For Bimolecular fluorescence complementation, HEK cells were transfected using jetOPTIMUS: 1.2x 10^5 HEK STIM1−/−;STIM2−/− cells were seeded in 6 well plates and transfected with a total amount of 2 μg DNA. For Fura2-based Ca2+ Imaging, jetPRIME was used: - MEF cells were seeded on 25mm coverslips 24 hours before transfection with 2μg of plasmids - SH-SY5Y cells were seeded in 35mm cell culture dishes 24 hours before transfection with 2μg of plasmids. Transfected SH-SY5Y were reseeded 4 hours post transfection on 25mm coverslips.

Store-operated Ca²⁺-entry (SOCE) regulates basal and receptor-triggered Ca²⁺ signaling with STIM proteins sensing the endoplasmic reticulum (ER) Ca²⁺ content and triggering Ca²⁺ entry by gating Orai channels. Although crucial for immune cells, STIM1's role in neuronal Ca²⁺ homeostasis is controversial. Here, we characterize a splice variant, STIM1B, which shows exclusive neuronal expression and protein content surpassing conventional STIM1 in cerebellum and of significant abundance in other brain regions. STIM1B expression results in a truncated protein with slower kinetics of ER-plasma membrane (PM) cluster formation and I_CRAC, as well as reduced inactivation. In primary wild-type neurons, STIM1B is targeted by its spliced-in domain B to presynaptic sites where it converts classic synaptic depression into Ca²⁺- and Orai-dependent short-term synaptic enhancement (STE) at high-frequency stimulation (HFS). In conjunction with altered STIM1 splicing in human Alzheimer disease, our findings highlight STIM1 splicing as an important regulator of neuronal calcium homeostasis and of synaptic plasticity.