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. Author manuscript; available in PMC: 2021 Sep 18.
Published in final edited form as: ACS Chem Biol. 2020 Sep 3;15(9):2539–2550. doi: 10.1021/acschembio.0c00577

Figure 2. Homology model of CaV1.3 and docking of cp-PYT reveals that cp-PYT uniquely engages Met1078 in CaV1.3-LTCCs.

Figure 2.

Proposed docking poses of isradipine (a) and cp-PYT (b) in the CaV1.3 structural model. Isradipine is represented in purple and cp-PYT is represented in orange, while CaV1.3 is represented by green ribbon diagrams. Key binding site residues and interactions with ligands are shown with dark green sticks and with yellow dash lines. Sequence numbers are based on rat CaV1.3 and CaV1.2 subunits. c. Schematic diagram of the CaV1 α1-subunit. The consensus amino acid sequence of the S5 segment of the third (blue) and the S6 segment of the fourth (green) transmembrane domains of nLTCCs are shown. The docking model predicts that methionine-1078 and valine-1198 (red) of the CaV1.3 α1-subunit underlie cp-PYT selective inhibition. d. Voltage-clamp currents evoked in HEK293 cells expressing CaV1.3 (left) or CaV1.2 (right) channels; CaV1.3 channels have a pore-forming α1 subunit with an M1078V mutation, whereas CaV1.2 channels had a pore-forming a1 subunit with an V1063M mutation. Current records are shown before and after bath application of cp-PYT (100 μM). Voltage protocol is shown at the bottom. e. Population data of the inhibition observed by application of either 100 μM cp-PYT (left) or 5 μM isradipine (right) to CaV1.3 (black, cp-PYT: n = 6, 60%, isradipine: n = 5, 90%), CaV1.3-M1078V (blue, cp-PYT: n = 6, 4%, isradipine: n = 5, 78%), and CaV1.3-V1198I (green, cp-PYT: n = 6, 45%, isradipine: n = 6, 94%) L-type calcium channels (LTCCs). The inhibition of CaV1.3 as compared to CaV1.3-M1078V LTCCs by application of 100 μM cp-PYT was deemed significant by the Mann-Whitney Rank Sum Test (**p < 0.001). f. Population data of the inhibition observed by application of either 100 μM cp-PYT (left) or 5 μM isradipine (right) to CaV1.2 (black, cp-PYT: n = 8, 21%, isradipine: n = 6, 78%), CaV1.2-V1063M (blue, cp-PYT: n = 6, 47%, isradipine: n = 6, 92%), CaV1.2-I1183V (green, cp-PYT: n = 8, 12%, isradipine: n = 8, 95%), and CaV1.2-V1063M+I1183V (gray, cp-PYT: n = 10, 32%, isradipine: n = 10, 93%) L-type calcium channels. The inhibition of CaV1.2 as compared to CaV1.2-V1063M and CaV1.2-V1063M+I1183V by application of 100 μM cp-PYT was deemed significant by the Mann-Whitney Rank Sum Test (*p < 0.05).