Cyclic Peptides as T-Type Calcium Channel Blockers: Characterization and Molecular Mapping of the Binding Site.

ABSTRACT

T-type calcium (CaV3) channels play a crucial role in the generation and propagation of action potentials in excitable cells and are considered potential drug targets for the treatment of neurological and cardiovascular diseases. Given the limited pharmacological repertoire for these channels, there is a great need for novel potent and selective CaV3 channel inhibitors. In this study, we used Xenopus oocytes to heterologously express CaV3.1 channels and characterized the interaction with a small cyclic peptide, PnCS1. Using molecular modeling, PnCS1 was docked into the cryo-electron microscopy structure of the human CaV3.1 channel and molecular dynamics were performed on the resultant complex. The binding site of the peptide was mapped with the involvement of critical amino acids located in the pore region and fenestrations of the channel. More specifically, we found that PnCS1 reclines in the central cavity of the pore domain of the CaV3.1 channel and resides stably between the selectivity filter and the intracellular gate, blocking the conduction pathway of the channel. Using Multiple Attribute Positional Scanning approaches, we developed a series of PnCS1 analogues. These analogues had a reduced level of inhibition, confirming the importance of specific residues and corroborating our modeling. In summary, functional studies of PnCS1 on the CaV3.1 channel combined with molecular dynamics results provide the basis for understanding the molecular interactions of PnCS1 with CaV3.1 and are fundamental to structure-based drug discovery for treating CaV3 channelopathies.