Biomaterial Database

Agonist-induced photoincorporation of a p-benzoylphenylalanine derivative of substance P into membrane-spanning region 2 of the Torpedo nicotinic acetylcholine receptor delta subunit. 1994

M P Blanton, and Y M Li, and E R Stimson, and J E Maggio, and J B Cohen

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115.

The neuropeptide substance P acts, at micromolar concentrations, as a noncompetitive antagonist of nicotinic acetylcholine receptors (AChRs) of both neuronal and muscle subtypes. The mechanism of this inhibition has been shown to be most consistent with stabilization of a nonconducting desensitized state of the AChR, via binding to a site distinct from both the agonist site and the high affinity noncompetitive antagonist site. We have used a radioiodinated photoreactive analogue of substance P, containing the amino acid p-benzoyl-L-phenylalanine in place of the Phe8 residue of substance P, to identify the sites of interaction of substance P within the Torpedo california AChR. AChR-rich membrane suspensions were photolabeled in the absence or presence of the agonist carbamylcholine and/or nonradioactive substance P, and incorporation into AChR subunits was assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of agonist 125I incorporation was detected in each subunit and was insensitive to substance P, whereas in the presence of carbamylcholine there was a 2-fold increase in photoincorporation into the AChR delta subunit that was inhibited by the addition of an excess of substance P. The sites of specific photoincorporation in the delta subunit were initially mapped by use of Staphylococcus aureus V8 protease to a 14-kDa fragment extending from delta Ile-192 to Glu-280. Further fragmentation of this 14-kDa fragment with trypsin and S. aureus V8 protease established that the sites of specific incorporation were restricted to the region delta Ser-253 to Glu-280, which contains the membrane-spanning region 2 that is known to form the lining of the ion channel. These results establish that in the presence of agonist at least a part of the undecapeptide substance P binds within the ion channel in the desensitized state of the AChR, and it is likely that the binding of substance P to this site is responsible for the action of substance P as a noncompetitive AChR antagonist.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010649	Phenylalanine	An essential aromatic amino acid that is a precursor of MELANIN; DOPAMINE; noradrenalin (NOREPINEPHRINE), and THYROXINE.	Endorphenyl,L-Phenylalanine,Phenylalanine, L-Isomer,L-Isomer Phenylalanine,Phenylalanine, L Isomer
D010777	Photochemistry	A branch of physical chemistry which studies chemical reactions, isomerization and physical behavior that may occur under the influence of visible and/or ultraviolet light.	Photochemistries
D011978	Receptors, Nicotinic	One of the two major classes of cholinergic receptors. Nicotinic receptors were originally distinguished by their preference for NICOTINE over MUSCARINE. They are generally divided into muscle-type and neuronal-type (previously ganglionic) based on pharmacology, and subunit composition of the receptors.	Nicotinic Acetylcholine Receptors,Nicotinic Receptors,Nicotinic Acetylcholine Receptor,Nicotinic Receptor,Acetylcholine Receptor, Nicotinic,Acetylcholine Receptors, Nicotinic,Receptor, Nicotinic,Receptor, Nicotinic Acetylcholine,Receptors, Nicotinic Acetylcholine
D000345	Affinity Labels	Analogs of those substrates or compounds which bind naturally at the active sites of proteins, enzymes, antibodies, steroids, or physiological receptors. These analogs form a stable covalent bond at the binding site, thereby acting as inhibitors of the proteins or steroids.	Affinity Labeling Reagents,Labeling Reagents, Affinity,Labels, Affinity,Reagents, Affinity Labeling
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D013373	Substance P	An eleven-amino acid neurotransmitter that appears in both the central and peripheral nervous systems. It is involved in transmission of PAIN, causes rapid contractions of the gastrointestinal smooth muscle, and modulates inflammatory and immune responses.	Euler-Gaddum Substance P,Hypothalamic Substance P,SP(1-11),Euler Gaddum Substance P,Substance P, Euler-Gaddum,Substance P, Hypothalamic
D014101	Torpedo	A genus of the Torpedinidae family consisting of several species. Members of this family have powerful electric organs and are commonly called electric rays.	Electric Rays,Torpedinidae,Rays, Electric
D018722	Nicotinic Agonists	Drugs that bind to and activate nicotinic cholinergic receptors (RECEPTORS, NICOTINIC). Nicotinic agonists act at postganglionic nicotinic receptors, at neuroeffector junctions in the peripheral nervous system, and at nicotinic receptors in the central nervous system. Agents that function as neuromuscular depolarizing blocking agents are included here because they activate nicotinic receptors, although they are used clinically to block nicotinic transmission.	Cholinergic Agonists, Nicotinic,Cholinergic Agonist, Nicotinic,Nicotinic Agonist,Agonist, Nicotinic,Agonist, Nicotinic Cholinergic,Agonists, Nicotinic,Agonists, Nicotinic Cholinergic,Nicotinic Cholinergic Agonist,Nicotinic Cholinergic Agonists