Pharmacological disruption of calcium channel trafficking by the α ₂ δ ligand gabapentin

Abstract

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 ␣2␦-1 and ␣2␦-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 ␣2␦ 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 ␣2␦ only when applied chronically, but not acutely, both in heterologous expression systems and in dorsal rootganglion 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 ␣2␦ subunits, being prevented by mutations in either ␣2␦-1 or ␣2␦-2 that abolish GBP binding, and is not observed for ␣2␦-3, which does not bind GBP. Furthermore, the trafficking of ␣2␦-2 and CaV2 channels is disrupted both by GBP and by the mutation in ␣2␦-2, which prevents GBP binding, and we find that GBP reduces cell-surface expression of ␣2␦-2 and CaV2.1 subunits. Our evidence indicates that GBP may act chronically by displacing an endogenous ligand that is normally a positive modulator of ␣2␦ subunit function, thereby impairing the trafficking function of the ␣2␦ subunits to which it binds. V oltage-gated Ca 2ϩ channels (VGCCs) are heteromeric complexes. The Ca V 1 and Ca V 2 subfamilies are made up of a pore-forming ␣1 subunit, associated with a membraneanchored, predominantly extracellular, ␣ 2 ␦ subunit (for review see ref. 1) and an intracellular ␤ subunit (for review see ref.2). Mammalian genes encoding four ␣ 2 ␦ subunits have been identified (for reviews see refs. 2 and 3). The topology of the ␣ 2 ␦ protein was first determined for ␣ 2 ␦-1 and is thought to generalize to all ␣ 2 ␦ subunits (for reviews see refs. 1 and 4). They are type I transmembrane proteins, the exofacial ␣ 2 subunit being disulfide-bonded to a transmembrane ␦ subunit, formed by posttranslational cleavage of the ␣ 2 ␦ preprotein (5).The mechanism of action of the antiepileptic and antinociceptive drugs of the gabapentinoid family has remained poorly understood. Gabapentin (GBP) itself was originally developed as an analog of ␥-amino-butyric acid (GABA), but is now believed to have no effect on GABA receptors or transporters (for review see ref. 6). The first key to understanding the mechanism of action of GBP came from purification of the GBP-binding protein from porcine brain (7), which was identified as the ␣ 2 ␦-1 auxiliary subunit of VGCCs. It is now known that GBP binds to an exofacial epitope present in both the ␣ 2 ␦-1 and ␣ 2 ␦-2 subunits (for reviews see refs. 1 and 8). However, although it was originally reported that GBP application results in a...

Gabapentinoid Insensitivity after Repeated Administration is Associated with Down-Regulation of the α2δ-1 Subunit in Rats with Central Post-Stroke Pain Hypersensitivity

“…Knock-down of a 2 d-1 reduces pain hypersensitivity in an animal model of nerve injury [30]. It has also been confirmed that the antinociceptive and anti-allodynic effects of GBP and pregabalin are due to disruption of trafficking of the VGCC a 2 d-1 subunit to the presynaptic terminal membrane induced by nerve injury [25][26][27]32]. Thus the pharmacological action of GBP against neuropathic pain could be dependent upon aberrant overexpression of the VGCC a 2 d-1 subunit in the presynaptic membrane.…”

“…channel (VGCC) in the CNS [21][22][23]. They inhibit presynaptic neurotransmitter release from hyperexcitable or abnormal neurons by blocking trafficking of the a 2 d-1 subunit to the presynaptic membrane [24][25][26][27]. Experimentally, expression of a 2 d-1, but not a 2 d-2 (another binding site of GBP), is significantly increased in both the dorsal root ganglia (DRG) and the dorsal horn of the spinal cord in animal models of peripheral neuropathic pain.…”

Acute augmentation of epoxygenated fatty acid levels rapidly reduces pain-related behavior in a rat model of type I diabetes

“…These effects were time-as well as dosedependent, indicating a specific interaction between the inhibition of sEH and the observed decrease in pain-related behavior. Strikingly, TUPS reduced allodynia at least 10-fold more efficaciously than gabapentin, a calcium channel trafficking disruptor drug prescribed to patients suffering diabetic neuropathy (38). Moreover, the efficacy of TUPS was longer in duration than gabapentin, which was administered daily to maintain a pain-free state (39).…”

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

“…Alterations in the cell surface density of Ca V ␣2␦1 caused by mutating glycosylation sites (our study), arrhythmogenic mutations (22), mutations within the "von Willebrand factor" structural domain (76,77), or following pharmacological modulation (e.g. gabapentin) (76,78,79) were shown to decrease channel function by altering the surface levels of Ca V ␣2␦1 (76,77). With the exception of the so-called R-domain (80), these manipulations altered the function of Ca V ␣2␦1 (and by extension the function of voltage-gated currents) through a decrease in the membrane expression of Ca V ␣2␦1.…”