Effects of LAs on 20S proteasome activity and binding to the 20S proteasome. (a) Effects of LAs and other related chemical agents (■: pilsicainide; ▼: lidocaine; ∆: MG132; ♦: bortezomib; □: verapamil; ○: E-4031; ◊: TTX; ▲: 4-F; ●: M-3) on 20S proteasome activity in vitro. Each point represents the mean ± SEM of more than 7 determinations. 20S proteasome activities were expressed relative to their values in the absence of drugs (100%). The calculated IC50 values were 4.1 ± 2.4 nM, 1.1 ± 1.8 nM, 3.5 ± 1.5 μM, 23 ± 0.2 μM, 20 ± 2.5 μM, 14 ± 6.3 μM for MG132, bortezomib, pilsicainide, lidocaine, 4F, and mexiletine, respectively. (b) Inhibitory action of pilsicainide, 2-F, 3-F, 4-F, 2,4-F, 2,6-F and M-3 at 10 μM (left) and 100 μM (right) on the chymotrypsin-like activity of the proteasome. (c) Inhibition of binding of [3H]-pilsicainide to the 20S proteasome by cold pilsicainide, 4-F, M-3 and lidocaine in vitro. Each point represents the mean ± SEM of more than 7 determinations. The binding fraction of [3H]-pilsicainide to the 20S proteasome was expressed relative to the values in the absence of cold pilsicainide, 4-F, M-3 or lidocaine (100%). K
d values for pilsicainide, lidocaine, and 4-F are1.0 ± 2.8 μM, 21.2 ± 3.5 μM, and 4-F, respectively, whereas no binding capacity of M-3 was observed.