Voltage-gated calcium channels (VGCCs) control and regulate numerous physiological processes, ranging from muscle contraction, heartbeat, neural communication and hormone secretion to cell differentiation, motility, growth and death. As such, their activity is tightly regulated by diverse molecules and pathways, including protein kinases and phosphatases, G proteins, calcium and calmodulin, SNAREs, and phospholipids. We have uncovered a new paradigm for VGCC regulation: The pore-forming a1 subunit of neuronal surface L-type VGCCs is proteolytically cleaved in the cytoplasmic loops (mainly the II-III loop but also the I-II loop) connecting the four homologous repeats, this cleavage is activity-dependent, and the cleaved fragments (called hemi-channels) physically separate. This novel form of channel regulation may significantly impact L-type calcium channel activity, calcium signaling, and gene transcription in neurons. We propose to study the signaling molecules and pathways, the dynamics, and the functional consequences of mid-channel proteolysis, and the fate and function of the resulting hemi-channels, using a combination of biochemistry, proteomics, fixed cell and live cell imaging, and electrophysiology. As intracellular calcium signaling is severely altered in aging neurons and age-related neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, we will investigate whether mid-channel proteolysis and hemi-channel separation are perturbed in these physiological and pathological processes. We will also expand our studies to N- and P/Q-type calcium channels, which mediate neurotransmitter release. These studies will provide mechanistic insights into a hitherto ill-studied form of VGCC regulation and may lead to the development of new therapeutic strategies for calcium homeostasis-related diseases. PUBLIC HEALTH RELEVANCE: Voltage-gated calcium channels (VGCCs) are essential for human physiology. Their mutations and malfunction cause a variety of diseases, including epilepsy, ataxia, cardiovascular diseases, and autism. By studying a novel form of activity-dependent fragmentation of VGCCs on the cell membrane, this work may lead to a better understanding of VGCC function and regulation in physiological and pathological conditions.