INVESTIGATING THE DYNAMIC REGULATION OF PRESYNAPTIC CA<SUB>V</SUB>2.1 CHANNELS IN SHORT-TERM PLASTICITY

Presynaptic CaV2.1 (P/Q-type) calcium channels mediate the fast and synchronous release of neurotransmitters at central synapses. Their function relies on auxiliary subunits that regulate channel properties, trafficking, and precise localization within active zones. The nanoscale organization of these channels is critical for tight coupling to synaptic vesicle fusion and for determining short-term synaptic plasticity. However, the molecular mechanisms controlling CaV2.1 channel abundance and spatial arrangement at mature synapses remain poorly understood.
Emerging evidence indicates that presynaptic calcium channels are not static entities, but dynamically regulated elements whose distribution may adapt to neuronal activity. A particularly intriguing hypothesis proposes that intracellular pools of CaV2.1 complexes reside in vesicular compartments and can be recruited to the presynaptic membrane in an activity-dependent manner, analogous to the trafficking of AMPA receptors in postsynaptic membranes. Such a mechanism could transiently enhance local calcium influx and fine-tune synaptic efficacy during periods of intense neuronal firing.
To investigate this, I engineered several tagged versions of human CaV2.1 channels fused to pHluorin, ALFA, or HALO tags, and verified their structural integrity through computational analysis. Expression in primary cortical neurons revealed that efficient channel trafficking requires co-expression with auxiliary subunits. Using pHluorin-tagged constructs, I am performing live-imaging experiments to visualize channel recruitment to the plasma membrane, complemented by STED super-resolution microscopy to resolve their nanoscale distribution. Additionally, biochemical immunoenrichment of vesicular and membrane fractions will assess whether neuronal activity modulates vesicle-associated CaV2.1 pools, unveiling a novel layer of presynaptic plasticity.