Ca_V 2.1 α₁  subunit motifs that control presynaptic Ca_V 2.1 subtype abundance are distinct from Ca_V 2.1 preference

Presynaptic voltage-gated Ca²⁺ channel (Ca_V ) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system (CNS), most presynaptic terminals are Ca_V 2.1 dominant with a developmental reduction in Ca_V 2.2 and Ca_V 2.3 levels, and Ca_V 2 subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic Ca_V 2 subtype levels are largely unsolved. Because the Ca_V 2 α₁  subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic Ca_V 2 subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric Ca_V 2.1 α₁  subunits containing swapped motifs with the Ca_V 2.2 and Ca_V 2.3 α₁  subunit on a Ca_V 2.1/Ca_V 2.2 null background at the calyx of Held presynaptic terminals. We found that expression of Ca_V 2.1 α₁  subunit chimeras containing the Ca_V 2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca²⁺ currents compared to the Ca_V 2.1 α₁  subunit. However, the Ca²⁺ current sensitivity to the Ca_V 2.1 blocker agatoxin-IVA was the same between the chimeras and the Ca_V 2.1 α₁  subunit. Additionally, we found no reduction in presynaptic Ca²⁺ currents with Ca_V 2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the Ca_V 2.1 loop II-III and CT do not individually regulate Ca_V 2.1 preference, although these motifs control Ca_V 2.1 levels and the Ca_V 2.3 CT contains motifs that negatively regulate presynaptic Ca_V 2.3 levels. We propose that the motifs controlling presynaptic Ca_V 2.1 preference are distinct from those regulating Ca_V 2.1 levels and may act synergistically to impact pathways regulating Ca_V 2.1 preference and abundance. KEY POINTS: Presynaptic Ca_V 2 subtype abundance regulates neuronal circuit properties, although the mechanisms regulating presynaptic Ca_V 2 subtype abundance and preference remain enigmatic. The Ca_V α₁  subunit determines subtype and contains multiple motifs implicated in regulating presynaptic subtype abundance and preference. The Ca_V 2.1 α₁  subunit domain II-III loop and cytoplasmic C-terminus are positive regulators of presynaptic Ca_V 2.1 abundance but do not regulate preference. The Ca_V 2.3 α₁  subunit cytoplasmic C-terminus negatively regulates presynaptic Ca_V 2 subtype abundance but not preference, whereas the Ca_V 2.2 α₁  subunit cytoplasmic C-terminus is not a key regulator of presynaptic Ca_V 2 subtype abundance or preference. The Ca_V 2 α₁  subunit motifs determining the presynaptic Ca_V 2 preference are distinct from abundance.