Neurotransmitter release at many central synapses is initiated by an influx of calcium ions through P/Q-type calcium channels, which are densely localized in nerve terminals. Because neurotransmitter release is proportional to the fourth power of calcium concentration, regulation of its entry can profoundly influence neurotransmission. N- and P/Q-type calcium channels are inhibited by G proteins, and recent evidence indicates feedback regulation of P/Q-type channels by calcium. Although calcium-dependent inactivation of L-type channels is well documented, little is known about how calcium modulates P/Q-type channels. Here we report a calcium-dependent interaction between calmodulin and a novel site in the carboxy-terminal domain of the alpha1A subunit of P/Q-type channels. In the presence of low concentrations of intracellular calcium chelators, calcium influx through P/Q-type channels enhances channel inactivation, increases recovery from inactivation and produces a long-lasting facilitation of the calcium current. These effects are prevented by overexpression of a calmodulin-binding inhibitor peptide and by deletion of the calmodulin-binding domain. Our results reveal an unexpected association of Ca2+/calmodulin with P/Q-type calcium channels that may contribute to calcium-dependent synaptic plasticity.

Summary At the presynaptic active zone, Ca2+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca2+-channel clustering and synaptic vesicle docking are organized. Here we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses - mostly lacking the ribbon - showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca2+-channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca2+-channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis were reduced notwithstanding normal coupling of the remaining Ca2+-channels to exocytosis. In-vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in-vivo data independently supported morphological and functional in-vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca2+-channels and vesicles, and (2) promote vesicle replenishment.

Ca2+-dependent facilitation and inactivation (CDF and CDI) of Cav2.1 channels modulate presynaptic P/Q-type Ca2+ currents and contribute to activity-dependent synaptic plasticity. This dual feedback regulation by Ca2+ involves calmodulin (CaM) binding to the α1 subunit (α12.1). The molecular determinants for Ca2+-dependent modulation of Cav2.1 channels reside in CaM and in two CaM-binding sites in the C-terminal domain of α12.1, the CaM-binding domain (CBD) and the IQ-like domain. In transfected tsA-201 cells, CDF and CDI were both reduced by deletion of CBD. In contrast, alanine substitution of the first two residues of the IQ-like domain (IM-AA) completely prevented CDF but had little effect on CDI, and glutamate substitutions (IM-EE) greatly accelerated voltage-dependent inactivation but did not prevent CDI. Mutational analyses of the Ca2+ binding sites of CaM showed that both the N- and C-terminal lobes of CaM were required for full development of facilitation, but only the N-terminal lobe was essential for CDI. In biochemical assays, CaM12 and CaM34 were unable to bind CBD, whereas CaM34 but not CaM12 retained Ca2+-dependent binding to the IQ-like domain. These findings support a model in which Ca2+ binding to the C-terminal EF-hands of preassociated CaM initiates CDF via interaction with the IQ-like domain. Further Ca2+ binding to the N-terminal EF-hands promotes secondary CaM interactions with CBD, which enhance facilitation and cause a conformational change that initiates CDI. This multifaceted mechanism allows positive regulation of Cav2.1 in response to local Ca2+ increases (CDF) and negative regulation during more global Ca2+ increases (CDI)

SUMMARY In the retina, rod and cone photoreceptors form distinct connections with different classes of downstream bipolar cells. However, the molecular mechanisms responsible for their selective connectivity are unknown. Here we identify a cell-adhesion protein, ELFN1, to be essential for the formation of synapses between rods and rod ON-bipolar cells in the primary rod pathway. ELFN1 is expressed selectively in rods where it is targeted to the axonal terminals by the synaptic release machinery. At the synapse, ELFN1 binds in trans to mGluR6, the postsynaptic receptor on rod ON-bipolar cells. Elimination of ELFN1 in mice prevents the formation of synaptic contacts involving rods, but not cones, allowing a dissection of the contributions of primary and secondary rod pathways to retinal circuit function and vision. We conclude that ELFN1 is necessary for the selective wiring of rods into the primary rod pathway and is required for high sensitivity of vision.