Ion channels control ion movement in and out of cells and play essential roles for neurons to perceive, process, and transmit signals. Mutations on ion channels have been identified as risk factors in schizophrenia, epilepsy, and autism. A T-type calcium channel gene (CACNA1I) has been associated with schizophrenia, a common disease with broad social implication and huge un-met medical needs. Many mutations on Cav3.3 channel, such as Cav3.3 R1346H, a missense loss-of-function mutation, exhibited defects in forward trafficking while the channel property is otherwise normal. Ion channel forward trafficking is a dynamic and mysterious process, which has been largely understudied due to the lack of quantifiable measurement of ion channel protein density on the membrane. The goal of this project is to develop a novel cellular trafficking assay to study the rate of movement of ion channels from ER-Golgi intermediate compartment to the plasma membrane. To develop the assay, we first need a specific non-permeable irreversible inhibitor for Cav3.3, and then carry out a pulse-chase experiment to determine the rate of Cav3.3 forward trafficking. We found that N-ethylmaleimide (NEM) irreversibly inhibited Cav3.3, but not voltage gated sodium channel (Nav1.2), using patch clamp technology. Next, we used the Syncropatch and Flipr assay to determine and compare the rate of ion channel forward trafficking in Cav3.3 WT cells and Cav3.3 R1346H mutants using pulse-chase approach. In the future, we hope to develop a forward trafficking assay to screen for compounds that may potentially correct the trafficking defective mutations such as Cav3.3 R1346H.
PROJECT: Development of a novel cellular trafficking assay for ion channels using electrophysiology
Coming to the Broad, I thought I had a clear idea of what my scientific interests were. But once here, I was exposed to cutting-edge research in a multitude of fields. With so many new possibilities to pursue research across disciplines opened to me, I began to think more deeply about my long-term scientific interests. The Broad gave me the tools to define and identify where my true passions lie. I was challenged to think critically not only about science but also about myself, allowing me to grow as a scientist and an individual.