Photoaffinity labeling of the ryanodine receptor/Ca2+ release channel with an azido derivative of ryanodine

Derrick R Witcher; Peter S Mcpherson; Steven D Kahl; Terence Lewis; Philip Bentley; Michael J Mullinnix; John D Windass; Kevin P Campbell

doi:10.1016/S0021-9258(17)36799-6

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Photoaffinity labeling of the ryanodine receptor/Ca2+ release channel with an azido derivative of ryanodine

Journal article

Open access

Peer reviewed

Photoaffinity labeling of the ryanodine receptor/Ca2+ release channel with an azido derivative of ryanodine

Derrick R Witcher, Peter S Mcpherson, Steven D Kahl, Terence Lewis, Philip Bentley, Michael J Mullinnix, John D Windass and Kevin P Campbell

The Journal of biological chemistry, Vol.269(18), pp.13076-13079

1994

DOI: 10.1016/S0021-9258(17)36799-6

PMID: 8175731

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Abstract

Ryanodine receptors/Ca2+ release channels play an important role in regulating the intracellular free calcium concentrations in both muscle and nonmuscle cells. Ryanodine, a neutral plant alkaloid, specifically binds to and modulates these Ca2+ release channels. In the work described here, we characterize the interaction of a tritium-labeled, photoactivable derivative of ryanodine (3H-labeled 10-O-[3-(4-azidobenzamido)propionyl]ryanodine ([3H]ABRy)) with the ryanodine receptor of skeletal, cardiac, and brain membranes. Scatchard analysis demonstrates that this ligand binds to a single class of high affinity sites in skeletal muscle triads. Furthermore, competition binding assays of [3H]ryanodine with skeletal, cardiac, and brain membranes in the presence of increasing concentrations of unlabeled ABRy illustrate that this azido derivative of ryanodine is able to specifically displace [3H]ryanodine from its binding site(s). Analysis of the effects of Ca2+, ATP, and KCl on [3H]ABRy binding in triad membranes shows a similar modulation of binding to that seen in these membranes with [3H]ryanodine. Photoaffinity labeling of triads with [3H]ABRy resulted in specific and covalent incorporation of [3H]ABRy into a 565-kDa protein that was shown to be the skeletal muscle ryanodine receptor. Digestion of the labeled ryanodine receptor revealed a [3H]ABRy-labeled 76-kDa tryptic fragment that was identified with an antibody directed against the COOH-terminal of the receptor. These results demonstrate that the 76-kDa COOH-terminal tryptic fragment contains the high affinity binding site for ryanodine.

Fundamental and applied biological sciences. Psychology

Cell receptors

Miscellaneous

Cell membranes. Ionic channels. Membrane pores

Biological and medical sciences

Molecular and cellular biology

Cell structures and functions

Details

Title: Subtitle: Photoaffinity labeling of the ryanodine receptor/Ca2+ release channel with an azido derivative of ryanodine
Creators: Derrick R Witcher - Univ. Iowa coll. medicine, Howard Hughes medical inst., dep. physiology, Iowa City IA 52242, United States
Peter S Mcpherson - Univ. Iowa coll. medicine, Howard Hughes medical inst., dep. physiology, Iowa City IA 52242, United States
Steven D Kahl - Univ. Iowa coll. medicine, Howard Hughes medical inst., dep. physiology, Iowa City IA 52242, United States
Terence Lewis - Univ. Iowa coll. medicine, Howard Hughes medical inst., dep. physiology, Iowa City IA 52242, United States
Philip Bentley
Michael J Mullinnix - Univ. Iowa coll. medicine, Howard Hughes medical inst., dep. physiology, Iowa City IA 52242, United States
John D Windass
Kevin P Campbell - Univ. Iowa coll. medicine, Howard Hughes medical inst., dep. physiology, Iowa City IA 52242, United States
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.269(18), pp.13076-13079
DOI: 10.1016/S0021-9258(17)36799-6
PMID: 8175731
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology; Bethesda, MD
Language: English
Date published: 1994
Academic Unit: Neurology; Molecular Physiology and Biophysics; Iowa Neuroscience Institute
Record Identifier: 9984068263202771

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