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.