Differentiation of the drug‐binding sites of calmodulin

Abstract

Calmodulin contains several binding sites for hydrophobic compounds. The apparent specificity of various 'calmodulin antagonists' for these sites was investigated. The Ki values for the inhibition of calmodulin-activated cyclic-nucleotide phosphodiesterase and myosin light-chain kinase was determined. In addition, the Kd values of the same compounds for binding to calmodulin were measured. The compounds could be separated into four groups. Group I and I1 compounds inhibited competitively the activation of the phosphodiesterase and myosin light-chain kinase by calmodulin. Group I compounds inhibited the activation of the phosphodiesterase and myosin light-chain kinase at identical concentrations. In contrast, group I1 compounds inhibited the activation of the phosphodiesterase at 5 -10-fold lower concentrations than that of myosin light-chain kinase. Group I11 compounds inhibited the activation of these enzymes by an uncompetitive mechanism. Group IV compounds inhibited the activation of the phosphodiesterase with Ki values above 10 pM and did not affect the activation of myosin light-chain kinase.Binding of I3H]bepridil to calmodulin under equilibrium conditions yielded one high-affinity site (apparent Kd 0.4 pM) and four low affinity sites (apparent Kd 44 pM). Group I compounds interfered with the binding of bepridil to the high and low-affinity sites in a competitive manner. Group I1 compounds interfered in a noncompetitive manner with the high-affinity site and apparently competed only with one of the low-affinity sites. These data show that 'calmodulin antagonists' can be differentiated into at least three distinct groups. Kinetic and binding data suggest that the three groups bind to at least three different sites on calmodulin. Selective occupation of these sites may inhibit specifically the activation of distinct enzymes.Calmodulin is a highly conserved intracellular calciumbinding protein, which activates more than 30 different proteins in a calcium-dependent manner 11, 21. At present it is not clear how calmodulin regulates so many protein functions with any degree of selectivity. Calmodulin has four calciumbinding sites and a number of hydrophobic sites, which are exposed on its surface upon calcium binding [3 -51. The exposure of these hydrophobic sites is thought to be essential for the binding of calmodulin to and activation of different proteins. A large number of compounds, known as calmodulin antagonists [6], bind in the vincinity of these hydrophobic sites. The binding of an antagonist to calmodulin interfers with the activation of many calmodulin-dependent enzymes suggesting that the binding sites for calmodulin antagonists are close to those sites which interact with the enzymes. The exact localization of the hydrophobic sites, calmodulin-antagonist-binding sites and sites for interaction with enzymes is presently unknown, although the approximate localization of these sites has been deduced from primary and tertiary structure data in connection with binding and NMR studies.

Blockade by antiarrhythmic drugs of glibenclamide‐sensitive K+ channels in Xenopus oocytes

“…Both ( + )-and (-)-propranolol blocked KRN2391 responses with similar potencies, and they are known to inhibit Ca2+-PDE with an IC5o value of 180 gM for each (Volpi et al, 1981). Diltiazem and verapamil also have calmodulin-antagonizing activities (Epstein et al, 1982;Zimmer & Hofmann, 1987). Typical local anaesthetics such as lignocaine and procaine' also possess very weak calmodulin-antagonizing activities (Muto et al, 1983).…”

“…Figure 7 and 3 gM, respectively (Hidaka et al, 1980;Itoh et al, 1984;Zimmer & Hofmann, 1987). Both ( + )-and (-)-propranolol blocked KRN2391 responses with similar potencies, and they are known to inhibit Ca2+-PDE with an IC5o value of 180 gM for each (Volpi et al, 1981).…”

“…Of all the antiarrhythmic drugs tested in this study, bepridil, prenylamine and aprindine, which showed relatively potent blocking effects on KRN2391 responses, are known to possess fairly strong antagonistic activities on calmodulin (Hidaka et al, 1980;-Itoh et al, 1984;Zimmer & Hofmann, 1987 (Haworth et al, 1989 (Vaughan Williams, 1984;1989;Woosley, 1991 (1I00 jAM) was applied for 20s (short bars), and W-7 (50 jAM) was applied for I min (long bar). Note slow recovery from inhibition by W-7.…”

Loss of cell surface microvilli on rat basophilic leukaemia cells precedes secretion and can be mimicked using the calmodulin antagonist trifluoperazine

“…Ⅵ In order to understand better the molecular process controlling cell surface changes a number of potential signal transduction inhibitors were screened for effects on cellular morphology. One of these inhibitors was TFP which has been widely used as a calmodulin antagonist, and thus would be expected to prevent calmodulin-dependent processes [20,21]. TFP at 20 M did not inhibit stimulated secretion in RBL cells, and neither did it cause secretion when used alone.…”

S100A1 Is a Novel Molecular Chaperone and a Member of the Hsp70/Hsp90 Multichaperone Complex

“…Effects of CaM Antagonists on the Chaperone Activity of S100A1-Phenothiazines and diphenylalkylamines were originally identified as CaM antagonists (36), which bind to CaM in a Ca 2ϩ -dependent manner and antagonize the interaction of CaM with its target proteins; however, some CaM antagonists also bind S100 proteins, including S100A1, and thus would be expected to antagonize S100 proteins (21). To investigate the possible antagonistic activity of two CaM antagonists against S100A1, effects of fluphenazine (a representative of the phenothiazines) and prenylamine (a representative of the diphenylalkylamines) on the chaperone activity of S100A1 by the CS aggregation assay were examined.…”