Ca²⁺channel and active zone protein abundance intersects with input-specific synapse organization to shape functional synaptic diversity

Synaptic heterogeneity is a hallmark of nervous systems that enables complex and adaptable communication in neural circuits. To understand circuit function, it is thus critical to determine the factors that contribute to the functional diversity of synapses. We investigated the contributions of voltage-gated calcium channel (VGCC) abundance, spatial organization, and subunit composition to synapse diversity among and between synapses formed by two closely relatedDrosophilaglutamatergic motor neurons with distinct neurotransmitter release probabilities (P_r). Surprisingly, VGCC levels are highly predictive of heterogeneous P_ramong individual synapses of either low- or high-P_rinputs, but not between inputs. We find that the same number of VGCCs are more densely organized at high-P_rsynapses, consistent with tighter VGCC-synaptic vesicle coupling. We generated endogenously tagged lines to investigate VGCC subunitsin vivoand found that the α2δ-3 subunit Straightjacket along with the CAST/ELKS active zone (AZ) protein Bruchpilot, both key regulators of VGCCs, are less abundant at high-P_rinputs, yet positively correlate with P_ramong synapses formed by either input. Consistently, both Straightjacket and Bruchpilot levels are dynamically increased across AZs of both inputs when neurotransmitter release is potentiated to maintain stable communication following glutamate receptor inhibition. Together, these findings suggest a model in which VGCC and AZ protein abundance intersects with input-specific spatial and molecular organization to shape the functional diversity of synapses.