Using a Functional Tagged Calcium Channel to Investigate Trafficking

3 Abstract N - type calcium channels belong to the family of voltage gated calcium channels , which open in response to membrane depolarisation. These channels are responsible for the calcium influx that triggers neurotransmitter release at presynaptic terminals. N - type channels are particularly important in mediating neurotransmitter release at the presynaptic terminals of peripheral sensory neurons and have been implicated i n neuropathic pain. Understanding the regulation of N - type channels is critical to the development of treatments that target these channels. Until now it has been difficult to directly investigate the regulation of their expression at the cell surface due to the inability to selectively visualise channels at the surface. Although N - type channels are thought to consist of a Ca v 2.2, α 2 δ and β subunits, there is uncertainty over whether the pore forming Ca v 2.2 subunit does indeed interact with α 2 δ at the cell surface. The work in this thesis has yielded two tagged functional Ca v 2.2 subunits that can be selectively visualised at th e plasma membrane , and has led to conclusive evidence that Ca v 2.2 and α 2 δ - 1 are closely associated at the cell surface. Furthermore, these tools have allowed the investigation of the mechanism by which α 2 δ - 1 enhances surface expression of Ca v 2.2 and which domains of α 2 δ - 1 are involved. Interestingly, this work implicates two poorly characterised chemosensory - like domains of α 2 δ - 1 as important for the interaction with Ca v 2.2, and also demonstrates that α 2 δ - 1 exerts its effect through increased forward traffi cking rather than increased surface stability. The application of gabapentin, a drug used to treat neuropathic pain, reduces surface expression of Ca v 2.2 through a mechanism that is wholly dependent on binding to α 2 δ - 1. T he tools generated in this work can be used to examine Ca v 2.2 regulation in neurons, val idating their potential for characterising the regulation of Ca v 2.2 in the nervous system.