Identification of the alpha2-delta-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin

Abstract

Neuropathic pain is a debilitating condition affecting millions of people around the world and is defined as pain that follows a lesion or dysfunction of the nervous system. This type of pain is difficult to treat, but the novel compounds pregabalin (Lyrica) and gabapentin (Neurontin) have proven clinical efficacy. Unlike traditional analgesics such as nonsteroidal antiinflammatory drugs or narcotics, these agents have no frank antiinflammatory actions and no effect on physiological pain. Although extensive preclinical studies have led to a number of suggestions, until recently their mechanism of action has not been clearly defined. Here, we describe studies on the analgesic effects of pregabalin in a mutant mouse containing a single-point mutation within the gene encoding a specific auxiliary subunit protein (alpha2-delta-1) of voltage-dependent calcium channels. The mice demonstrate normal pain phenotypes and typical responses to other analgesic drugs. We show that the mutation leads to a significant reduction in the binding affinity of pregabalin in the brain and spinal cord and the loss of its analgesic efficacy. These studies show conclusively that the analgesic actions of pregabalin are mediated through the alpha2-delta-1 subunit of voltage-gated calcium channels and establish this subunit as a therapeutic target for pain control.

Fig. 1.

Construct of R217A mutant mouse and its effect on α₂-δ protein levels and pregabalin binding. (A) Strategy for mutating arginine 217 to alanine in mouse α₂-δ-1. (B) Sequence of exon 8 of α₂-δ-1 mutant and WT RNA showing R217A mutation. (C–F) Effect of mutation on α₂-δ protein levels (C and D) and [3H]pregabalin binding (E and F). Analyses were carried out in murine tissue (C and E) and cells expressing α₂-δ-1 WT or α₂-δ-1 R217A (D and F). The membranes from HEK293 cells overproducing human α₂-δ-2 protein were used as control for anti-α₂-δ-1 antibody specificity (D). The same membranes (40–76 μg) were used in 20 nM [³H]pregabalin binding assays. Values are expressed as mean ± SD.

Fig. 2.

Pregabalin binding in mutant and WT mice. Shown is autoradiographic localization of [³H]pregabalin binding to WT brain (A) and lumbar spinal cord (C) and R217A mutant brain (B) and lumbar dorsal horn (D). (E) Quantitative densitometry of [³H]pregabalin binding values were expressed as mean ± SEM.

Fig. 3.

Effect of WT α₂-δ-1 and R217A α₂-δ-1 on Ca_V2.2/β1b currents. Ca_V2.2/β1b was expressed either alone or with WT α₂-δ-1 or R217A α₂-δ-1 in tsA-201 cells (A and B) or Xenopus oocytes (C) and recorded by using 10 mM Ba²⁺as charge carrier. (A) Representative current traces elicited by steps to test potentials (TP) between −30 and +15 mV in 10-mV steps from a holding potential of −90 mV for Ca_V2.2/β1b (Left), Ca_V2.2/β1b/α₂-δ-1 (Center), Ca_V2.2/β1b/R217A α₂-δ-1 (Right). The scale bar refers to all traces. (B) I–V relationships for the three experimental conditions following expression in tsA-201 cells. Open triangles, Ca_V2.2/β1b (n = 11); open squares, Ca_V2.2/β1b/α₂-δ-1 (n = 9); filled circles, Ca_V2.2/β1b/R217A α₂-δ-1 (n = 11). (C) I–V relationships for the three experimental conditions after expression in Xenopus oocytes. Open triangles, Ca_V2.2/β1b (n = 9); open squares, Ca_V2.2/β1b/α₂-δ-1 (n = 15); filled circles, Ca_V2.2/β1b/R217A α₂-δ-1 (n = 14).

Fig. 4.

Formalin test in mutant and WT mice. (A) Effect of formalin induced nocifensive response in knockin and WT mice. (B) Effect of pregabalin and morphine in the formalin test in mutant and WT mice. Results are expressed as mean ± SEM time spent licking and biting the hind paw for late phase (n = 6–8 for each group). ∗, P < 0.05; ∗∗, P < 0.01 (one-way ANOVA followed by Dunnett's t test vs. vehicle at each time point).

Fig. 5.

Development of CCI-induced punctate allodynia (A) and effect of pregabalin (B), gabapentin (C), and amitriptyline (D) on CCI-induced punctate allodynia in the mutant and WT mice. Baseline (BL) paw withdrawal thresholds (PWT) to von Frey hairs were assessed. After drug administration, PWTs were reassessed for up to 4 h. The punctate allodynia data are expressed as median force (g) required to induce a paw withdrawal in six to seven animals per group (vertical bars represent first and third quartiles). ∗, P < 0.05; ∗∗, P < 0.01 (Mann–Whitney U test) from vehicle-treated group at each time point.