Pharmacological disruption of calcium channel trafficking by the alpha2delta ligand gabapentin.

The mechanism of action of the antiepileptic and antinociceptive drugs of the gabapentinoid family has remained poorly understood. Gabapentin (GBP) binds to an exofacial epitope of the alpha(2)delta-1 and alpha(2)delta-2 auxiliary subunits of voltage-gated calcium channels, but acute inhibition of calcium currents by GBP is either very minor or absent. We formulated the hypothesis that GBP impairs the ability of alpha(2)delta subunits to enhance voltage-gated Ca(2+)channel plasma membrane density by means of an effect on trafficking. Our results conclusively demonstrate that GBP inhibits calcium currents, mimicking a lack of alpha(2)delta only when applied chronically, but not acutely, both in heterologous expression systems and in dorsal root-ganglion neurons. GBP acts primarily at an intracellular location, requiring uptake, because the effect of chronically applied GBP is blocked by an inhibitor of the system-L neutral amino acid transporters and enhanced by coexpression of a transporter. However, it is mediated by alpha(2)delta subunits, being prevented by mutations in either alpha(2)delta-1 or alpha(2)delta-2 that abolish GBP binding, and is not observed for alpha(2)delta-3, which does not bind GBP. Furthermore, the trafficking of alpha(2)delta-2 and Ca(V)2 channels is disrupted both by GBP and by the mutation in alpha(2)delta-2, which prevents GBP binding, and we find that GBP reduces cell-surface expression of alpha(2)delta-2 and Ca(V)2.1 subunits. Our evidence indicates that GBP may act chronically by displacing an endogenous ligand that is normally a positive modulator of alpha(2)delta subunit function, thereby impairing the trafficking function of the alpha(2)delta subunits to which it binds.

Functional biology of the alpha(2)delta subunits of voltage-gated calcium channels.

In this review, we examine what is known about the mechanism of action of the auxiliary alpha2delta subunits of voltage-gated Ca(2+) (Ca(v)) channels. First, to provide some background on the alpha2delta proteins, we discuss the genes encoding these channels, in addition to the topology and predicted structure of the alpha2delta subunits. We then describe the effects of alpha2delta subunits on the biophysical properties of Ca(v) channels and their physiological function. All alpha2delta subunits increase the density at the plasma membrane of Ca(2+) channels activated by high voltage, and we discuss what is known about the mechanism underlying this trafficking. Finally, we consider the link between alpha2delta subunits and disease, both in terms of spontaneous and engineered mouse mutants that show cerebellar ataxia and spike-wave epilepsy, and in terms of neuropathic pain and the mechanism of action of the gabapentinoid drugs - small-molecule ligands of the alpha2delta-1 and alpha2delta-2 subunits.

The calcium channel alpha2delta-2 subunit partitions with CaV2.1 into lipid rafts in cerebellum: implications for localization and function.

The accessory alpha2delta subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel alpha1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar alpha2delta-2 subunits with alpha2delta-2 stably expressed in tsA-201 cells. We identified that alpha2delta-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel alpha1 subunit CaV2.1, although CaV2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, alpha2delta-2 is not completely proteolytically processed into alpha2-2 and delta-2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which alpha2delta-2 also colocalizes with CaV2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced CaV2.1/alpha2delta-2/beta4 currents. Furthermore, alpha2delta-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of alpha2delta-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable alpha2delta-2 cell line. In contrast, alpha2delta-2 containing a point mutation (R282A) has a much lower affinity for gabapentin, and this is not enhanced in the lipid raft fraction. This R282A mutant alpha2delta-2 shows reduced functionality in terms of enhancement of CaV2.1/beta4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of alpha2delta-2. Together, these results indicate that both alpha2delta-2 and CaV2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality.