A sensitive and rapid method for identification and characterization of low abundance receptors

doi:10.1016/0003-2697(90)90269-F

Analytical Biochemistry

Volume 185, Issue 1, 15 February 1990, Pages 143-146

https://doi.org/10.1016/0003-2697(90)90269-F Get rights and content

Abstract

An improved method for detection of low intensity radioligand-receptor complexes resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. [³H]Azidopine-labeled 1,4-dihydropyridine (DHP) receptor from skeletal muscle resolved by SDS-PAGE was transferred to nitrocellulose and cut into strips and individual slices were analyzed for radioincorporation by liquid scintillation counting. [³H]Azidopine-labeled DHP binding subunit migrated as a single entity with a mass of 170 kDa and was confirmed using conventional methods. Results were obtained within 4 h after resolution by SDS-PAGE compared to 3–40 days using conventional methods. In addition, detection of extremely low signals (less than 50 cpm/lane), otherwise overwhelmed by background noise using conventional methods, was possible due to removal of free ligand during electrotransfer to nitrocellulose. This technique offers a rapid sensitive, cost effective alternative to fluorography or other conventional gel slice analysis methods for detecting low intensity radiolabeled complexes resolved by SDS-PAGE.

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Cited by (6)

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The dihydropyridine (DHP) and ryanodine (RY) receptors play a critical role in depolarization-induced calcium release in skeletal muscle, yet the factors which govern their expression remain unknown. We investigated the roles of electrical activity and trophic factors in the regulation of the genes encoding the α₁, α₂, and β subunits of the DHP receptor as well as the RY receptor in rat skeletal muscle in vivo. Muscle paralysis, induced by denervation, had no effect on the DHP receptor mRNA levels while the RY receptor mRNA was decreased. In contrast, chronic superfusion of tetrodotoxin onto the sciatic nerve resulted in a marked increase in mRNA levels and transcriptional activity of both DHP and RY receptor genes. Since nerve can induce changes in second messenger pathways which modulate muscle gene expression, we attempted to identify factors which regulate DHP and RY receptor expression using cultured myotubes. Elevated cAMP levels specifically inhibited the expression of RY receptor mRNA while 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, increased the transcripts encoding the RY receptor and the α₁ subunit of the DHP receptor. Changes in the level of mRNAs were paralleled by altered receptor numbers. Neither cAMP nor protein kinase C altered transcriptional activity of the DHP and RY receptor genes. These results demonstrate that neural factor(s) regulate DHP and RY receptor mRNA levels in vivo via transcriptional mechanisms while protein kinase C and cAMP can modulate DHP and RY receptor transcript levels by a transcription-independent process.
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Identification of 1,4-dihydropyridine binding domains within the primary structure of the α<inf>1</inf> subunit of the skeletal muscle L-type calcium channel
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A sensitive and rapid method for identification and characterization of low abundance receptors

Abstract

Trends Pharmacol. Sci

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

FEBS Lett

J. Mol. Cell. Cardiol