Ca V 2.1 α 1  subunit motifs that control presynaptic Ca V 2.1 subtype abundance are distinct from Ca V 2.1 preference.

Presynaptic voltage-gated Ca 2+ 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 α 1  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 α 1  subunits containing swapped motifs with the Ca V 2.2 and Ca V 2.3 α 1  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 α 1  subunit chimeras containing the Ca V 2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca 2+ currents compared to the Ca V 2.1 α 1  subunit. However, the Ca 2+ current sensitivity to the Ca V 2.1 blocker agatoxin-IVA was the same between the chimeras and the Ca V 2.1 α 1  subunit. Additionally, we found no reduction in presynaptic Ca 2+ 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 α 1  subunit determines subtype and contains multiple motifs implicated in regulating presynaptic subtype abundance and preference. The Ca V 2.1 α 1  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 α 1  subunit cytoplasmic C-terminus negatively regulates presynaptic Ca V 2 subtype abundance but not preference, whereas the Ca V 2.2 α 1  subunit cytoplasmic C-terminus is not a key regulator of presynaptic Ca V 2 subtype abundance or preference. The Ca V 2 α 1  subunit motifs determining the presynaptic Ca V 2 preference are distinct from abundance.