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Neurotransmitter is released from presynaptic nerve terminals at fast-transmitting synapses by the action potential-gating of voltage dependent calcium channels (CaV), primarily of the CaV2.1 and CaV2.2 types. Entering Ca2+ diffuses to a nearby calcium sensor associated with a docked synaptic vesicle (SV) and initiates its fusion and discharge. Our previous findings that single CaVs can gate SV fusion argued for one or more tethers linking CaVs to docked SVs but the molecular nature of these tethers have not been established. We recently developed a cell-free, in vitro biochemical assay, termed SV pull-down (SV-PD), to test for SV binding proteins and used this to demonstrate that CaV2.2 or the distal third of its C-terminal can capture SVs. In subsequent reports we identified the binding site and characterized an SV binding motif. In this study, we set out to test if a similar SV-binding mechanism exists in the primary presynaptic channel type, CaV2.1. We cloned the chick variant of this channel and to our surprise found that it lacked the terminal third of the C-terminal, ruling out direct correlation with CaV2.2. We used SV-PD to identify an SV binding site in the distal half of the CaV2.1 C-terminal, a region that corresponds to the central third of the CaV2.2 C-terminal. Mutant fusion proteins combined with motif-blocking peptide strategies identified two domains that could account for SV binding; one in an alternatively spliced region (E44) and a second more distal site. Our findings provide a molecular basis for CaV2.1 SV binding that can account for recent evidence of C-terminal-dependent transmitter release modulation and that may contribute to SV tethering within the CaV2.1 single channel Ca2+ domain.

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Coastal wetlands are restored to regenerate lost ecosystem services. Accurate and frequent representations of the distribution and area of coastal wetland communities are critical for evaluating restoration success. Typically, such data are acquired through laborious, intensive and expensive field surveys or traditional remote sensing methods that can be erroneous. Recent advances in remote sensing techniques such as high-resolution sensors (<2 m resolution), object-based image analysis and shallow learning classifiers provide promising alternatives but have rarely been applied in a restoration context. We measured the changes to wetland communities at a 200 ha restoring coastal wetland in eastern Australia, using remotely sensed Worldview-2 imagery, object-based image analysis and random forest classification. Our approach used structural rasters (digital elevation and canopy height models) and a multi-temporal technique to distinguish between spectrally similar land cover. The accuracy of our land cover maps was high, with overall accuracies ranging between 91 and 95%, and this supported early detection of increases in the area of key ecosystems, including mixed she-oak and paperbark (10 ha), mangroves (0.91 ha) and saltmarsh (4.31 ha), over a 5-year monitoring period. Our approach provides coastal managers with an accurate and frequent method for quantifying early responses of coastal wetlands to restoration, which is essential for informing adaptive management in the regeneration of ecosystem services.