Table 1. Effects of important CaM-inhibitors on particular CaM-target interactions.
| CaM-target and reference | Assays used | Analyzed substances, used concentraction ranges a and IC50 values b | Most important findings |
|---|---|---|---|
| AC in rat cerebellar membranes [70] | AC activity assay | CDZ (1–80 μM) IC 50: 3 μM; TFP (10–100 μM) IC 50: 30 μM; W-7 (10–100 μM) | CDZ and TFP inhibit AC in an apparently competitive manner, W-7 in a non-competitive. Potency of antagonists is dependent upon CaM-concentration. CaM-AC complex is relatively refractory to inhibition. |
| AC2, AC9 and recombinant AC fusion proteins [51] | AC activity assay | CDZ (1–1000 μM) IC 50 (AC5-AC2 fusion protein): 20 μM | CDZ is a non-competitive inhibitor of AC activity. Effect of CDZ is mediated by direct interaction with the catalytic core of AC in an apparently different manner than inhibition by adenosine analogues. CDZ has biphasic effects on AC activity: at low concentrations of CDZ, AC activity increases. |
| Bordetella pertussis AC toxin CyaA [21] | AC activity assay, fluorescence studies | CDZ (0.001–100 μM) IC 50: 0.66 μM; TFP (0.001–100 μM); W-7 (0.001–100 μM) | Inhibition of CyaA by CDZ is CaM-independent. Data suggest that CDZ binds to one or two hydrophobic binding sites in CyaA preventing conformational changes required for catalytic activity of CyaA. TFP and W-7 do not inhibit CyaA. |
| NO-activated sGC (cerebellar cells and purified enzyme) [52] | Ca2+-imaging, NO and sGC activity assay | CDZ (1–100 μM) IC 50: 11.3 μM; TFP (3–300 μM) IC 50: 177 μM | Inhibitory effect of CDZ on cGMP accumulation does not depend on Ca2+-signaling. CDZ directly inhibits purified sGC in an uncompetitive and CaM-independent manner. CDZ inhibits purified sGC with similar potency to its effect on cerebellar astrocytes. |
| SERCA [49] | Ca2+-ATPase activity assay, fluorescence studies, Ca2+-binding and phosphoryla-tion studies | Various concentrations up to 200 μM were used of CaM-binding peptide IC 50: 7.0 μM; CDZ IC 50: 0.5 μM; chlorpromazine IC 50: 23 μM; fluphenazine IC 50: 15 μM; TFP IC 50: 45 μM; W-7 IC 50: 125 μM | Effects of CaM-antagonists are independent of CaM and they inhibit the SERCA in an isoform-specific manner. CaM-antagonists not only reduce the maximal activity, they also increase the Km for Ca2+-binding of the high-affinity (stimulatory) side. CDZ and CaM-binding peptide are the most potent inhibitors of SR c Ca2+-ATPase activity, W-7 is the least potent inhibitor. |
| Skeletal muscle SR c Ca2+-ATPase [50] | Ca2+-ATPase activity assay, steady-state phosphoryla-tion assay | CDZ (1–10 μM) IC 50: 4.1 μM; TFP (10–100 μM) IC 50: ~ 200 μM | CDZ inhibits skeletal SR c Ca2+-ATPase with high affinity and in a non-competitive manner. Results suggest that inhibition is not a specific antagonism of enzyme activation by CaM but depends on binding to the membrane phospholipids. |
| a) Ca2+/CaM-dependent PDE; b) trypsin-treated PDE: lost its sensitivity to Ca2+/CaM [17] | Radiometric PDE activity assay, binding study with W-7-coupled sepharose | Chlorpromazine a) IC 50: 35 μM, IC 50: 210 μM; TFP (10–1000 μM) a) IC 50: 7 μM, IC 50: 110 μM; W-7 (10–1000 μM) a) IC 50: 28 μM, b) IC 50: 375 μM | CaM-antagonists inhibit also trypsin-treated PDE. Binding sites for CaM-antagonists on trypsin-treated PDE have structural similarities to Ca2+/CaM. CaM-antagonists binding site is at or near the active site. |
| Connexin50 gap junctions (expressed in the lens of the eye) [78] | Whole cell patch clamp experiments, NMR studies, fluorescence studies, CD, mass spectrometry | Various concentrations of CDZ and Cx50-peptide (Cx50p141-166 out of the CaM-binding domain of Cx50) d | Shown by using CDZ and Cx50p141-166: Ca2+-dependent inhibition of Cx50 gap junctions is mediated by CaM. |
| MLCK [18] | MLCK activity assay, fluorescence studies | CDZ (0.3–500 μM) IC 50: 18 μM e ; TFP (0.3–500 μM) IC 50: 144 μM e ; W-7 (0.3–500 μM) IC 50: 300 μM e | CDZ, TFP and W-7 inhibit the MLCK CaM-dependently and CaM-independently. All antagonists bind to MLCK. CaM-independent inhibition of antagonists occurred by binding to MLCK. |
aConcentration ranges studied are given in parentheses.
bIf available, IC50 values are indicated in italics.
cSR, sarcoplasmatic reticulum.
dIC50 values were not determined.
eIC50 values obtained from experiments using 115 nM CaM and 8.0 mg/ml MLCK.