Localization of a felodipine (dihydropyridine) binding site on calmodulin.
The fluorescent dihydropyridine calcium antagonist drug felodipine binds to calmodulin (CaM) in a Ca2+-dependent manner. Its binding can be regulated by the interaction of CaM antagonist drugs through allosteric mechanisms [Mills, J.S., & Johnson, J.D. (1985) Biochemistry 24, 4897]. Here, we have examined the binding of a nonspecific hydrophobic fluorescent probe molecule TNS (toluidinylnaphthalenesulfonate) and of felodipine to CAM and several of its proteolytic fragments. While TNS interacts with sites on both the amino-terminal half of the protein [proteolytic fragment TR1C (1-77)] and carboxy-terminal half [proteolytic fragment TR2C (78-148)], felodipine binding shows more selectivity. It binds in a Ca2+-dependent manner to the proteolytic fragments TM1 (1-106) and TR2E (1-90) but exhibits only weak affinity for TR1C (1-77) and TR2C (78-148). Furthermore, felodipine exhibits selectivity over TNS and trifluoperazine (TFP) in blocking the tryptic cleavage between residues 77 and 78. These studies indicate a selective binding of felodipine to a hydrophobic site existing in residues 1-90 and suggest that productive binding requires amino acids in the region 78-90. Although the felodipine binding site is preserved in fragment 1-106, the allosteric interactions between the prenylamine and the felodipine binding sites that are observed with intact CaM are not observed in this fragment. Rather, prenylamine simply displaces felodipine from its binding site on this fragment. Our results are consistent with calmodulin containing not less than two allosterically related hydrophobic drug binding sites. One of these sites (felodipine) appears to be localized in region 1-90 and the other one in region 78-148.
J D
JD
Johnson
L A
LA
Wittenauer
E
E
Thulin
S
S
Forsén
H J
HJ
Vogel
D000818:Animals; D000959:Antihypertensive Agents / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D015736:Felodipine; D007700:Kinetics; D008297:Male; D009543:Nifedipine / Q000031:analogs & derivatives* / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D013737:Testis / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D000959:Antihypertensive Agents; D002147:Calmodulin; D010446:Peptide Fragments; D014357:Trypsin; D009543:Nifedipine; D015736:Felodipine; D002118:Calcium