemical properties to be useful in identifying the site of etomidate-induced anesthesia.

Role of individual disulfide bridges in the conformation and activity of spinoxin (α-KTx6.13), a potassium channel toxin from Heterometrus spinifer scorpion venom.

Spinoxin (SPX; α-KTx6.13), isolated from venom of the scorpion Heterometrus spinifer, is a K+ channel-specific peptide toxin (KTx), which adopts a cysteine-stabilized α/ß scaffold that is cross-linked by four disulfide bridges (Cys1-Cys5, Cys2-Cys6, Cys3-Cys7, and Cys4-Cys8). To investigate the role of the individual disulfide bonds in the structure-activity relationship of SPX, we synthesized four SPX analogs in which each pair of cysteine residues was replaced by alanine residues. The analysis of circular dichroism spectra and inhibitory activity against Kv1.3 channels showed that the SPX analogs lacking any of three specific disulfide bonds (Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7) were unable to form the native secondary structure and completely lost inhibitory activities. Thus, we conclude that Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 are required for the inhibition of the Kv1.3 channel by SPX. In contrast, the analog lacking Cys4-Cys8 retained both native secondary structure and inhibitory activity. Interestingly, one of the isomers of the analog lacking Cys1-Cys5 also showed inhibitory activities, although its inhibition was ∼18-fold weaker than native SPX. This isomer had an atypical disulfide bond pairing (Cys3-Cys4 and Cys7-Cys8) that corresponds to that of maurotoxin (MTX), another α-KTx6 family member. These results indicate that the Cys1-Cys5 and Cys2-Cys6 bonds are important for restricting the toxin from forming an atypical (MTX-type) disulfide bond pairing among the remaining four cysteine residues (Cys3, Cys4, Cys7, and Cys8) in native SPX.

Assignment of voltage-gated potassium channel blocking activity to kappa-KTx1.3, a non-toxic homologue of kappa-hefutoxin-1, from Heterometrus spinifer venom.

A new family of weak K(+) channel toxins (designated kappa-KTx) with a novel "bi-helical" scaffold has recently been characterized from Heterometrus fulvipes (Scorpionidae) venom. Based on the presence of the minimum functional dyad (Y5 and K19), kappa-hefutoxin-1 (kappa-KTx1.1) was investigated and found to block Kv 1.2 (IC(50) approximately 40 microM) and Kv 1.3 (IC(50) approximately 150 microM) channels. In the present study, kappa-KTx1.3, that shares approximately 60% identity with kappa-hefutoxin 1, has been isolated from Heterometrus spinifer venom. Interestingly, despite the presence of the functional dyad (Y5 and K19), kappa-KTx1.3 failed to reproduce the K(+) channel blocking activity of kappa-hefutoxin-1. Since the dyad lysine in kappa-KTx1.3 was flanked by another lysine (K20), it was hypothesized that this additional positive charge could hinder the critical electrostatic interactions known to occur between the dyad lysine and the Kv 1 channel selectivity filter. Hence, mutants of kappa-KTx1.3, substituting K20 with a neutral (K20A) or a negatively (K20E) or another positively (K20R) charged amino acid were synthesized. kappa-KTx1.3 K20E, in congruence with kappa-hefutoxin 1 with respect to subtype selectivity and affinity, produced blockade of Kv 1.2 (IC(50) = 36.8+/-4.9 microM) and Kv 1.3 (IC(50)=53.7+/-6.7 microM) but not Kv 1.1 channels. kappa-KTx1.3 K20A produced blockade of both Kv 1.2 (IC(50) = 36.9+/-4.9 microM) and Kv 1.3 (IC(50)=115.7+/-7.3 microM) and in addition, acquired affinity for Kv 1.1 channels (IC(50) =1 10.7+/-7.7 microM). kappa-KTx1.3 K20R failed to produce any blockade on the channel subtypes tested. These data suggest that the presence of an additional charged residue in a position adjacent to the dyad lysine impedes the functional block of Kv 1 channels produced by kappa-KTx1.3.

Role of cadaveric dissections in modern medical curricula: a study on student perceptions.

The shift from traditional medical curricula to newer teaching and learning approaches such as problem-based learning has often resulted in omission or significant reduction of cadaveric dissections as a method of learning anatomy. The objective of this study was to evaluate students' perception of dissection in a graduate-entry, problem-based learning-based medical curriculum. At the end of the musculoskeletal dissection program in second year, a Likert-type questionnaire was used to explore medical student perceptions of the perceived advantages and challenges of cadaveric dissections in comparison with other anatomy teaching methods. Overall, a majority of students had a positive perception of dissections. Students who attended dissections regularly had significantly more positive perceptions about their experience and were in agreement with statements such as "dissections make learning more interesting" and "I would be disadvantaged if I did not attend dissection classes." Non-regular attendance was associated with statements about dissections such as "I do not like the smell," "time consuming," and "bored with the way it is carried-out." A follow-up study after completion of the medical program revealed a significant improvement of positive perception about dissection. Student perceptions appear to favour a role for cadaveric dissection in learning anatomy in modern medical curricula. However, optimal and effective integration of dissections is important, with consideration given to its structure and extent of content weighed against logistics and availability of resources; while addressing negative perceptions of dissection-based teaching.

Biochemical and pharmacological characterization of the venom of the black scorpion Heterometrus spinifer.

The sting of the black scorpion Heterometrus spinifer, which can cause intense localized pain, has not been reported to produce lethal cardiovascular complications, which are well known to result from scorpion envenomation as a consequence of a massive release of catecholamines. Therefore, we have undertaken a biochemical and pharmacological characterization of the venom of H. spinifer. Pharmacologically, the venom (0.125 microL/mL) produced a marked, reversible contracture in the chick biventer cervicis muscle that was blocked by d-tubocurarine (2 microM) but not by tetrodotoxin (5 microM) and omega-conotoxin GVIA (3 microM). The anticholinesterase neostigmine (1 microM) potentiated the contracture by 5.3-fold. An ultra-filtrate fraction of MW < 3000 (F3K) of the venom produced a similar contracture in the biventer muscle, whereas the retentate of MW > 3000 did not. In the rat anococcygeus muscle, the venom produced a contractile response that was partially (37.4 +/- 1.6%) blocked by atropine (5 microM); phentolamine (5 microM) blocked the remaining response. Tetrodotoxin (5 microM) did not block the contractile response of the venom on the anococcygeus muscle. Electrospray ionization-mass spectrometry/mass spectrometry confirmed the presence of high concentrations of acetylcholine (79.8 +/- 1.7 microM) and norepinephrine (146.7 +/- 19.8 microM) in H. spinifer venom, which can fully account for the observed cholinergic and adrenergic effects. In contrast to scorpion venoms that selectively target neuronal ion channels in mediating transmitter release, our data show that H. spinifer venom does not possess such activity, which likely explains the apparent lack of lethality of black scorpion envenomation.

Characterization of the rat isolated retractor penis muscle as a model for the study of nitrergic transmission.

INTRODUCTION: The anococcygeus and retractor penis muscles are part of the erectile machinery in male rodents. The rat anococcygeus muscle is a widely used smooth muscle preparation for the study of the effects of test substances on adrenergic, nitrergic, and cholinergic transmission. There is, however, little information available on the process of autonomic transmission in the rat retractor penis muscle, although its autonomic innervation has generally been assumed to be similar to that of the anococcygeus muscle because of the contiguous nature of the two muscles. The present study investigated the involvement of nitrergic transmission in mediating relaxant responses of the rat retractor penis muscle to electrical field stimulation. METHODS: The retractor penis muscle was isolated from Sprague-Dawley rats and mounted in Krebs solution. Phentolamine (5 microM) was added to the bath to block the adrenergic responses of the muscle, which was then precontracted with carbachol (10 microM). RESULTS: Electrical field stimulation (20-30 V, 1 ms pulse width, at 0.5-20 Hz for 10 s) of the carbachol precontracted muscle elicited frequency-dependent relaxant responses (0.9-68%). Tetrodotoxin (1 microM), N(G)-nitro-L-arginine (L-NOARG) (50 microM), N(G)-nitro-L-arginine methylester (L-NAME) (100 microM), and haemoglobin (100 microM) inhibited these relaxant responses by 99.3%, 93.9%, 86.9%, and 77.5%, respectively. L-Arginine (250 microM) (but not its D-isomer) reversed the blockade produced by L-NOARG (72.7%) and L-NAME (81.5%). DISCUSSION: Our results provide clear evidence that the inhibitory (relaxant) responses of the rat retractor penis muscle to electrical field stimulation are mediated by nitric oxide involving the L-arginine-nitric oxide synthase-nitric oxide pathway. The rat retractor penis muscle is a versatile preparation that can replace the cumbersome preparations from the pig, ox, and horse, hitherto used as pharmacological models for the study of the retractor penis muscle.

Identification of binding sites in the nicotinic acetylcholine receptor for TDBzl-etomidate, a photoreactive positive allosteric effector.

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 microm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 microm) increased the submaximal response to acetylcholine (10 microm) with a 2.5-fold increase at 60 microm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [(3)H]tetracaine and [(3)H]phencyclidine to Torpedo nAChR-rich membranes (IC(50) values of 0. 8 mm). nAChR-rich membranes were photolabeled with [(3)H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (deltaLeu-265) and M2-13 (alphaVal-255 and deltaVal-269), whereas labeling of alphaM2-10 (alphaSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gammaM3 (gammaMet-299), a residue that contributes to the same pocket in the nAChR structure as alphaM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the alpha and gamma subunits (labeling of alphaM2-10 and gammaMet-299) likely to be a site for positive allosteric modulation.

[3H]Benzophenone photolabeling identifies state-dependent changes in nicotinic acetylcholine receptor structure.

Interactions of benzophenone (BP) with the Torpedo nicotinic acetylcholine receptor (nAChR) were characterized by electrophysiological analyses, radioligand binding assays, and photolabeling of nAChR-rich membranes with [3H]BP to identify the amino acids contributing to its binding sites. BP acted as a low potency noncompetitive antagonist, reversibly inhibiting the ACh responses of nAChRs expressed in Xenopus oocytes (IC50 = 600 microM) and the binding of the noncompetitive antagonist [3H]tetracaine to nAChR-rich membranes (IC50 = 150 microM). UV irradiation at 365 nm resulted in covalent incorporation of [3H]BP into the nAChR subunits (delta > alpha approximately beta > gamma), with photoincorporation limited to the nAChR transmembrane domain. Comparison of nAChR photolabeling in the closed state (absence of agonist) and desensitized state (equilibrated with agonist) revealed selective desensitized state labeling in the delta subunit of deltaPhe-232 in deltaM1 and deltaPro-286/deltaIle-288 near the beginning of deltaM3 that are within a pocket at the interface between the transmembrane and extracellular domains. There was labeling in the closed state within the ion channel at position M2-13 (alphaVal-255, betaVal-261, and deltaVal-269) that was reduced by 90% upon desensitization and labeling in the transmembrane M3 helices of the beta and gamma subunits (betaMet-285, betaMet-288, and gammaMet-291) that was reduced by 50-80% in the desensitized state. Labeling at the lipid interface (alphaMet-415 in alphaM4) was unaffected by agonist. These results provide a further definition of the regions in the nAChR transmembrane domain that differ in structure between the closed and desensitized states.

Site specificity of agonist-induced opening and desensitization of the Torpedo californica nicotinic acetylcholine receptor.

Agonist-binding kinetics to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were measured using sequential-mixing stopped-flow fluorescence methods to determine the contribution of each individual site to agonist-induced opening and desensitization. Timed dansyl-C6-choline (DC6C) binding followed by its dissociation upon mixing with high, competing agonist concentrations revealed four kinetic components: an initial, fast fluorescence decay, followed by a transient increase, and then two characteristic decays that reflect dissociation from the desensitized agonist sites. The transient increase resulted from DC6C binding to the open-channel based on its prevention by proadifen, a noncompetitive antagonist. Further characterization of DC6C channel binding by the inhibition of [3H]phencyclidine binding and by equilibrium measurements of DC6C fluorescence yielded KD values of 2-4 microM for the desensitized AChR and approximately 600 microM for the closed state. At this site, DC6C displayed a strongly blue-shifted emission spectrum, higher intrinsic fluorescence, and weaker energy transfer from tryptophans than when bound to either agonist site. The initial, fast fluorescence decay was assigned to DC6C dissociation from the alphadelta site of the AChR in its closed conformation, on the basis of inhibition with the site-selective antagonists d-tubocurarine and alpha-conotoxin MI. Fast decay amplitude data indicated an apparent affinity of 0.9 microM for the closed-state alphadelta site; the closed-state alphagamma-site affinity is inferred to be near 100 microM. These values and the known affinities for the desensitized conformation show that the alphagamma site drives AChR desensitization to a approximately 40-fold greater extent than the alphadelta site, undergoes energetically larger conformational changes, and is the primary determinant of agonist potency.

Photolabeling the Torpedo nicotinic acetylcholine receptor with 4-azido-2,3,5,6-tetrafluorobenzoylcholine, a partial agonist.

The interactions of a photoreactive analogue of benzoylcholine, 4-azido-2,3,5,6-tetrafluorobenzoylcholine (APFBzcholine), with nicotinic acetylcholine receptors (nAChRs) were studied using electrophysiology and photolabeling. APFBzcholine acted as a low-efficacy partial agonist, eliciting maximal responses that were 0.3 and 0.1% of that of acetylcholine for embryonic mouse and Torpedo nAChRs expressed in Xenopus oocytes, respectively. Equilibrium binding studies of [3H]APFBzcholine with nAChR-rich membranes from Torpedo electric organ revealed equal affinities (K(eq) = 12 microM) for the two agonist binding sites. Upon UV irradiation at 254 nm, [3H]APFBzcholine was photoincorporated into the nAChR alpha, gamma, and delta subunits in an agonist-inhibitable manner. Photolabeled amino acids in the agonist binding sites were identified by Edman degradation of isolated, labeled subunit fragments. [3H]APFBzcholine photolabeled gammaLeu-109/deltaLeu-111, gammaTyr-111, and gammaTyr-117 in binding site segment E as well as alphaTyr-198 in alpha subunit binding site segment C. The observed pattern of photolabeling is examined in relation to the predicted orientation of the azide when APFBzcholine is docked in the agonist binding site of a homology model of the nAChR extracellular domain based upon the structure of the snail acetylcholine binding protein.