Reporter epitopes: A novel approach to examine transmembrane topology of integral membrane proteins applied to the .alpha.1 subunit of the nicotinic acetylcholine receptor

Biochemistry ◽  
1993 ◽  
Vol 32 (38) ◽  
pp. 9975-9984 ◽  
Author(s):  
Rene Anand ◽  
Lynn Bason ◽  
Mohammad S. Saedi ◽  
Volodymyr Gerzanich ◽  
Xiao Peng ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Integral Membrane Proteins ◽  
Nicotinic Acetylcholine ◽  
Transmembrane Topology ◽  
Novel Approach

scholarly journals Novel Approach for the Search for Chemical Scaffolds with Activity at Both Acetylcholinesterase and the α7 Nicotinic Acetylcholine Receptor: A Perspective on Scaffolds with Dual Activity for the Treatment of Neurodegenerative Disorders

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 446 ◽  
Author(s):  
Natalia Kowal ◽  
Dinesh Indurthi ◽  
Philip Ahring ◽  
Mary Chebib ◽  
Elin Olafsdottir ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Neurodegenerative Disorders ◽  
Homology Modelling ◽  
Colorimetric Method ◽  
Α7 Nicotinic Acetylcholine Receptor ◽  
Nicotinic Acetylcholine ◽  
Α7 Nachr ◽  
Novel Approach

Neurodegenerative disorders, including Alzheimer’s disease, belong to the group of the most difficult and challenging conditions with very limited treatment options. Attempts to find new drugs in most cases fail at the clinical stage. New tactics to develop better drug candidates to manage these diseases are urgently needed. It is evident that better understanding of the neurodegeneration process is required and targeting multiple receptors may be essential. Herein, we present a novel approach, searching for dual active compounds interacting with acetylcholinesterase (AChE) and the α7 nicotinic acetylcholine receptor (nAChR) using computational chemistry methods including homology modelling and high throughput virtual screening. Activities of identified hits were evaluated at the two targets using the colorimetric method of Ellman and two-electrode voltage-clamp electrophysiology, respectively. Out of 87,250 compounds from a ZINC database of natural products and their derivatives, we identified two compounds, 8 and 9, with dual activity and balanced IC50 values of 10 and 5 µM at AChE, and 34 and 14 µM at α7 nAChR, respectively. This is the first report presenting successful use of virtual screening in finding compounds with dual mode of action inhibiting both the AChE enzyme and the α7 nAChR and shows that computational methods can be a valuable tool in the early lead discovery process.

Halothane Interactions with Nicotinic Acetylcholine Receptor Membranes

1997 ◽  
Vol 86 (2) ◽  
pp. 476-486 ◽  
Author(s):  
Douglas E. Raines ◽  
Katie B. McClure
Keyword(s):  
Steady State ◽  
Membrane Proteins ◽  
Sodium Dodecyl Sulfate ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Sodium Dodecyl ◽  
Tryptophan Fluorescence ◽  
Stopped Flow ◽  
Nicotinic Acetylcholine ◽  
Dodecyl Sulfate

Background Although it has been suggested that anesthetics alter protein conformational states by binding to nonpolar sites within the interior regions of proteins, the rate and extent to which anesthetics penetrate membrane proteins has not been characterized. The authors report the use of steady-state and stopped-flow spectroscopy to characterize the interactions of halothane with receptor membranes. Methods Steady-state and stopped-flow fluorescence spectroscopy was used to characterize halothane quenching of nicotinic acetylcholine receptor (nAcChoR)-rich membrane intrinsic fluorescence and the rate of isoflurane-induced nAcChoR desensitization. Results At equilibrium, halothane quenched only 54 +/- 1.4% of all tryptophan fluorescence. Diethyl ether failed to reduce fluorescence quenching by halothane, suggesting that it does not bind to the same protein sites as halothane. Stopped-flow fluorescence traces defined two kinetic components of quenching: a fast component that occurred in less than 1 ms followed by a slower biphasic fluorescence decay. Protein unfolding with sodium dodecyl sulfate reduced halothane's Stern-Volmer quenching constant, eliminated the biphasic decay, and rendered fluorescence accessible to quenching by halothane within 1 ms. Functional studies indicate that anesthetic-induced desensitization of nAcChoR occurs in less than 2 ms. Conclusions Unquenchable fluorescence arises from tryptophan residues that are buried within the protein and protected from halothane. Sodium dodecyl sulfate unfolds membrane proteins and allows previously buried fluorescence protein residues to be rapidly and homogeneously quenched by halothane. Halothane quenches protein components of nAcChoR membranes over the same concentration range and time scale that it exerts its functional effects, a finding that is generally consistent with a protein site of action.

scholarly journals Surface and intracellular distribution of a putative neuronal nicotinic acetylcholine receptor.

1986 ◽  
Vol 103 (1) ◽  
pp. 205-214 ◽  
Author(s):  
M H Jacob ◽  
J M Lindstrom ◽  
D K Berg
Keyword(s):  
Membrane Proteins ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Dorsal Root Ganglion Neurons ◽  
Synaptic Membrane ◽  
Coated Pits ◽  
Nicotinic Acetylcholine ◽  
Ganglion Neurons

Chick ciliary ganglion neurons have a membrane component that shares an antigenic determinant with the main immunogenic region (MIR) of nicotinic acetylcholine receptors from skeletal muscle and electric organ. Previous studies have shown that the component has many of the properties expected for a ganglionic nicotinic acetylcholine receptor, and that its distribution on the neuron surface in vivo is restricted predominantly to synaptic membrane. Here we report the presence of a large intracellular pool of the putative receptor in embryonic neurons and demonstrate that it is associated with organelles known to comprise the biosynthetic and regulatory pathways of integral plasma membrane proteins. Embryonic chick ciliary ganglia were lightly fixed, saponin-permeabilized, incubated with an anti-MIR monoclonal antibody (mAb) followed by horseradish peroxidase-conjugated secondary antibody, reacted for peroxidase activity, and examined by electron microscopy. Deposits of reaction product were associated with synaptic membrane, small portions of the pseudodendrite surface membrane, most of the rough endoplasmic reticulum, small portions of the nuclear envelope, some Golgi complexes, and a few coated pits, coated vesicles, multivesicular bodies, and smooth-membraned vacuoles. No other labeling was present in the neurons. The labeling was specific in that it was not present when the anti-MIR mAb was replaced with either nonimmune serum or mAbs of different specificity. Chick dorsal root ganglion neurons thought to lack nicotinic acetylcholine receptors were not labeled by the anti-MIR mAb. Substantial intracellular populations have also been reported for the muscle acetylcholine receptor and brain voltage-dependent sodium channel alpha-subunit. This may represent a general pattern for multisubunit membrane proteins during development.

Sign in / Sign up

Export Citation Format

Share Document