Preferential Localization of STIM1 to Dendritic Subsurface ER Structures in Mouse Purkinje Cells

The endoplasmic reticulum (ER) is the largest intracellular Ca²⁺ store, serving as the source and sink of intracellular Ca²⁺ The ER Ca²⁺ store is continuous yet organized into distinct subcompartments with spatial and functional heterogeneity. In cerebellar Purkinje cells (PCs), glutamatergic inputs trigger Ca²⁺ release from specific ER domains via inositol 1,4,5-trisphosphate receptors (IP₃Rs) or ryanodine receptors (RyRs). Upon ER store depletion, refilling occurs through store-operated Ca²⁺ entry mediated by stromal interaction molecule-1 (STIM1). Although the significance of STIM1-mediated Ca²⁺ regulation within PCs is established, STIM1 localization in ER subcompartments in PCs for Ca²⁺ release and refilling remains elusive. Using validated antibodies, we demonstrated that STIM1 was predominantly localized as intense puncta along dendritic shafts in male and female mice, colocalizing with IP₃R1 but not with RyR1. Immunoelectron microscopy revealed that STIM1 was accumulated in the subsurface ER in the dendritic shaft but excluded from those in the dendritic spine, the primary site of metabotropic glutamate receptor 1 (mGluR1)-IP₃R-mediated Ca²⁺ signaling. Ca²⁺ imaging from control and STIM1-knockdown (STIM1-KD) PCs demonstrated that mGluR1-mediated Ca²⁺ release is more critically dependent on STIM1 than RyR-mediated Ca²⁺ release. These findings reveal a spatially organized ER network in PCs, where specialized ER subcompartments differentially regulate Ca²⁺ release and refilling. These findings suggest that STIM1 preferentially regulates Ca²⁺ dynamics associated with mGluR1-IP₃R signaling, supporting specialized ER subcompartments for Ca²⁺ release and refilling. These findings highlight the intricate molecular-anatomical organization of dendritic ER Ca²⁺ signaling in PCs, which is crucial for synaptic plasticity and motor learning.<b>Significance statement</b> Intracellular calcium (Ca²⁺) signaling is essential for neuronal function, yet the organization of endoplasmic reticulum (ER) subcompartments that coordinate Ca²⁺ release and refilling remains unclear. This study demonstrates that stromal interaction molecule-1 (STIM1), a key regulator of store-operated Ca²⁺ entry, is predominantly localized to the subsurface ER in Purkinje cell dendrites, which had not been previously identified. STIM1 colocalizes with inositol 1,4,5-trisphosphate receptor type 1 (IP₃R1) and sarco/endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2) but is segregated from ryanodine receptor 1 (RyR1), highlighting specialized ER subdomains for Ca²⁺ release and refilling. These findings provide new insights into the molecular-anatomical organization of Ca²⁺ signaling in Purkinje cells, which plays key roles in synaptic plasticity, motor learning, and the pathophysiology of neurodegenerative diseases.