Localization of felodipine (dihydropyridine) binding site on calmodulin

Abstract

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.

Differentiation of the drug‐binding sites of calmodulin

“…The exact localization of this site is unknown, but recent evidence suggests that the connecting c( helix is part of the binding site [27]. Direct binding experiments carried out in this study show that this site does not recognize other calmodulin antagonists as suggested by Johnson and colleagues [27,29]. However, the kinetic experments indicate that occupancy of a second lowaffinity site is necessary to inhibit the activation of the myosin light-chain kinase.…”

“…10) suggesting that binding of a dihydropyridine to this site inhibits the activation of the phosphodiesterase. The exact localization of this site is unknown, but recent evidence suggests that the connecting c( helix is part of the binding site [27]. Direct binding experiments carried out in this study show that this site does not recognize other calmodulin antagonists as suggested by Johnson and colleagues [27,29].…”

“…A slightly different affinity or hydrophobicity for these two sites has been noted [27] suggesting that these sites may select slightly different compounds. An additional interaction site is present between calmodulin antagonists, which have a positive charge in the side chain and residues 82 -92 [28].…”

“…The dihydropyridine felodipine binds to a specific site on calmodulin [27,291. The binding to this site is allosterically modified by occupancy of a phenothiazine-binding site [29].…”

Effects of Myosin Light Chain Kinase and Peptides on Ca2+ Exchange with the N- and C-terminal Ca2+ Binding Sites of Calmodulin

“…2ϩ Binding Sites-The hydrophobic polarity probe TNS undergoes a large fluorescence increase when it binds to the Ca 2ϩ -dependent hydrophobic pockets in both the N-and Cterminal halves of CaM (Suko et al, 1985;Johnson et al, 1986).…”