Photoaffinity labelling of the phenylalkylamine receptor of the skeletal muscle transverse‐tubule calcium channel

FEBS Letters

Seitz, Werner¹  Knaus, Hans-Günther³  Lietz, Helmut²  Glossmann, Hartmut³  Striessnig, Jörg³  Moosburger, Kurt³  Grabner, Manfred³ 

[1] Hauptlaboratorium der BASF AG, D-6700 LudwigshafenFRG;Forschung und Entwicklung, Knoll AG, D-6700 Ludwigshafen, FRG;Institut für Biochemische Pharmakologie, Peter-Mayr Str. I, A-6020 Innsbruck, Austria

关键词: Ca2+ channel;    Purification;    Phenylalkylamine;    Photoaffinity label;   

DOI  :  10.1016/0014-5793(87)81354-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The tritiated arylazido phenylalkylamine (-)-5-[(3-azidophenethyl)[N-methyl-³H]methylamino]-2-(3,4,5-tri-methoxyphenyl)-2-isopropylvaleronitrile was synthesized and used to photoaffinity label the phenylalkylamine receptor of the membrane-bound and purified calcium channel from guinea-pig skeletal muscle trans-verse-tubule membranes. The photoaffinity ligand binds reversibly to partially purified membranes with a K _d of 2.0 ± 0.5 nM and a B _max of 17.0 ± 0.9 protein. Binding is stereospecifically regulated by all three classes of organic calcium channel drugs. A 155 kDa band was specifically photolabelled in transverse-tubule particulate and purified calcium channel preparations after ultraviolet irradiation. Additional minor labelled polypeptides (92, 60 and 33 kDa) were only observed in membranes. The heterogeneous 155 kDa region of the purified channel was resolved into two distinct silver-stained polypeptides after reduction (i.e. 155 and 135 kDa). Only the 155 kDa polypeptide carries the photoaffinity label and it is concluded that the 135 kDa polypeptide (which migrates as a 165 kDa band under alkylating conditions) is not a high-affinity drug receptor carrying subunit of the skeletal muscle transverse-tubule L-type calcium channel.

【 授权许可】

Unknown