Abstract
N-type voltage-gated calcium channels (CaV2.2) are predominantly expressed at presynaptic terminals, and their function is regulated by auxiliary α2δ and β subunits. All four mammalian α2δ subunits enhance calcium currents through CaV1 and CaV2 channels, and this increase is attributed, in part, to increased CaV expression at the plasma membrane. In the present study we provide evidence that α2δ-1, like α2δ-2, is recycled to the plasma membrane through a Rab11a-dependent endosomal recycling pathway. Using a dominant-negative Rab11a mutant, Rab11a(S25N), we show that α2δ-1 increases plasma membrane CaV2.2 expression by increasing the rate and extent of net forward CaV2.2 trafficking in a Rab11a-dependent manner. Dominant-negative Rab11a also reduces the ability of α2δ-1 to increase CaV2.2 expression on the cell-surface of hippocampal neurites. In contrast, α2δ-3 does not enhance rapid forward CaV2.2 trafficking, regardless of whether Rab11a(S25N) is present. In addition, whole-cell CaV2.2 currents are reduced by co-expression of Rab11a(S25N) in the presence of α2δ-1, but not α2δ-3. Taken together these data suggest that α2δ subtypes participate in distinct trafficking pathways which in turn influence the localisation and function of CaV2.2.
Highlights
N-type voltage-gated calcium channels (CaV2.2) are predominantly expressed at presynaptic terminals, and their function is regulated by auxiliary α2δ and β subunits
In our previous study[16] we found no effect of co-expressing WT Rab11a on any aspects of α2δ trafficking or calcium channel function, and it was not included as an additional control in the present study
This result was supported by measurement of the intracellular α2δ levels which were strongly reduced by Rab11a(S25N) in the case of α2δ-1 but not α2δ-3 (Fig. 1A,C)
Summary
N-type voltage-gated calcium channels (CaV2.2) are predominantly expressed at presynaptic terminals, and their function is regulated by auxiliary α2δ and β subunits. The development of functional exofacially-tagged C aV2 constructs[10], has allowed us to define populations of plasma membrane-inserted CaV2 channels using imaging approaches This method was previously used to demonstrate that heterologous expression of α2δ-1 increases cell-surface C aV2.2 expression, consistent with earlier models of α2δ-1 function[10]. These studies provide convincing evidence that the α2δ-mediated increase in CaV currents is dependent on an increase in cell-surface C aV2.2 expression, and requires a molecular activation switch, triggered by the proteolytic cleavage of α2δ into α2 and δ It is unclear whether α2δ isoforms differ significantly in their ability to enhance CaV currents, and it remains to be seen if α2δ subtypes have uniform trafficking mechanisms with regard to their effect on CaV localisation. Since α2δ-3 is widely distributed in the nervous system, if there were functional differences, for example between α2δ-1 and α2δ-3, this might influence the trafficking and localisation of associated C aV channels
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