Annette C. Dolphin

Identification of the α2-δ-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin

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 (α2-δ-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 α2-δ-1 subunit of voltage-gated calcium channels and establish this subunit as a therapeutic target for pain control.

Pharmacological disruption of calcium channel trafficking by the α2δ 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 α2δ-1 and α2δ-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 α2δ subunits to enhance voltage-gated Ca2+channel plasma membrane density by means of an effect on trafficking. Our results conclusively demonstrate that GBP inhibits calcium currents, mimicking a lack of α2δ 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 α2δ subunits, being prevented by mutations in either α2δ-1 or α2δ-2 that abolish GBP binding, and is not observed for α2δ-3, which does not bind GBP. Furthermore, the trafficking of α2δ-2 and CaV2 channels is disrupted both by GBP and by the mutation in α2δ-2, which prevents GBP binding, and we find that GBP reduces cell-surface expression of α2δ-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 α2δ subunit function, thereby impairing the trafficking function of the α2δ subunits to which it binds.

Mechanisms of modulation of voltage-dependent calcium channels by G proteins.

Voltage-dependent calcium channels (VDCCs) are key components in the complex functioning of excitable cells. Because of this, their regulation is of paramount importance to the control of cellular activity. VDCCs consist of a pore-forming α1 subunit, which has four domains each containing six putative transmembrane segments. From purification studies, these are associated with an intracellular β subunit. They also co-purify with an α2 subunit, which is entirely extracellular, linked into the membrane by S-S bonding to a transmembrane δ subunit (Witcher et al. 1993; Liu, De Waard, Scott, Gurnett, Lennon & Campbell, 1996) (Fig. 1). A number of different α1 subunits have been cloned; α1C, D and S all form 1,4-dihydropyridine (DHP)-sensitive L-type calcium channels, whereas α1A, B and E form P/Q-, N- and possibly R- or T-type channels, respectively. There are several means by which these channels may be modulated, but for neuronal channels, particularly N and P/Q, a major mechanism involves inhibitory modulation via the activation of heterotrimeric G proteins by seven transmembrane (7TM) receptors (for review see Dolphin, 1995). The key features of this inhibition are that it is always partial and is typified by a slowing of the current activation kinetics, which is thought to be due to a time-dependent recovery from voltage-dependent inhibition (Bean, 1989). The voltage dependence is manifested by a shift to more depolarized potentials of the current activation-voltage relationship, and the loss of inhibition at large depolarizations (Bean, 1989). There may also be additional mechanisms that are not voltage dependent, manifested by an incomplete ability of a depolarizing prepulse to reverse the inhibition (Diverse-Pierluissi & Dunlap, 1993), and the continuing presence of inhibition at large depolarizations measured from the tail current amplitude. Figure 1 The VDCC oligomeric complex Calcium channels are present in many tissues, where they fulfil a number of different specialized roles. In neurons the distribution of the channels is non-uniform, with α1B and α1A being particularly concentrated at synaptic terminals (Westenbroek, Hell, Warner, Dubel, Snutch & Catterall, 1992; Westenbroek et al. 1995). G protein-mediated modulation of these channels has been shown to occur at presynaptic terminals (Toth, Bindokas, Bleakman, Colmers & Miller, 1993). Evidence suggests that this mechanism may be responsible for at least some of the presynaptic inhibition of synaptic transmission mediated by a wide variety of 7TM receptors in many areas of the nervous system (Man-Son-Hing, Zoran, Lukowiak & Haydon, 1989; Hille, 1992; Toth et al. 1993; Dolphin, 1995). Activation of such receptors will reduce calcium entry into presynaptic terminals via VDCCs, but the effect should be frequency dependent. Inhibition will be reduced during a high frequency train as a result of the voltage dependence of the inhibitory modulation, providing a gain-setting mechanism. Relief of inhibition of calcium currents, evoked by action potential-like voltage waveforms, has been reported during high frequency trains (Williams, Serafin, Muhlethaler & Bernheim, 1997), and might contribute to the modulation of presynaptic inhibition depending on input frequency. The search for the molecular mechanism of this modulation is hotting up. Nevertheless, it should not be forgotten that indirect mechanisms such as presynaptic hyperpolarization, or direct mechanisms involving inhibition of exocytosis, are also likely to play a role in the modulation of presynaptic release of transmitter (Man-Son-Hing et al. 1989).

The Increased Trafficking of the Calcium Channel Subunit α2δ-1 to Presynaptic Terminals in Neuropathic Pain Is Inhibited by the α2δ Ligand Pregabalin

Neuropathic pain results from damage to the peripheral sensory nervous system, which may have a number of causes. The calcium channel subunit α2δ-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of neuropathic pain, and this is causally related to the onset of allodynia, in which a non-noxious stimulus becomes painful. The therapeutic drugs gabapentin and pregabalin (PGB), which are both α2δ ligands, have antiallodynic effects, but their mechanism of action has remained elusive. To investigate this, we used an in vivo rat model of neuropathy, unilateral lumbar spinal nerve ligation (SNL), to characterize the distribution of α2δ-1 in DRG neurons, both at the light- and electron-microscopic level. We found that, on the side of the ligation, α2δ-1 was increased in the endoplasmic reticulum of DRG somata, in intracellular vesicular structures within their axons, and in the plasma membrane of their presynaptic terminals in superficial layers of the dorsal horn. Chronic PGB treatment of SNL animals, at a dose that alleviated allodynia, markedly reduced the elevation of α2δ-1 in the spinal cord and ascending axon tracts. In contrast, it had no effect on the upregulation of α2δ-1 mRNA and protein in DRGs. In vitro, PGB reduced plasma membrane expression of α2δ-1 without affecting endocytosis. We conclude that the antiallodynic effect of PGB in vivo is associated with impaired anterograde trafficking of α2δ-1, resulting in its decrease in presynaptic terminals, which would reduce neurotransmitter release and spinal sensitization, an important factor in the maintenance of neuropathic pain.

G Protein Modulation of Voltage-Gated Calcium Channels

Calcium influx into any cell requires fine tuning to guarantee the correct balance between activation of calcium-dependent processes, such as muscle contraction and neurotransmitter release, and calcium-induced cell damage. G protein-coupled receptors play a critical role in negative feedback to modulate the activity of the CaV2 subfamily of the voltage-dependent calcium channels, which are largely situated on neuronal and neuro-endocrine cells. The basis for the specificity of the relationships among membrane receptors, G proteins, and effector calcium channels will be discussed, as well as the mechanism by which G protein-mediated inhibition is thought to occur. The inhibition requires free Gβγ dimers, and the cytoplasmic linker between domains I and II of the CaV2 α1 subunits binds Gβγ dimers, whereas the intracellular N terminus of CaV2 α1 subunits provides essential determinants for G protein modulation. Evidence suggests a key role for the β subunits of calcium channels in the process of G protein modulation, and the role of a class of proteins termed “regulators of G protein signaling” will also be described.

Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension.

At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs. We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa. We performed exome sequencing of ten zona glomerulosa–like APAs and identified nine with somatic mutations in either ATP1A1, encoding the Na+/K+ ATPase α1 subunit, or CACNA1D, encoding Cav1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis.

PI3K promotes voltage-dependent calcium channel trafficking to the plasma membrane

Phosphatidylinositol 3-kinase (PI3K) has been shown to enhance native voltage-dependent calcium channel (Cav) currents both in myocytes and in neurons; however, the mechanism(s) responsible for this regulation were not known. Here we show that PI3K promotes the translocation of GFP-tagged Cav channels to the plasma membrane in both COS-7 cells and neurons. We show that the effect of PI3K is mediated by Akt/PKB and specifically requires Cavβ2 subunits. The mutations S574A and S574E in Cavβ2a prevented and mimicked, respectively, the effect of PI3K/Akt-PKB, indicating that phosphorylation of Ser574 on Cavβ2a is necessary and sufficient to promote Cav channel trafficking.

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential

Voltage-gated calcium channels are required for many key functions in the body. In this review, the different subtypes of voltage-gated calcium channels are described and their physiologic roles and pharmacology are outlined. We describe the current uses of drugs interacting with the different calcium channel subtypes and subunits, as well as specific areas in which there is strong potential for future drug development. Current therapeutic agents include drugs targeting L-type CaV1.2 calcium channels, particularly 1,4-dihydropyridines, which are widely used in the treatment of hypertension. T-type (CaV3) channels are a target of ethosuximide, widely used in absence epilepsy. The auxiliary subunit α2δ-1 is the therapeutic target of the gabapentinoid drugs, which are of value in certain epilepsies and chronic neuropathic pain. The limited use of intrathecal ziconotide, a peptide blocker of N-type (CaV2.2) calcium channels, as a treatment of intractable pain, gives an indication that these channels represent excellent drug targets for various pain conditions. We describe how selectivity for different subtypes of calcium channels (e.g., CaV1.2 and CaV1.3 L-type channels) may be achieved in the future by exploiting differences between channel isoforms in terms of sequence and biophysical properties, variation in splicing in different target tissues, and differences in the properties of the target tissues themselves in terms of membrane potential or firing frequency. Thus, use-dependent blockers of the different isoforms could selectively block calcium channels in particular pathologies, such as nociceptive neurons in pain states or in epileptic brain circuits. Of important future potential are selective CaV1.3 blockers for neuropsychiatric diseases, neuroprotection in Parkinson’s disease, and resistant hypertension. In addition, selective or nonselective T-type channel blockers are considered potential therapeutic targets in epilepsy, pain, obesity, sleep, and anxiety. Use-dependent N-type calcium channel blockers are likely to be of therapeutic use in chronic pain conditions. Thus, more selective calcium channel blockers hold promise for therapeutic intervention.

Facilitation of Ca2+current in excitable cells

Voltage-dependent Ca2+ channels are one of the main routes for the entry of Ca2+ into excitable cells. These channels are unique in cell-signalling terms in that they can transduce an electrical signal (membrane depolarization) via Ca2+ entry into a chemical signal, by virtue of the diverse range of intracellular Ca(2+)-dependent enzymes and processes. In a variety of cell types, currents through voltage-dependent Ca2+ channels can be increased in amplitude by a number of means. Although the term facilitation was originally defined as an increase of Ca2+ current resulting from one or a train of prepulses to depolarizing voltages, there is a great deal of overlap between facilitation by this means and enhancement by other routes, such as phosphorylation.

Calcium channel diversity: multiple roles of calcium channel subunits.

Until recently we held the simple view that voltage-gated calcium channels consisted of an alpha1 subunit, usually associated with auxiliary beta subunits and alpha(2)delta subunits and that skeletal muscle calcium channels were also associated with a gamma subunit. However, as discussed here, there is now evidence that the auxiliary subunits may also perform other roles unrelated to voltage-gated calcium entry. In the past students were taught the simplistic view that second messenger signaling to voltage-gated calcium channels involved mainly phosphorylation of L-type calcium channels, Ca(2+)-dependent inactivation via calmodulin, and direct G-protein-mediated inhibition of the neuronal N and P/Q channels. However, it is now clear that there are many other means of modulating calcium channel activity, including receptor-mediated internalization, proteolytic cleavage, phosphorylation of beta subunits, and interaction of calcium channels with other proteins, including enzymes masquerading as scaffold proteins.