α-Bungarotoxin

In vivo injection of alpha-bungarotoxin to improve the efficiency of motor endplate labeling.[Pubmed:27186443]

Brain Behav. 2016 Apr 27;6(6):e00468.

INTRODUCTION: Motor endplates are composed of a motor neuron terminal and muscle fiber and are distributed in skeletal muscle, causing muscle contraction. However, traditional motor endplate staining methods are limited to the observation of partial skeletal muscle. The procedure was time-consuming due to strict incubation conditions, and usually provided unsatisfactory results. We explored a novel method to label motor endplate rapidly by in vivo injection of fluorescent alpha-bungarotoxin. METHODS: Fifty-two mice were randomly divided into two groups, an experiment group (n = 50), and a contrast group (n = 2). In experiment group, alpha-bungarotoxin was injected via the caudal vein. The injection dosages were designated as 0.1, 0.2, 0.3, 0.4, and 0.5 mug/g. The experimental mice were divided into five subgroups of ten mice per group. The contrast group was only injected with 200 muL normal saline solution. Bilateral gastrocnemius were acquired for microscope analysis and optical clearing to seek specific fluorescent signal. RESULTS: A dose of 0.3 mug/g of alpha-bungarotoxin with 1 h conjugation time could display the number and structure of motor endplate in plane view. Compared with the traditional procedure, this method was rapid, convenient, and time-saving. Combined with the optical clearing technique, spatial distribution could also be seen, helping to better understand the stereoscopic view of motor endplate position in skeletal muscle. CONCLUSIONS: In vivo injection of alpha-bungarotoxin proved effective for studying motor endplate in skeletal muscle.

cGMP/cGMP-dependent protein kinase pathway modulates nicotine-induced currents through the activation of alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors from insect neurosecretory cells.[Pubmed:27059649]

J Neurochem. 2016 Jun;137(6):931-8.

Insect neurosecretory cells, called dorsal unpaired median neurons, are known to express two alpha-bungarotoxin-insensitive nicotinic acetylcholine receptor (nAChR) subtypes, nAChR1 and nAChR2. It was demonstrated that nAChR1 was sensitive to cAMP/cAMP-dependent protein kinase (PKA) regulation, resulting in a modulation of nicotine currents. In this study, we show that cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) pathway modulates nicotine-induced currents, as increased cGMP affects the second compound of the biphasic current-voltage curve, corresponding to the nAChR2 receptors. Indeed, maintaining the guanosine triphosphate level with 100 muM guanosine triphosphate-gamma-S increased nicotine currents through nAChR2. We also demonstrated that inhibition of PKG activity with 0.2 muM (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H, 11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c,d,e)-trinden-1-one (KT5823), a PKG specific inhibitor, reduced nicotine-induced current amplitudes. KT5823 effect on nicotine currents is associated with calcium (Ca(2+) ) activity because inhibition of Ca(2+) concentration with cadmium chloride (CdCl2 ) abolished KT5823-induced inhibition mediated by nAChR2. However, specific inhibition of nitric oxide-guanylyl cyclase (GC) complex by 10 muM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) significantly increased nicotine-induced current amplitudes on both nAChR1 and nAChR2. These results suggest that nicotine-induced currents mediated by both alpha-bungarotoxin-insensitive nAChR1 and nAChR2 are coupled to the cGMP/PKG pathway. We propose that nicotinic acetylcholine receptor activation induces an increase in intracellular calcium (Ca(2+) ) concentration. Elevation of intracellular Ca(2+) results in the formation of Ca(2+) -calmodulin (CaM) complex, which activates guanylyl cyclase (GC) and/or adenylyl cyclase (AC). Ca(2+) -CaM complex could activate Ca(2+) calmodulin kinase II which could directly or indirectly modulate the nicotinic response. The mechanisms by which cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) interact remain unclear. We demonstrate that nicotine-induced currents are coupled to the cGMP/PKG pathway.

Stoichiometry for alpha-bungarotoxin block of alpha7 acetylcholine receptors.[Pubmed:26282895]

Nat Commun. 2015 Aug 18;6:8057.

alpha-Bungarotoxin (alpha-Btx) binds to the five agonist binding sites on the homopentameric alpha7-acetylcholine receptor, yet the number of bound alpha-Btx molecules required to prevent agonist-induced channel opening remains unknown. To determine the stoichiometry for alpha-Btx blockade, we generate receptors comprised of wild-type and alpha-Btx-resistant subunits, tag one of the subunit types with conductance mutations to report subunit stoichiometry, and following incubation with alpha-Btx, monitor opening of individual receptor channels with defined subunit stoichiometry. We find that a single alpha-Btx-sensitive subunit confers nearly maximal suppression of channel opening, despite four binding sites remaining unoccupied by alpha-Btx and accessible to the agonist. Given structural evidence that alpha-Btx locks the agonist binding site in an inactive conformation, we conclude that the dominant mechanism of antagonism is non-competitive, originating from conformational arrest of the binding sites, and that the five alpha7 subunits are interdependent and maintain conformational symmetry in the open channel state.

DNA Aptamers against Taiwan Banded Krait alpha-Bungarotoxin Recognize Taiwan Cobra Cardiotoxins.[Pubmed:26959062]

Toxins (Basel). 2016 Mar 5;8(3). pii: toxins8030066.

Bungarus multicinctus alpha-bungarotoxin (alpha-Bgt) and Naja atra cardiotoxins (CTXs) share a common structural scaffold, and their tertiary structures adopt three-fingered loop motifs. Four DNA aptamers against alpha-Bgt have been reported previously. Given that the binding of aptamers with targeted proteins depends on structural complementarity, in this study, we investigated whether DNA aptamers against alpha-Bgt could also recognize CTXs. It was found that N. atra cardiotoxin 3 (CTX3) reduced the electrophoretic mobility of aptamers against alpha-Bgt. Analysis of the changes in the fluorescence intensity of carboxyfluorescein-labeled aptamers upon binding toxin molecules revealed that CTX3 and alpha-Bgt could bind the tested aptamers. Moreover, the aptamers inhibited the membrane-damaging activity and cytotoxicity of CTX3. In addition to CTX3, other N. atra CTX isotoxins also bound to the aptamer against alpha-Bgt. Taken together, our data indicate that aptamers against alpha-Bgt show cross-reactivity with CTXs. The findings that aptamers against alpha-Bgt also suppress the biological activities of CTX3 highlight the potential utility of aptamers in regard to the broad inhibition of snake venom three-fingered proteins.

Neuronal acetylcholine receptors that bind alpha-bungarotoxin with high affinity function as ligand-gated ion channels.[Pubmed:7507338]

Neuron. 1994 Jan;12(1):167-77.

Neuronal membrane components that bind alpha-bungarotoxin with high affinity can increase intracellular levels of free calcium, demonstrating the components function as nicotinic receptors. Though such receptors often contain the alpha 7 gene product, which by itself can produce ionotropic receptors in Xenopus oocytes, numerous attempts have failed to demonstrate an ion channel function for the native receptors on neurons. Using rapid application of agonist, we show here that the native receptors are ligand-gated ion channels which are cation selective, prefer nicotine over acetylcholine, and rapidly desensitize. Much of the calcium increase caused in neurons by the receptors under physiological conditions appears to result from their depolarizing the membrane sufficiently to trigger calcium influx through voltage-gated channels.