The trafficking of the α2δ-2 subunit of voltage-gated calcium channels.
Doctoral thesis, UCL (University College London).
Voltage-gated calcium channels (VGCC) are essential actors for many physiological functions of excitable cells. The properties of VGCC are modulated by association with auxiliary subunits: β, α2δ and in some cases γ. The α2δ subunits increase the functional expression of calcium channels, via a trafficking mechanism. The α2δ-1 and α2δ-2 subunits of VGCC are the binding sites of gabapentin, an anti-epileptic and anti-hyperalgesic drug. The goals of this study were: 1) to characterise the trafficking properties of the α2δ- 2 subunit; 2) to elucidate the controversial mechanism of action of gabapentin upon binding to α2δ-1 and α2δ-2 subunits. These questions were addressed by the biochemical and imaging study of calcium channel subunits in heterologous expression systems. A Bungarotoxin Binding Site epitope was introduced in the extracellular region of α2δ-2, and the resulting tagged subunit was used in combination with fluorescent conjugates of αBungarotoxin, in order to study the internalization and insertion into the plasma membrane of the α2δ-2 subunit. Consistent with the previous identification of α2δ-2 as a lipid raft protein, the alteration of the cholesterol content of the plasma membrane was found to modify its cell-surface expression and trafficking. Cholesterol depletion and loading were found to respectively decrease, and increase the endocytosis of α2δ-2. The chronic, but not the acute application of gabapentin caused a reduction in the cell surface expression of α2δ-2 and Cav2.1 subunits. This effect was shown to be due to a reduction in the forward trafficking of α2δ-2, by preventing its recycling from Rab11-associated endosomes to the plasma membrane. These studies have led to the identification of some of the elements regulating the trafficking of α2δ-2, and their alteration by gabapentin. These mechanisms might be crucial for the modulation of the cell surface expression of calcium channels, in physiological and pathological conditions.
|Title:||The trafficking of the α2δ-2 subunit of voltage-gated calcium channels|
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