EXTRACELLULAR ASSOCIATION OF THE ALPHA2DELTA-1 SUBUNIT WITH CAV2.1 CHANNELS INFLUENCES PRESYNAPTIC CALCIUM CHANNEL ORGANIZATION AND FUNCTION

At central synapses, Cav2.1 channels mediate fast glutamatergic neurotransmitter release. Here, the precise yet dynamic organization of the channel is crucial to ensure functional transmission while remaining adaptable to activity changes. The association of the pore-forming with its extracellular alpha2delta-1 subunit promotes forward trafficking of the channel to the cell surface and modulates its biophysical properties in addition to the subunits impact on synaptogenesis. Although the importance of alpha2delta subunits in regulating neurotransmission is evident, it remains elusive how channel associated alpha2delta-1 subunits regulate the position, density and function of cell surface Cav2.1 channels. We used endogenous CRISPR/Cas9-mediated tagging of Cav2.1 and alpha2delta-1 in mouse hippocampal cultures and their Drosophila melanogaster homologues cacophony and straightjacket to decipher the organization of presynaptic calcium channel complexes. Single particle tracking reveals distinct dynamics with confined and mobile calcium channels in synapses and suggests rather transient interactions with the alpha2delta-1 subunit. Acute pharmacological disruption of these interactions using gabapentinoids induces rapid dissociation of the subunits and causes synaptic Cav2.1 reorganization with reduced channel density. These effects are accompanied by altered neurotransmission, affecting glutamate release, increasing synaptic failure rate and reducing overall network activity. We propose that beside the intracellular interactions of Cav2.1 with scaffold proteins, the transient extracellular interaction with the alpha2delta-1 subunit determines the organization of susceptible calcium channels. Our results suggest that the dynamic association of calcium channel subunits can modulate the dwell-time, abundance and organization of the channel within the synapse and thus may represent an activity-dependent mechanism for fine-tuning neurotransmission.