Regulation of spatial distribution of neuronal calcium channels and SNARE proteins by G-protein-coupled receptors

Regulation of the timing of voltage-dependent calcium channel activity is traditionally thought to involve multiple heterotrimeric G protein signaling pathways. These complex timing events are controlled by interactions between components of several signaling pathways and a dynamic cytoskeletal structure that can serve as the scaffold for interaction between these components and the functional effector, the calcium channel. In neurons, voltage-dependent calcium channels are clustered in active zones in the plasma membrane where they are in close proximity to components of the exocytic machinery such as SNARE proteins, synaptic vesicles and the cytoskeletal matrix. Numerous studies have shown that, after transmitter release, components of exocytic machinery are endocytosed in clathrin-coated vesicles and recycled. While the regulatory trafficking of ionotropic receptors at the post-synaptic density has been previously described, it is not known whether voltage-gated calcium channels of the presynaptic active zone are targets of regulation by trafficking. Here we present evidence that in embryonic chick dorsal root ganglion neurons, calcium channels undergo spatio-temporal redistribution upon activation of G-protein-coupled receptors that are known to inhibit calcium influx and therefore, exocytosis. Experiments in which Ca_v2.2 (N-type, α_1B ) calcium channels were labeled with tetramethylrhodamine-conjugated ω-conotoxin GVIA (an N-type channel antagonist) have shown that upon exposure to transmitter the channels move away from the surface of the plasma membrane. The time course of the rearrangement parallels that of transmitter-mediated inhibition of Ca_v2.2 current. Immunofluorescence studies show that, upon transmitter application, channels are sequestered into clathrin-coated vesicles. Pretreatment with pertussis toxin and inhibition of PI-3 kinase decrease the clustering of the channels. As calcium channels play a pivotal role in synaptic transmission, a change in the number of channels available in the plasma membrane could have important physiological consequences.