Plasma membrane association of a2δ subunit of voltage-gated calcium channels

Voltage-gated calcium (Cav) channels are essential components of excitable cells. The function of neurons is highly dependent on the plasma membrane availability of Cav channels, which allow entry of Ca2+. Cav channels control a variety of processes, including synaptic transmission and muscle contraction. Alpha-2-delta (α2δ), an auxiliary subunit of Cav channels increases the current amplitude of by about 4-fold through supporting membrane trafficking of the ionconducting Cav alpha-1 subunit. Furthermore, α2δ modulates the biophysical channel properties. The α2δ-1 isoform has an important pharmacological role of binding antiepileptic gabapentinoid drugs. Additionally, α2δ regulation is enhanced in neuropathic pain models. Until recently, α2δ was characterized as a Type I transmembrane protein. We have identified that α2δ is instead anchored to the extracellular leaflet of the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor. This type of membrane attachment plays an important role in sorting proteins to specialized domains of the lipid bilayer, termed lipid rafts, allowing lateral movement, interactions and signal transduction. The first part of this thesis examines the regulatory properties of neuron-specific α2δ-3 in association with the N-type Cav channel. The results obtained strongly support the hypothesis that α2δ-3 is attached to the membrane via a GPI-anchor. This was demonstrated by enzymatic PI-PLC cleavage, along with proteoglycan NotumWT hydrolytic activity, both of which resulted in a significant reduction in the Ba2+ current. These findings are further corroborated through the use of recombinant constructs of α2δ-3, mutated at the GPI-attachment site. Together with immunofluorescence results, which demonstrate reduced cellular transport of the two α2δ-3 constructs, it was concluded that less channels reach the plasma membrane in the absence of an intact GPI-signal sequence. Moreover, effects of acute and chronic cholesterol modulation on Cav channels plasma membrane targeting were examined. In both instances, N-type Cav/α2δ-3 function and distribution were impaired, providing additional support for the importance of Cav channel localization to lipid raft regions. The second part of this thesis examines the prediction that the α2δ-1 isoform is GPIanchored. A C-terminal truncated form of α2δ-1 (DGPI α2δ-1) shows altered subcellular and surface localization, as well as functional P/Q-type Cav/α2δ-1 channel reduction, providing independent evidence that the GPI anchor of α2δ-1 facilitates Cav channel functional expression. In the final section of the thesis, the WT and DGPI α2δ-1 were used to assess cellular prion protein (PrPC) modulation on P/Q-type Cav/α2δ-1 trafficking using electrophysiological and immunocytochemical methods. An interaction between the WT α2δ-1 and PrPC was proposed, based on the findings that co-expression of the two proteins resulted in a strong colocalization as well as altered channel function. The implications of these results are highlighted by established findings that cellular PrP is required for susceptibility to prion infections and for prion toxicity, and that mice expressing anchorless PrP show reduced infectivity with the pathogenic scrapie PrP isoform