L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI-DII domains

Voltage-dependent facilitation (VDF) is a unique phenomenon observed in L-type voltage-gated calcium (CaV) channels, in which depolarized prepulse voltages increase inward current by several fold, contributing to neuronal firing, neurotransmitter release, cardiac automaticity, and muscle contraction. Despite three decades of research, the molecular origin and biophysical mechanisms underlying VDF in L-type (CaV1.1-CaV1.4) channels remain elusive. A serendipitous observation that solutions stored in polypropylene tubes eliminated CaV1.2 and CaV1.3 L-type channel VDF led us to identify the leachable 2,4-di-tert-butylphenol (2,4-DTBP) as a selective VDF inhibitor. Docking and molecular dynamics simulations of 2,4-DTBP revealed a previously unreported {pi}-H and {pi}-{pi} interdomain quadrangle interaction, asymmetrically positioned at the DI-DII pore-domain (PD) interface of L-type channels. Point mutagenesis disrupted these quadrangle interactions and abolished the VDF, as confirmed by whole-cell and single-channel recordings of CaV1.2. Remarkably, introducing this {pi}-H and {pi}-{pi} quadrangle interaction into the DIIS6 segment of non-VDF-displaying CaV2.1 channels induced robust VDF. In conclusion, this study provides the first molecular evidence for the VDF endpoint: L-type channel-specific interdomain quadrangle interactions asymmetrically positioned at the DI-DII PD interface.