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.     

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.     

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.     

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.     

Contrasting the effects of nifedipine on subtypes of endogenous and recombinant T-type Ca2+ channels

There is evidence that nifedipine (Nif) - a dihydropyridine (DHP) Ca(2+)-channel antagonist mostly known for its L-type-specific action--is capable of blocking low voltage-activated (LVA or T-type) Ca(2+) channels as well. However, the discrimination by Nif of either various endogenous T-channel subtypes, evident from functional studies, or cloned Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 T-channel alpha 1 subunits have not been determined. Here, we investigated the effects of Nif on currents induced by Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 expression in Xenopus oocytes or HEK-293 cells (I(alpha 1G), I(alpha 1H) and I(alpha 1I), respectively) and two kinetically distinct, "fast" and "slow", LVA currents in thalamic neurons (I(LVA,f) and I(LVA,s)). At voltages of the maximums of respective currents the drug most potently blocked I(alpha 1H) (IC(50)=5 microM, max block 41%) followed by I(alpha 1G) (IC(50)=109 microM, 23%) and I(alpha 1I) (IC(50)=243 microM, 47%). The mechanism of blockade included interaction with Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 open and inactivated states. Nif blocked thalamic I(LVA,f) and I(LVA,s) with nearly equal potency (IC(50)=22 microM and 28 microM, respectively), but with different maximal inhibition (81% and 51%, respectively). We conclude that Ca(v)3.2 is the most sensitive to Nif, and that quantitative characteristics of drug action on T-type Ca(2+) channels depend on cellular system they are expressed in. Some common features in the voltage- and state-dependence of Nif action on endogenous and recombinant currents together with previous data on T-channel alpha 1 subunits mRNA expression patterns in the thalamus point to Ca(v)3.1 and Ca(v)3.3 as the major contributors to thalamic I(LVA,f) and I(LVA,s), respectively.     

Mechanisms of the antinociceptive action of gabapentin

Gabapentin, a gamma-aminobutyric acid (GABA) analogue anticonvulsant, is also an effective analgesic agent in neuropathic and inflammatory, but not acute, pain systemically and intrathecally. Other clinical indications such as anxiety, bipolar disorder, and hot flashes have also been proposed. Since gabapentin was developed, several hypotheses had been proposed for its action mechanisms. They include selectively activating the heterodimeric GABA(B) receptors consisting of GABA(B1a) and GABA(B2) subunits, selectively enhancing the NMDA current at GABAergic interneurons, or blocking AMPA-receptor-mediated transmission in the spinal cord, binding to the L-alpha-amino acid transporter, activating ATP-sensitive K(+) channels, activating hyperpolarization-activated cation channels, and modulating Ca(2+) current by selectively binding to the specific binding site of [(3)H]gabapentin, the alpha(2)delta subunit of voltage-dependent Ca(2+) channels. Different mechanisms might be involved in different therapeutic actions of gabapentin. In this review, we summarized the recent progress in the findings proposed for the antinociceptive action mechanisms of gabapentin and suggest that the alpha(2)delta subunit of spinal N-type Ca(2+) channels is very likely the analgesic action target of gabapentin.     

Calcium channel alpha(2)delta subunits-structure and Gabapentin binding

High-voltage activated calcium channels are modulated by a series of auxiliary proteins, including those of the alpha(2)delta family. Until recently, only a single alpha(2)delta subunit was known, but two further members, alpha(2)delta-2 and -3, have since been identified. In this study, the structure of these two novel subunits has been characterized and binding of the antiepileptic drug gabapentin investigated. Using antibodies directed against the amino terminal portion of the proteins, the gross structure of the subunits could be analyzed by Western blotting. Similar to alpha(2)delta-1, both alpha(2)delta-2 and -3 subunits consist of two proteins-a larger alpha(2) and a smaller delta that can be separated by reduction. The subunits are also highly N-glycosylated with approximately 30 kDa of their mass consisting of oligosaccharides. alpha(2)delta-1 was detected in all mouse tissues studied, whereas alpha(2)delta-2 was found at high levels in brain and heart. The alpha(2)delta-3 subunit was observed only in brain. alpha(2)delta-1 and alpha(2)delta-2, but not alpha(2)delta-3, were found to bind gabapentin. The K(d) value of gabapentin binding to alpha(2)delta-2 was 153 nM compared with the higher affinity binding to alpha(2)delta-1 (K(d) = 59 nM).     

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.     

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 recombinant low-voltage-activated Ca(2+) channels by the neuroprotective agent BW619C89 (Sipatrigine)

T-type Ca(2+) currents were recorded in 2 mM Ca(2+) from HEK 293 cells stably expressing recombinant low-voltage-activated Ca(2+) channel subunits. Current-voltage relationships revealed that these currents were low-voltage activated in nature and could be reversibly antagonised by mibefradil, a known T-type channel blocker. At a test potential of -25 mV alpha(1I)-mediated Ca(2+) currents were rapidly and reversibly inhibited by 1-100 microM BW619C89 (IC(50)=14 microM, Hill coefficient 1.3). In contrast to its actions on N-type Ca(2+) channels, a near IC(50) dose (10 microM) of BW619C89 produced no alterations in either the kinetics or voltage-dependence of T-type currents. In additional single dose experiments, currents mediated by rat alpha(1G), human alpha(1H) or human alpha(1I) channel subunits were also inhibited by BW619C89. Overall our data indicate that T-type Ca(2+) channels are more potently blocked by BW619C89 than either type-II Na(+) channels or N-type Ca(2+) channels. It seems, therefore, that inhibition of low-voltage-activated Ca(2+) channels is likely to contribute to the anticonvulsant and neuroprotective actions of this and related compounds.     

Vascular calcium channels and high blood pressure: pathophysiology and therapeutic implications

Long-lasting Ca(2+) (Ca(L)) channels of the Ca(v)1.2 gene family are heteromultimeric structures that are minimally composed of a pore-forming alpha(1C) subunit and regulatory beta and alpha(2)delta subunits in vascular smooth muscle cells. The Ca(L) channels are the primary pathways for voltage-gated Ca(2+) influx that trigger excitation-contraction coupling in small resistance vessels. Notably, vascular smooth muscle cells of hypertensive rats show an increased expression of Ca(L) channel alpha(1C) subunits, which is associated with elevated Ca(2+) influx and the development of abnormal arterial tone. Indeed, blood pressure per se appears to promote Ca(L) channel expression in small arteries, and even short-term rises in pressure may alter channel expression. Membrane depolarization has been shown to be one stimulus associated with elevated blood pressure that promotes Ca(L) channel expression at the plasma membrane. Future studies to define the molecular processes that regulate Ca(L) channel expression in vascular smooth muscle cells will provide a rational basis for designing antihypertensive therapies to normalize Ca(L) channel expression and the development of anomalous vascular tone in hypertensive pathologies.     

Lysophosphatidylcholine augments Ca(v)3.2 but not Ca(v)3.1 T-type Ca(2+) channel current expressed in HEK-293 cells

Lysophosphatidylcholine (LPC) has been shown to induce electrophysiological disturbances to arrhythmogenesis. However, the effects of LPC on the low-voltage-activated T-type Ca(2+) channels in the heart are not understood yet. We found that LPC increases the T-type Ca(2+) channel current (I(Ca.T)) in neonatal rat cardiomyocytes. To further investigate the underlying modulatory mechanism of LPC on T-type Ca(2+) channels, we utilized HEK-293 cells stably expressing alpha1G and alpha1H subunits (HEK-293/alpha1G and HEK-293/alpha1H), by use of patch-clamp techniques. A low concentration of LPC (10 micromol/l) significantly increased Ca(v)3.2 I(Ca.T) (alpha1H) that were similar to those observed in neonatal rat cardiomyocytes. Activation and steady-state inactivation curves were shifted in the hyperpolarized direction by 5.1 +/- 0.2 and 4.6 +/- 0.4 mV, respectively, by application of 10 micromol/l LPC. The pretreatment of cells with a protein kinase C inhibitor (chelerythrine) attenuated the effects of LPC on I(Ca.T) (alpha1H). However, the application of LPC failed to modify Ca(v)3.1 (alpha1G) I(Ca.T) at concentrations of 10-50 micromol/l. In conclusion, these data demonstrate that extracellularly applied LPC augments Ca(v)3.2 I(Ca.T) (alpha1H) but not Ca(v)3.1 I(Ca.T) (alpha1G) in a heterologous expression system, possibly by modulating protein kinase C signaling.     

Molecular determinants of frequency dependence and Ca2+ potentiation of verapamil block in the pore region of Cav1.2

Verapamil block of Ca(v)1.2 is frequency-dependent and potentiated by Ca(2+). We examined the molecular determinants of these characteristics using mutations that effect Ca(2+) interactions with Ca(v)1.2. Mutant and wild-type Ca(v)1.2 channels were transiently expressed in tsA 201 cells with beta(1b) and alpha(2)delta subunits. The four conserved glutamates that compose the Ca(2+) selectivity filter in Ca(v)1.2 were mutated to Gln (E363Q, E709Q, E1118Q, E1419Q) and the adjacent conserved threonine in each domain was mutated to Ala (T361A, T707A, T1116A, T1417A). The L-type-specific residues in the domain III pore region (F1117G) and the C-terminal tail (I1627A) were also mutated and assayed for block by verapamil using whole-cell voltage-clamp recordings in 10 mM Ba(2+) or 10 mM Ca(2+). In Ba(2+), none of the pore-region mutations reduced the fraction of current blocked by 30 microM verapamil at 0.05 Hz stimulation. However, all of the pore-region mutations abolished Ca(2+) potentiation of verapamil block at 0.05 Hz. The T1116A, F1117G, E1118Q, and E1419Q mutations all significantly reduced frequency-dependent verapamil block (1-Hz stimulation) in both Ba(2+) and Ca(2+). The I1627A mutation, which disrupts Ca(2+)-dependent inactivation, increased the fraction of closed channels blocked by 30 microM verapamil in Ba(2+) but did not affect frequency-dependent block in Ba(2+) or Ca(2+). Our data suggest that the pore region of domain III may contribute to a high affinity verapamil binding site accessed during 1-Hz stimulation and that Ca(2+) binding to multiple sites may be required for potentiation of verapamil block of closed channels.     

Contrasting anesthetic sensitivities of T-type Ca2+ channels of reticular thalamic neurons and recombinant Ca(v)3.3 channels

Reticular thalamocortical neurons express a slowly inactivating T-type Ca(2+) current that is quite similar to that recorded from recombinant Ca(v)3.3b (alpha1Ib) channels. These neurons also express abundant Ca(v)3.3 mRNA, suggesting that it underlies the native current. Here, we test this hypothesis by comparing the anesthetic sensitivities of recombinant Ca(v)3.3b channels stably expressed in HEK 293 cells to native T channels in reticular thalamic neurons (nRT) from brain slices of young rats. Barbiturates completely blocked both Ca(v)3.3 and nRT currents, with pentobarbital being about twice more potent in blocking Ca(v)3.3 currents. Isoflurane had about the same potency in blocking Ca(v)3.3 and nRT currents, but enflurane, etomidate, propofol, and ethanol exhibited 2-4 fold higher potency in blocking nRT vs Ca(v)3.3 currents. Nitrous oxide (N(2)O; laughing gas) blocked completely nRT currents with IC(50) of 20%, but did not significantly affect Ca(v)3.3 currents at four-fold higher concentrations. In addition, we observed that in lower concentration, N(2)O reversibly increased nRT but not Ca(v)3.3 currents. In conclusion, contrasting anesthetic sensitivities of Ca(v)3.3 and nRT T-type Ca(2+) channels strongly suggest that different molecular structures of Ca(2+) channels give rise to slowly inactivating T-type Ca(2+) currents. Furthermore, effects of volatile anesthetics and ethanol on slowly inactivating T-type Ca(2+) channel variants may contribute to the clinical effects of these agents.     

N-type Ca2+ channel

Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCCs) regulates various aspects of physiological function, including neurotransmitter release, regulation of cell membrane excitability, and control of gene expression. VDCCs are classified into several sub-types (L-, N-, P/Q-, R-, and T-types) based on electrophysiological and pharmacological properties. Each type of channels except the T-type is composed of at least four subunits, designated alpha(1), alpha(2), beta, and delta. During the past decade, a number of genes encoding these subunits have been cloned, and cDNA expression studies using heterologous expression systems have revealed the intricate nature of subunit interaction and many biophysical aspects of channel function. In recent years, an entirely new strategy has been introduced in attempts to clarify the physiological role of each of the VDCCs, and this has proven to be very useful in defining previously unknown in vivo functions of VDCCs. In this article, we briefly review the recent advances in our understanding of VDCCs with special emphasis on the N-type channel, which is mainly expressed in neural tissues and is the essential component of neurotransmitter release. We will mainly discuss the subunit composition, channel regulation by G proteins and exocytotic proteins, and the mouse phenotypes in which N-type channel subunits have been deleted by gene targeting technology.     

Molecular determinants of diltiazem block in domains IIIS6 and IVS6 of L-type Ca(2+) channels

The benzothiazepine diltiazem blocks ionic current through L-type Ca(2+) channels, as do the dihydropyridines (DHPs) and phenylalkylamines (PAs), but it has unique properties that distinguish it from these other drug classes. Wild-type L-type channels containing alpha(1CII) subunits, wild-type P/Q-type channels containing alpha(1A) subunits, and mutants of both channel types were transiently expressed in tsA-201 cells with beta(1B) and alpha(2)delta subunits. Whole-cell, voltage-clamp recordings showed that diltiazem blocks L-type Ca(2+) channels approximately 5-fold more potently than it does P/Q-type channels. Diltiazem blocked a mutant P/Q-type channel containing nine amino acid changes that made it highly sensitive to DHPs, with the same potency as L-type channels. Thus, amino acids specific to the L-type channel that confer DHP sensitivity in an alpha(1A) background also increase sensitivity to diltiazem. Analysis of single amino acid mutations in domains IIIS6 and IVS6 of alpha(1CII) subunits confirmed the role of these L-type-specific amino acid residues in diltiazem block, and also indicated that Y1152 of alpha(1CII), an amino acid critical to both DHP and PA block, does not play a role in diltiazem block. Furthermore, T1039 and Y1043 in domain IIIS5, which are both critical for DHP block, are not involved in block by diltiazem. Conversely, three amino acid residues (I1150, M1160, and I1460) contribute to diltiazem block but have not been shown to affect DHP or PA block. Thus, binding of diltiazem to L-type Ca(2+) channels requires residues that overlap those that are critical for DHP and PA block as well as residues unique to diltiazem.     

CaVbeta subunit-mediated up-regulation of CaV2.2 currents triggered by D2 dopamine receptor activation

Voltage-dependent Ca(2+) channels (VDCCs) are subject to modulation by a number of pathways, including membrane-delimited inhibition by heterotrimeric G-proteins and modulation through phosphorylation by diverse kinases. Here we report that in the Xenopus oocyte expression system Ca(V)2.2 channels undergo a sustained, linear and irreversible run-up lasting up to 30 min, which is potentiated during G-protein-mediated inhibition by activation of co-expressed G-protein coupled receptors (GPCRs). This up-regulation is not a result of receptor desensitization, but is associated with a hyperpolarization of the voltage for activation and depends on the presence of accessory subunits such that beta subunits promote, and alpha2delta subunits oppose the current increase. We have investigated the involvement of G-proteins and found that over-expression of Galpha(o) subunits or Galpha-transducin reduced the amount of agonist-mediated up-regulation. However, we have found no evidence for the involvement of any second messenger pathways in the increase of current run-up in the presence of a GPCR agonist. Taken together, our data suggest that the effect reported herein involves an enhancement of the GTPase activity of endogenous Galpha subunits, which is triggered by GPCR activation and mediated by accessory Ca(V)beta subunits. It may involve an increased association of Ca(V)beta subunits with alpha1 subunits in the plasma membrane or trafficking of channels to the plasma membrane.     

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.     

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.     

Functional proteins involved in regulation of intracellular Ca(2+) for drug development: chronic nicotine treatment upregulates L-type high voltage-gated calcium channels

Neurochemical mechanisms underlying drug dependence and withdrawal syndrome remain unclear. In this review, we discuss how chronic nicotine exposure to neurons affects expression of diazepam binding inhibitor (DBI), an endogenous anxiogenic neuropeptide supposed to be a common substance participating drug dependence, and function of L-type high voltage-gated Ca(2+) channels (HVCCs). We also discuss the functional interaction between DBI and L-type HVCCs in nicotine dependence. Both DBI levels and [(45)Ca(2+)] influx significantly increased in the brain from mice treated with nicotine for long term, which was further enhanced after abrupt cessation of nicotine and was abolished by nicotinic acetylcholine receptor (nAChR) antagonists. Similar responses of DBI expression and L-type HVCC function were observed in cerebral cortical neurons after sustained exposure to nicotine. In addition, increased DBI expression was inhibited by antagonists of nAChR and L-type HVCCs. Sustained exposure of neurons to nicotine significantly enhanced expression of alpha(1) and alpha(2)/delta(1) subunits for L-type HVCCs and caused an increase in the B(max) value of [(3)H]verapamil binding to the particulate fractions. Therefore, it is concluded that the alterations in DBI expression is mediated via increased influx of Ca(2+) through upregulated L-type HVCCs and these neurochemical changes have a close relationship with development of nicotine dependence and/or its withdrawal syndrome.