RECONSTITUTION OF THE HUMAN R-TYPE Cav2.3 CHANNEL INTO PROTEOLIPOSOMES FOR CRYO-EM STRUCTURAL ANALYSIS

Abstract

Voltage-gated calcium (Cav) channels are a family of transmembrane proteins that mediate calcium entry into the cell by responding to changes in membrane potential. They are found in many types of excitable cells and regulate key physiological processes through membrane-dependent channel gating. In particular, the human R-type Cav2.3 channel is strongly expressed in neuronal and neuroendocrine cells, elevating its pharmacological importance as a therapeutic target for neurological diseases like epilepsy, pain response, and Parkinson’s disease. However, due to the resistance of Cav2.3 channels to most selective inhibitors, little is known about the modulation and inactivation of these channels for rational drug design. Moreover, the molecular mechanisms of Cav channel gating processes are unclear, as detailed structures of most channel states have not yet been elucidated. Recently, high-resolution structures of the human Cav2.3 complex were determined by the Yan lab, and correlated functional studies were performed to gain insight into the inactivation gating mechanism of Cav channels. The goal of the current study was to further image Cav2.3 channels in native-like environments, through the development of a reproducible method for reconstituting human Cav2.3 into proteoliposome systems and preparing high-quality cryo-samples for 3D structural reconstruction. Our method lays a strong foundation for uncovering the complete working mechanisms of Cav channels through structural elucidation of their various physiological states.