Structural analysis of the calcium channel by photoaffinity labelling and limited proteolysis.
The L-type calcium channel of rabbit skeletal muscle triads, purified from digitonin extracts, was photolabelled with the dihydropyridine (+)[3H]PN 200-110. This photolabelled form was then subjected to limited proteolysis with papain and staphylococcus V-8 protease and analyzed by polyacrylamide gel electrophoresis. In the absence of proteolysis, the photolabelled channel was represented by a single protein with an apparent molecular weight of 160 kDa in the presence or absence of reducing agents. Following proteolysis, numerous photoadducts were observed with smaller molecular weights. The V-8 protease digestion pattern indicated that photoinsertion occurred in at least two distinct domains of 33 and 28 kDa. Papain digests were more extensive, generating smaller fragments of 28 and ca. 10 kDa. The results suggest that at least two distinct regions of the calcium channel interface at or near the dihydropyridine binding site, and that the binding site for these calcium antagonists resides within the channel proper, thereby modulating calcium influx.
Johnson
D F
DF
Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201.
Kuo
T H
TH
Giacomelli
F
F
Wiener
J
J
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism*; D017275:Isradipine; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D010069:Oxadiazoles / Q000378:metabolism*; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D011817:Rabbits
D000345:Affinity Labels; D002121:Calcium Channel Blockers; D007473:Ion Channels; D008565:Membrane Proteins; D010069:Oxadiazoles; D010446:Peptide Fragments; D010206:Papain; D002118:Calcium; D017275:Isradipine