CaV2.1 α1 subunit motifs that control presynaptic CaV2.1 subtype abundance are distinct from CaV2.1 preference

Presynaptic voltage-gated Ca2+ channels (CaV) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system, most presynaptic terminals are CaV2.1 dominant with a developmental reduction in CaV2.2 and CaV2.3 levels, and CaV2 subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic CaV2 subtype levels are largely unsolved. Since the CaV2 1 subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic CaV2 subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric CaV2.1 1 subunits containing swapped motifs with the CaV2.2 and CaV2.3 1 subunit on a CaV2.1/CaV2.2 null background at the calyx of Held presynaptic terminal. We found that expression of CaV2.1 1 subunit chimeras containing the CaV2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca2+ currents compared to the CaV2.1 1 subunit. However, the Ca2+ current sensitivity to the CaV2.1 blocker Agatoxin-IVA, was the same between the chimeras and the CaV2.1 1 subunit. Additionally, we found no reduction in presynaptic Ca2+ currents with CaV2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the CaV2.1 loop II-III and CT do not individually regulate CaV2.1 preference, but these motifs control CaV2.1 levels and the CaV2.3 CT contains motifs that negatively regulate presynaptic CaV2.3 levels. We propose that the motifs controlling presynaptic CaV2.1 preference are distinct from those regulating CaV2.1 levels and may act synergistically to impact pathways regulating CaV2.1 preference and abundance. Key points summaryO_LIPresynaptic CaV2 subtype abundance regulates neuronal circuit properties, however the mechanisms regulating presynaptic CaV2 subtype abundance and preference remains enigmatic. C_LIO_LIThe CaV 1 subunit determines subtype and contains multiple motifs implicated in regulating presynaptic subtype abundance and preference. C_LIO_LIThe CaV2.1 1 subunit domain II-III loop and cytoplasmic C-terminus are positive regulators of presynaptic CaV2.1 abundance but do not regulate preference. C_LIO_LIThe CaV2.3 1 subunit cytoplasmic C-terminus negatively regulates presynaptic CaV2 subtype abundance but not preference while the CaV2.2 1 subunit cytoplasmic C-terminus is not a key regulator of presynaptic CaV2 subtype abundance or preference. C_LIO_LIThe CaV2 1 subunit motifs determining the presynaptic CaV2 preference are distinct from abundance. C_LI