Gabapentin-mediated inhibition of voltage-activated Ca2+ channel currents in cultured sensory neurones is dependent on culture conditions and channel subunit expression

We have used the whole cell patch clamp method and fura-2 fluorescence imaging to study the actions of gabapentin (1-(aminoethyl) cyclohexane acetic acid) on voltage-activated Ca(2+) entry into neonatal cultured dorsal root ganglion (DRG) neurones and differentiated F-11 (embryonic rat DRG x neuroblastoma hybrid) cells. Gabapentin (2.5 microM) in contrast to GABA (10 microM) did not influence resting membrane potential or input resistance. In current clamp mode gabapentin failed to influence the properties of evoked single action potentials but did reduce the duration of action potentials prolonged by Ba(2+). Gabapentin attenuated high voltage-activated Ca(2+) channel currents in a dose- and voltage- dependent manner in DRG neurones and reduced Ca(2+) influx evoked by K(+) depolarisation in differentiated F-11 cells loaded with fura-2. The sensitivity of DRG neurones to gabapentin was not changed by the GABA(B) receptor antagonist saclofen but pertussis toxin pre-treatment reduced the inhibitory effects of gabapentin. Experiments following pre-treatment of DRG neurones with a PKA-activator and a PKA-inhibitor implicated change in phosphorylation state as a mechanism, which influenced gabapentin action. Sp- and Rp-analogues of cAMP significantly increased or decreased gabapentin-mediated inhibition of voltage-activated Ca(2+) channel currents. Culture conditions used to maintain DRG neurones and passage number of differentiated F-11 cells also influenced the sensitivity of Ca(2+) channels to gabapentin. We analysed the Ca(2+) channel subunits expressed in populations of DRG neurones and F-11 cells that responded to gabapentin had low sensitivity to gabapentin or were insensitive to gabapentin, by Quantitative TaqMan PCR. The data obtained from this analysis suggested that the relative abundance of the Ca(2+) channel beta(2) and alpha(2)delta subunit expressed was a key determinant of gabapentin sensitivity of both cultured DRG neurones and differentiated F-11 cells. In conclusion, gabapentin inhibited part of the high voltage-activated Ca(2+) current in neonatal rat cultured DRG neurones via a mechanism that was independent of GABA receptor activation, but was sensitive to pertussis toxin. Gabapentin responses identified in this study implicated Ca(2+) channel beta(2) subunit type as critically important to drug sensitivity and interactions with alpha(1) and alpha(2)delta subunits may be implicated in antihyperalgesic therapeutic action for this compound.     

Inhibition of neuronal Ca(2+) influx by gabapentin and pregabalin in the human neocortex.

Gabapentin and pregabalin (S-(+)-3-isobutylgaba) produced concentration-dependent inhibitions of the K(+)-induced [Ca(2+)](i) increase in fura-2-loaded human neocortical synaptosomes (IC(50)=17 microM for both compounds; respective maximal inhibitions of 37 and 35%). The weaker enantiomer of pregabalin, R-(-)-3-isobutylgaba, was inactive. These findings were consistent with the potency of these drugs to inhibit [(3)H]-gabapentin binding to human neocortical membranes. The inhibitory effect of gabapentin on the K(+)-induced [Ca(2+)](i) increase was prevented by the P/Q-type voltage-gated Ca(2+) channel blocker omega-agatoxin IVA. The alpha 2 delta-1, alpha 2 delta-2, and alpha 2 delta-3 subunits of voltage-gated Ca(2+) channels, presumed sites of gabapentin and pregabalin action, were detected with immunoblots of human neocortical synaptosomes. The K(+)-evoked release of [(3)H]-noradrenaline from human neocortical slices was inhibited by gabapentin (maximal inhibition of 31%); this effect was prevented by the AMPA receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide). Gabapentin and pregabalin may bind to the Ca(2+) channel alpha 2 delta subunit to selectively attenuate depolarization-induced Ca(2+) influx of presynaptic P/Q-type Ca(2+) channels; this results in decreased glutamate/aspartate release from excitatory amino acid nerve terminals leading to a reduced activation of AMPA heteroreceptors on noradrenergic nerve terminals.     

Gabapentin inhibits high-threshold calcium channel currents in cultured rat dorsal root ganglion neurones.

1. This study examined the action of gabapentin (gabapentin,1-(aminomethyl) cyclohexane acetic acid (Neurontin) on voltage-gated calcium (Ca(2+)) channel influx recorded in cultured rat dorsal root ganglion (DRG) neurones. 2. Voltage-gated Ca(2+) influx was monitored using both fura-2 based fluorescence Ca(2+) imaging and the whole-cell patch clamp technique. 3. Imaging of intracellular Ca(2+) transients revealed that gabapentin inhibited KCl (30 mM)-evoked voltage-dependent Ca(2+) influx. Both the duration for 50% of the maximum response (W50) and total Ca(2+) influx were significantly reduced by approximately 25-30% in the presence of gabapentin (25 microM). 4. Gabapentin potently inhibited the peak whole-cell Ca(2+) channel current (I(Ba)) in a dose-dependent manner with an estimated IC(50) value of 167 nM. Block was incomplete and saturated at a maximal concentration of 25 microM. 5. Inhibition was significantly decreased in the presence of the neutral amino acid L-isoleucine (25 microM) but unaffected by application of the GABA(B) antagonist, saclofen (200 microM), suggesting a direct action on the alpha(2)delta subunit of the Ca(2+) channel. 6. Gabapentin inhibition was voltage-dependent, producing an approximately 7 mV hyperpolarizing shift in current voltage properties and reducing a non-inactivating component of whole-cell current activated at relatively depolarized potentials. 7. The use of specific Ca(2+) channel antagonists revealed a mixed pharmacology of the gabapentin-sensitive current (N-, L- and P/Q-type), which is dominated by N-type current. 8. The present study is the first to demonstrate that gabapentin directly mediates inhibition of voltage-gated Ca(2+) influx in DRG neurones, providing a potential means for gabapentin to effectively mediate spinal anti-nociception.     

Alternative splicing of the Ca2+ channel beta4 subunit confers specificity for gabapentin inhibition of Cav2.1 trafficking

Gabapentin is well established as an effective treatment for neuropathic pain; however, little is known about its mechanism of action. It binds with high affinity to Ca2+ channel alpha2delta subunits that are expressed in dorsal root ganglia. Mutation of a single alpha2delta amino acid, R217A, eliminates both gabapentin binding and analgesic efficacy. Gabapentin does not seem to have direct Ca2+ channel blocking properties but does affect overall levels of Ca2+channel surface expression in some circumstances. In this report, we examined gabapentin effects on trafficking and voltage-dependent gating properties of recombinant Ca(v)2.1 Ca2+ channel complexes transiently expressed in Xenopus laevis oocytes. We also determined electrophysiologically whether gabapentin causes displacement of beta subunits from Ca(v)2.1 complexes. Our principal findings are as follows: 1) gabapentin inhibits trafficking of recombinant Ca(v)2.1 Ca2+ channels in X. laevis oocytes; 2) gabapentin inhibition occurs in the presence of the Ca2+ channel beta4a subunit but not in the presence of beta4b; 3) gabapentin does not affect Ca(v)2.1 voltage-dependent gating parameters; 4) inhibition of Ca(v)2.1 trafficking is highly dependent on beta-subunit concentration; and 5) gabapentin inhibition of Ca(v)2.1 trafficking can be reversed by the alpha2delta R217A mutation. Overall, our results suggest that gabapentin reduces the number of beta4a-bound Ca(v)2.1 complexes that are successfully trafficked to the plasma membrane. This mechanism may help to explain why gabapentin is both effective and selective in the treatment of neuropathic pain states that involve up-regulation of alpha2delta subunits.     

Further evidence for the role of the alpha(2)delta subunit of voltage dependent calcium channels in models of neuropathic pain

1. Current analgesic therapy is dominated by NSAIDs and opiates, however these agents have limited efficacy in the treatment of neuropathic pain. The novel anticonvulsant agent gabapentin (Neurontin) has been shown to be an effective treatment for neuropathic pain in the clinic. Recent studies have demonstrated that gabapentin selectively interacts with the alpha(2)delta subunit of voltage dependent calcium channels (VDCCs) which may be important in its mechanism of action. 2. Previous studies have identified a gabapentin analogue, 3-methyl gabapentin, that stereoselectively interacts with the alpha(2)delta subunit of VDCCs. Thus, whilst (1S, 3R) 3-methyl gabapentin binds to the alpha(2)delta protein with high affinity (IC(50)=42 nM), the corresponding (1R,3R) isomer is 300 times weaker (Bryans et al., 1998: J. Med. Chem., 41, 1838 - 1845). The present study examines the activity of diastereoisomers of 3-methyl gabapentin in two rat models of neuropathic pain to assess the importance of an interaction with the alpha(2)delta subunit of VDCCs. 3. (1S,3R) 3-methyl-gabapentin dose-dependently (10 - 100 mg kg(-1), p.o.) blocked the maintenance of static allodynia in the rat streptozocin and Chung models of neuropathic pain with MEDs of 30 mg kg(-1). This isomer also dose-dependently blocked the maintenance of dynamic allodynia in both models with respective MEDs of 30 and 100 mg kg(-1). In contrast, (1R,3R) 3-methyl gabapentin (100 mg kg(-1), p.o.) failed to block either static or dynamic allodynia in the streptozocin model. 4. It is concluded that these data further support the hypothesis that the alpha(2)delta subunit of VDCCs plays an important role in the maintenance of mechanical hypersensitivity in models of neuropathic pain.     

Ca2+ channel alpha2delta ligands: novel modulators of neurotransmission

The term 'Ca2+ channel alpha2delta ligands' has recently been applied to an evolving drug class that includes gabapentin (Neurontin) and pregabalin (Lyrica), and reflects significant progress over the past decade in elucidating the mechanism of action of these drugs: a novel, specific action at one of the subunits constituting voltage-sensitive Ca2+ channels. Binding of these ligands to the alpha2delta subunit is considered to explain their usefulness in treating several clinical disorders, including epilepsy, pain from diabetic neuropathy, postherpetic neuralgia and fibromyalgia, and generalized anxiety disorder. The evidence indicates a relationship between alpha2delta subunit binding and the modulation of processes that subserve neurotransmission. This modulation is characterized by a reduction of the excessive neurotransmitter release that is observed in certain neurological and psychiatric disorders.     

Characteristics of block by Pb2+ of function of human neuronal L-, N-, and R-type Ca2+ channels transiently expressed in human embryonic kidney 293 cells

Lead (Pb(2+)) is a well-known inhibitor of voltage-dependent Ca(2+) channels in their native environments in several types of cells. However, its effects on discrete Ca(2+) channel phenotypes in isolation have not been well studied. We compared how specific subtypes of human neuronal high-voltage-activated Ca(2+) channels were affected by acute exposure to Pb(2+). Expression cDNA clones of human alpha(1C), alpha(1B), or alpha(1E) subunit genes encoding neuronal L-, N-, and R-subtypes of Ca(2+) channels, respectively, along with a constant alpha(2)delta and beta(3) subunits were transfected into human embryonic kidney 293 cells. Currents through the respective transiently expressed channels were measured using whole-cell recording techniques with Ba(2+) (20 mM) as charge carrier. Extracellular bath applications of Pb(2+) significantly reduced current amplitude through all three types of Ca(2+) channels in a concentration-dependent manner. The order of potency was: alpha(1E) (IC(50) = 0.10 microM), followed by alpha(1C) (IC(50) = 0.38 microM) and alpha(1B) (IC(50) = 1.31 microM). Pb(2+)-induced perturbation of function of alpha(1C) and alpha(1B) containing Ca(2+) channels was more easily reversed than for alpha(1E)-containing Ca(2+) channels after washing with Pb(2+) free solution. The current-voltage relationships were not altered after 3-min exposure to Pb(2+) for any of the three types. However, the steady-state inactivation relationships were shifted to more negative potentials for channels containing alpha(1B) and alpha(1E) subunits, but not for those containing alpha(1C) subunits. Pb(2+) accelerated the inactivation time of current in all three subtypes of Ca(2+) channels in a concentration- and voltage-dependent manner. Therefore, different subtypes of Ca(2+) channels exhibit differential susceptibility to Pb(2+) even when expressed in the same cell type. Current expressed by alpha(1E)-containing channels is more sensitive to Pb(2+) than that expressed by alpha(1C)- or alpha(1B)-containing channels. Several Ca(2+) channel phenotypes are quite sensitive to the inhibitory action of Pb(2+). Furthermore, it seems that Pb(2+) is more likely to combine with Ca(2+) channels in the closed state.     

Recombinant GABA(B) receptors formed from GABA(B1) and GABA(B2) subunits selectively inhibit N-type Ca(2+) channels in NG108-15 cells

Efficient transfection of NG108-15 cells with GABA(B) receptor subunits was achieved using polyethylenimine. Baclofen modulated high voltage-activated Ca(2+) current in differentiated cells transfected with GABA(B1) and GABA(B2) receptor subunits or with the GABA(B2) subunit alone, but not with the GABA(B1) subunit alone. Characteristics of the current modulation were very similar for cells transfected with GABA(B1/2) and GABA(B2) subunits. Using antisense oligonucleotides against GABA(B1) subunits and also western immunoblotting, we are able to show that NG108-15 cells contain endogenous GABA(B1) subunits. Therefore, functional receptors can be formed by the combination of native GABA(B1) subunits with transfected GABA(B2) subunits, in agreement with the proposed heteromeric structure of GABA(B) receptors. Finally, we used selective channel blockers to identify the subtypes of Ca(2+) channels that are modulated by GABA(B) receptors. In fact, in differentiated NG108-15 cells, the recombinant GABA(B) receptors couple only to N-type Ca(2+) channels.     

Pharmacological comparison of anticonvulsant drugs in animal models of persistent pain and anxiety

Signs and symptoms of persistent pain are associated with neuronal hyperexcitability within nociceptive pathways. This manifests behaviourally as a decrease in the nociceptive threshold to sensory stimulation, and is closely correlated with altered affective pain processing and increased expression of anxiety-like symptoms. Anticonvulsant drugs can have marked analgesic actions in animals and humans, and some have also been reported to possess anxiolytic-like properties in animals. In the current study, we have compared the antinociceptive actions of diazepam (allosteric GABA(A) receptor modulator), gabapentin (binds to alpha(2)delta Ca(2+) channel subunit), lamotrigine, riluzole and phenytoin (Na(+) channel blockers), levetiracetam (unknown mechanism), sodium valproate (potentiates GABA-mediated inhibition), ethosuximide (T-type Ca(2+) channel blocker) and retigabine (K(v)7 channel opener) in the rat formalin test, with their anxiolytic actions in the rat conditioned emotional response (CER) model of anxiety. Lamotrigine, gabapentin, riluzole, retigabine and ethosuximide attenuated second phase nociceptive responses in the formalin test. Lamotrigine, gabapentin and riluzole also displayed an anxiolytic-like profile in the CER model. Notably, the minimum doses of these drugs required to attenuate anxiety behaviour were similar to, or considerably lower than those needed to reverse pain-like behaviours. Diazepam was anxiolytic but only attenuated pain-like behaviours at sedative doses. The other drugs tested were inactive in both models. Our data suggests: (i) an antiepileptic mechanism of action per se is not necessarily sufficient for a compound to display antinociceptive and/or anxiolytic actions; and (ii) the combined antinociceptive and anxiolytic-like profiles of lamotrigine, gabapentin and riluzole suggests that these compounds likely modulate both sensory and affective dimensions of pain.     

Effects of diltiazem on human nicotinic acetylcholine and GABA(A) receptors

Effects of the L-type calcium channel antagonist diltiazem on recombinant human GABA(A) receptor (alpha1beta2gamma2s) or on muscle (alpha1beta1deltagamma and alpha1beta1delta(epsilon)) or neuronal (alpha7 and alpha4beta2) nicotinic acetylcholine receptors expressed in Xenopus oocytes were examined using two-electrode voltage-clamp. Diltiazem inhibited the function of both muscle and neuronal nicotinic receptors, but it had no effect on GABA(A) receptors. The extent of functional inhibition of nicotinic receptors depended on the receptor subtype, and the order of inhibition potency by diltiazem was alpha7>alpha4beta2 approximately alpha1beta1deltagamma approximately alpha1beta1delta(epsilon). Inhibition of alpha7 receptor function was non-competitive and voltage-independent, and it occurred at concentrations far lower than those needed to inhibit (never completely) binding of (125)I-alpha-bungarotoxin to heterologously expressed alpha7 receptors in mammalian cells. Pre-incubation in diltiazem before concomitant application with acetylcholine increased inhibition of function and slowed recovery from inhibition. Verapamil, a phenylalkylamine antagonist of L-type Ca(2+) channels also fully inhibited alpha7 receptor function and partially inhibited (125)I-alpha-bungarotoxin binding to alpha7 receptors, but was less potent than diltiazem. Effects on both alpha7 receptor function and (125)I-alpha-bungarotoxin binding by verapamil plus diltiazem suggest separate sites for verapamil and diltiazem on alpha7 receptors. These results provide further evidence that L-type Ca(2+) channel drugs inhibit ligand-gated cationic channels and suggest that caution should be applied when using these compounds to study systems in which L-type Ca(2+) channels and ligand-gated cationic channels co-exist.     

The inhibition of release by mGlu7 receptors is independent of the Ca2+ channel type but associated to GABAB and adenosine A1 receptors

Neurotransmitter release is inhibited by G-protein coupled receptors (GPCRs) through signalling pathways that are negatively coupled to Ca(2+) channels and adenylyl cyclase. Through Ca(2+) imaging and immunocytochemistry, we have recently shown that adenosine A(1), GABA(B) and the metabotropic glutamate type 7 receptors coexist in a subset of cerebrocortical nerve terminals. As these receptors inhibit glutamate release through common intracellular signalling pathways, their co-activation occluded each other responses. Here we have addressed whether the occlusion of receptor responses is restricted to the glutamate release mediated by N-type Ca(2+) channels by analysing this process in nerve terminals from mice lacking the alpha(1B) subunit (Ca(v) 2.2) of these channels. We found that glutamate release from cerebrocortical nerve terminals without these channels, in which release relies exclusively on P/Q type Ca(2+) channels, is not modulated by mGlu7 receptors. Furthermore, there is no occlusion of the release inhibition by GABA(B) and adenosine A(1). Hence, in the cerebrocortical preparation, these three receptors only appear to coexist in N-type channel containing nerve terminals. In contrast, in hippocampal nerve terminals lacking this subunit, where mGlu7 receptors modulate glutamate release via P/Q type channels, the occlusion of inhibitory responses by co-stimulation of adenosine A(1), GABA(B) and mGlu7 receptors was observed. Thus, occlusion of the responses by the three GPCRs is independent of the Ca(2+) channel type but rather, it is associated to functional mGlu7 receptors.     

Inhibition of recombinant N-type and native high voltage-gated neuronal Ca2+ channels by AdGABA: mechanism of action studies

High-voltage activated Ca(2+) (Ca(V)) channels play a key role in the regulation of numerous physiological events by causing transient changes in the intracellular Ca(2+) concentration. These channels consist of a pore-forming Ca(V)α(1) protein and three auxiliary subunits (Ca(V)β, Ca(V)α(2)δ and Ca(V)γ). Ca(V)α(2)δ is an important component of Ca(V) channels in many tissues and of great interest as a drug target. It is well known that anticonvulsant agent gabapentin (GBP) binds to Ca(V)α(2)δ and reduces Ca(2+) currents by modulating the expression and/or function of the Ca(V)α(1) subunit. Recently, we showed that an adamantane derivative of GABA, AdGABA, has also inhibitory effects on Ca(V) channels. However, the importance of the interaction of AdGABA with the Ca(V)α(2)δ subunit has not been conclusively demonstrated and the mechanism of action of the drug has yet to be elucidated. Here, we describe studies on the mechanism of action of AdGABA. Using a combined approach of patch-clamp recordings and molecular biology we show that AdGABA inhibits Ca(2+) currents acting on Ca(V)α(2)δ only when applied chronically, both in a heterologous expression system and in dorsal root-ganglion neurons. AdGABA seems to require uptake and be acting intracellularly given that its effects are prevented by an inhibitor of the L-amino acid transport system. Interestingly, a mutation in the Ca(V)α(2)δ that abolishes GBP binding did not affect AdGABA actions, revealing that its mechanism of action is similar but not identical to that of GBP. These results indicate that AdGABA is an important Ca(V)α(2)δ ligand that regulates Ca(V) channels.     

Gabapentin may inhibit synaptic transmission in the mouse spinal cord dorsal horn through a preferential block of P/Q-type Ca2+ channels

Gabapentin is a lipophilic analog of gamma-amino butyric acid (GABA) with therapeutic activity against certain forms of epilepsy and neuropathic pain. Despite its structural similarity to GABA, it does not bind GABAA or GABAB receptors and the mechanism, especially of its analgesic action, has remained elusive. Here, we have studied its effects on synaptic transmission mediated by the major spinal fast excitatory and inhibitory neurotransmitters, L-glutamate and glycine, in the superficial layers of the spinal cord dorsal horn, a CNS area, which is critically involved in nociception. Gabapentin reversibly reduced evoked excitatory postsynaptic currents mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA-EPSCs) and inhibitory postsynaptic currents mediated by glycine (gly-IPSCs). Inhibition of AMPA-EPSCs and gly-IPSCs occurred with similar potencies (approximately 10-50 nM) and by about the same degree (approximately 40% at 1 microM). Gabapentin did not affect membrane currents elicited by exogenously applied glutamate or glycine arguing against a postsynaptic site of action. Selective blockade of N-type Ca2+ channels with omega-conotoxin GVIA dramatically increased and blockade of P/Q-type channels with omega-agatoxin IVA strongly attenuated inhibition of evoked synaptic transmission by gabapentin. These results show that gabapentin affects both excitatory and inhibitory spinal neurotransmission via a presynaptic mechanism which preferentially involves P/Q-type Ca2+ channels.     

Gamma-aminobutyric acid type B receptors with specific heterodimer composition and postsynaptic actions in hippocampal neurons are targets of anticonvulsant gabapentin action

Gamma-aminobutyric acid (GABA) activates two qualitatively different inhibitory mechanisms through ionotropic GABA(A) multisubunit chloride channel receptors and metabotropic GABA(B) G protein-coupled receptors. Evidence suggests that pharmacologically distinct GABA(B) receptor subtypes mediate presynaptic inhibition of neurotransmitter release by reducing Ca2+ conductance, and postsynaptic inhibition of neuronal excitability by activating inwardly rectifying K+ (Kir) conductance. However, the cloning of GABA(B) gb1 and gb2 receptor genes and identification of the functional GABA(B) gb1-gb2 receptor heterodimer have so far failed to substantiate the existence of pharmacologically distinct receptor subtypes. The anticonvulsant, antihyperalgesic, and anxiolytic agent gabapentin (Neurontin) is a 3-alkylated GABA analog with an unknown mechanism of action. Here we report that gabapentin is an agonist at the GABA(B) gb1a-gb2 heterodimer coupled to Kir 3.1/3.2 inwardly rectifying K+ channels in Xenopus laevis oocytes. Gabapentin was practically inactive at the human gb1b-gb2 heterodimer, a novel human gb1c-gb2 heterodimer and did not block GABA agonism at these heterodimer subtypes. Gabapentin was not an agonist at recombinant GABA(A) receptors as well. In CA1 pyramidal neurons of rat hippocampal slices, gabapentin activated postsynaptic K+ currents, probably via the gb1a-gb2 heterodimer coupled to inward rectifiers, but did not presynaptically depress monosynaptic GABA(A) inhibitory postsynaptic currents. Gabapentin is the first GABA(B) receptor subtype-selective agonist identified providing proof of pharmacologically and physiologically distinct receptor subtypes. This selective agonism of postsynaptic GABA(B) receptor subtypes by gabapentin in hippocampal neurons may be its key therapeutic advantage as an anticonvulsant.     

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.     

Molecular and pharmacological bases for the gating regulation of L-type voltage-dependent Ca2+ channels

The voltage-dependent L-type Ca(2+) channel plays a key role in the spacial and temporal regulation of Ca(2+). In cardiac excitation-contraction coupling, Ca(2+)-induced Ca(2+) release (CICR) from ryanodine receptors (RyRs), triggered by Ca(2+) entry through the nearby L-type Ca(2+) channel, induces the Ca(2+)-dependent inactivation (CDI) of the Ca(2+) channel. We demonstrated that the CICR-dependent CDI of L-type Ca(2+) channels, under control of the privileged cross-signaling between L-type Ca(2+) channels and RyRs, plays important roles for monitoring and tuning the SR Ca(2+) content via changes of AP waveform and the amount of Ca(2+)-influx during AP in ventricular myocytes. L-type Ca(2+) channels are modulated by the binding of Ca(2+) channel antagonists and agonists to the pore-forming alpha(1C) subunit. We identified Phe(1112) and Ser(1115) in the pore-forming IIIS5-S6 linker region of the alpha(1C) subunit as critical determinants of the binding of dihydropyridines (DHP). Interestingly, double mutant Ca(2+) channel (F1112A/S1115A) failed to discriminate between a DHP Ca(2+) channel agonist and antagonist stereoisomers. We proposed that Phe(1112) and Ser(1115) in the pore-forming IIIS5-S6 linker region is required for the stabilization of the Ca(2+) channel in the open state by Ca(2+) channel agonists and further proposed a novel model for the DHP-binding pocket of the alpha(1C) subunit. These integrative studies on the gating regulation of cardiac L-type Ca(2+) channels will provide the molecular basis for the pharmacology of Ca(2+) channel modulators.     

Auxiliary Ca(2+) channel subunits: lessons learned from muscle

Voltage-gated Ca(2+) channels are multi-subunit complexes involved in many key functions of excitable cells. A multitude of studies in heterologous cells demonstrated that coexpression of the pore-forming alpha(1) subunits with auxiliary alpha(2)delta and beta subunits promotes membrane expression and modulates the biophysical channel properties. New null-mutant animal models and shRNA based knockdown experiments in skeletal muscle cells for the first time demonstrated the physiological roles and possible pathological effects of the alpha(2)delta-1 and beta(1a) subunits in a differentiated excitable cell. The alpha(2)delta-1 subunit is the determinant of the typical current properties of skeletal and cardiac muscle Ca(2+) channels. The beta(1a) subunit links the skeletal muscle Ca(2+) channel to the Ca(2+) release channel in the sarcoplasmic reticulum. Whether these specific functions in muscle indicate similar roles of alpha(2)delta and beta subunits as functional modulator and structural organizer, respectively, in neurons is being discussed.     

A comparison of Ca2+ channel blocking mode between gabapentin and verapamil: implication for protection against hypoxic injury in rat cerebrocortical slices

1 The mode of Ca(2+) channel blocking by gabapentin [1-(aminomethyl)cyclohexane acetic acid] was compared to those of other Ca(2+) channel blockers, and the potential role of Ca(2+) channel antagonists in providing protection against hypoxic injury was subsequently investigated in rat cerebrocortical slices. 2 mRNA for the alpha(2)delta subunits of Ca(2+) channels was found in rat cerebral cortex. 3 Nitric oxide (NO) synthesis estimated from cGMP formation was enhanced by KCl stimulation, which was mediated primarily by the activation of N- and P/Q-type Ca(2+) channels. Gabapentin blocked both types of Ca(2+) channels, and preferentially reversed the response to 30 mM K(+) stimulation compared with 50 mM K(+) stimulation. In contrast, verapamil preferentially inhibited the response to depolarization by the higher concentration (50 mM) of K(+). 4 Gabapentin inhibited KCl-induced elevation of intracellular Ca(2+) in primary neuronal culture. 5 Hypoxic injury was induced in cerebrocortical slices by oxygen deprivation in the absence (severe injury) or presence of 3 mM glucose (mild injury). Gabapentin preferentially inhibited mild injury, while verapamil suppressed only severe injury. omega-Conotoxin GVIA (omega-CTX) and omega-agatoxin IVA (omega-Aga) were effective in both models. 6 NO synthesis was enhanced in a manner dependent on the severity of hypoxic insults. Gabapentin reversed the NO synthesis induced by mild insults, while verapamil inhibited that elicited by severe insults. omega-CTX and omega-Aga were effective in both the cases. 7 Therefore, the data suggest that gabapentin and verapamil cause activity-dependent Ca(2+) channel blocking by different mechanisms, which are associated with their cerebroprotective actions and are dependent on the severity of hypoxic insults.     

Molecular cloning and characterization of the human voltage-gated calcium channel alpha(2)delta-4 subunit

The voltage-gated calcium channel is composed of a pore-forming alpha(1) subunit and several regulatory subunits: alpha(2)delta, beta, and gamma. We report here the identification of a novel alpha(2)delta subunit, alpha(2)delta-4, from the expressed sequence tag database followed by its cloning and characterization. The novel alpha(2)delta-4 subunit gene contains 39 exons spanning about 130 kilobases and is co-localized with the CHCNA1C gene (alpha(1C) subunit) on human chromosome 12p13.3. Alternative splicing of the alpha(2)delta-4 gene gives rise to four potential variants, a through d. The open reading frame of human alpha(2)delta-4a is composed of 3363 base pairs encoding a protein with 1120 residues and a calculated molecular mass of 126 kDa. The alpha(2)delta-4a subunit shares 30, 32, and 61% identity with the human calcium channel alpha(2)delta-1, alpha(2)delta-2, and alpha(2)delta-3 subunits, respectively. Primary sequence comparison suggests that alpha(2)delta-4 lacks the gabapentin binding motifs characterized for alpha(2)delta-1 and alpha(2)delta-2; this was confirmed by a [(3)H]gabapentin-binding assay. In human embryonic kidney 293 cells, the alpha(2)delta-4 subunit associated with Ca(V)1.2 and beta(3) subunits and significantly increased Ca(V)1.2/beta(3)-mediated Ca(2+) influx. Immunohistochemical study revealed that the alpha(2)delta-4 subunit has limited distribution in special cell types of the pituitary, adrenal gland, colon, and fetal liver. Whether the alpha(2)delta-4 subunit plays a distinct physiological role in select endocrine tissues remains to be demonstrated.