Inhibitory Mechanism of the Isoflavone Derivative Genistein in the Human Ca V 3.3 Channel

• Published in: ACS chemical neuroscience 2021-02, Vol.12 (4), p.651-659
• Main Authors: Rangel-Galván, Maricruz, Rangel, Azahel, Romero-Méndez, Catalina, Dávila, Eliud Morales, Castro, María Eugenia, Caballero, Norma A, Meléndez Bustamante, Francisco J, Sanchez-Gaytan, Brenda L, Meza, Ulises, Perez-Aguilar, Jose Manuel
• Format: Article
• Language: English
• ISSN: 1948-7193; 1948-7193
• DOI: 10.1021/acschemneuro.0c00684

Regulation of cellular excitability and oscillatory behavior of resting membrane potential in nerve cells are largely mediated by the low-voltage activated T-type calcium channels. This calcium channel family is constituted by three isoforms, namely, Ca 3.1, Ca 3.2, and Ca 3.3, that are largely distributed in the nervous system and other parts of the body. Dysfunction of T-type calcium channels is associated with a wide range of pathophysiologies including epilepsy, neuropathic pain, cardiac problems, and major depressive disorders. Due to their pharmacological relevance, finding molecular agents able to modulate the channel's function may provide therapeutic means to ameliorate their related disorders. Here we used electrophysiological experiments to show that genistein, a canonical tyrosine kinase inhibitor, reduces the activity of the human Ca 3.3 channel in a concentration-dependent manner. The inhibitory effect of genistein is independent of tyrosine kinase modulation and does not affect the voltage-dependent gating of the channel. Subsequently, we used computational methods to identify plausible molecular poses for the interaction of genistein and the Ca 3.3 channel. Starting from different molecular poses, we carried out all-atom molecular dynamics (MD) simulations to identify the interacting determinants for the Ca 3.3/genistein complex formation. Our extensive (microsecond-length) simulations suggest specific binding interactions that seem to stabilize the protein/inhibitor complex. Furthermore, our results from the unbiased MD simulations are in good agreement with the recently solved cryoelectron microscopy structure of the Ca 3.1/Z944 complex in terms of both the location of the ligand binding site and the role of several equivalent amino acid residues. Proposed interacting complex loci were subsequently tested and corroborated by electrophysiological experiments using another naturally occurring isoflavone derivative, daidzein. Thus, by using a combination of and techniques, we have identified interacting determinants relevant to the Ca 3.3/genistein complex formation and propose that genistein directly blocks the function of the human Ca 3.3 channel as a result of such interaction. Specifically, we proposed that a combination of polar interactions involving the three hydroxyl groups of genistein and an aromatic interaction with the fused rings are the main binding interactions in the complex formation. Our results pave the way for the rationa