The R-Domain: Identification of an N-terminal Region of the α2δ-1 Subunit Which is Necessary and Sufficient for its Effects on Cav2.2 Calcium Currents.

Voltage-gated calcium channels (Cav) and their associated proteins are pivotal signalling complexes in excitable cell physiology. In nerves and muscle, Cav tailor calcium influx to processes including neurotransmission, muscle contraction and gene expression. Cav comprise a pore-forming α1 and modulatory ß and α2δ subunits - the latter targeted by anti-epileptic and anti-nociceptive gabapentinoid drugs. However, the mechanisms of gabapentinoid action are unclear, not least because detailed structure-function mapping of the α2δ subunit remains lacking. Using molecular biology and electrophysiological approaches we have conducted the first systematic mapping of α2δ subunit structure-function. We generated a series of cDNA constructs encoding chimera, from which successive amino acids from the rat α2δ-1 subunit were incorporated into a Type 1 reporter protein - PIN-G, to produce sequential extensions from the transmembrane (TM) region towards the N-terminus. By successive insertion of a TGA stop codon, a further series of N- to C-terminal extension constructs lacking the TM region, were also generated. Using this approach we have defined the minimal region of α2δ-1 - we term the R-domain (Rd), that appears to contain all the machinery necessary to support the electrophysiological and trafficking effects of α2δ-1 on Cav. Structural algorithms predict that Rd is conserved across all four α2δ subunits, including RNA splice variants, and irrespective of phyla and taxa. We suggest, therefore, that Rd likely constitutes the major locus for physical interaction with the α1 subunit and may provide a target for novel Cav therapeutics.

Changes in the mRNA levels of α2A and α2C adrenergic receptors in rat models of Parkinson's disease and L-DOPA-induced dyskinesia.

The changes in the mRNA levels of α(2A) and α(2C) adrenoceptors were investigated in unilateral 6-OHDA-lesioned rat model of Parkinson's disease and L: -DOPA-induced dyskinesia using in situ hybridization. In the untreated 6-OHDA-lesioned rats, α(2A) expression was elevated in the locus coeruleus (160 ± 8% and 142 ± 8% in lesioned and unlesioned sides compared to the comparable side in sham-operated rats). Following long-term (21 days, twice daily) treatment with L: -DOPA (25 mg/kg L: -DOPA methyl ester plus benserazide 6.25 mg/kg) in 6-OHDA-lesioned rats, levels of α(2A) adrenoceptor mRNA in the locus coeruleus were decreased, compared to the 6-OHDA-lesioned rats, returning to the levels of α(2A) mRNA in the sham-operated rats. α(2A) adrenoceptor expression was not changed in other brain regions in any treatment group. There was no change in α(2C) expression in the rostral or caudal striatum in which the highest density of α(2C) mRNA is present. In conclusion, the data presented in this study demonstrate an increase in α(2A) adrenoceptor mRNA in the locus coeruleus in the 6-OHDA-lesioned rat model of Parkinson's disease. In addition, the data show that repeated treatment with L: -DOPA in 6-OHDA-lesioned rats, which induces dyskinesia, restores α(2A) mRNA levels. These changes of α(2A) mRNA expression, observed in the locus coeruleus, might be of importance to basal ganglia transmission and motor function.

Targeting of voltage-gated calcium channel α2δ-1 subunit to lipid rafts is independent from a GPI-anchoring motif.

Voltage-gated calcium channels (Ca(v)) exist as heteromultimers comprising a pore-forming α(1) with accessory ß and α(2)δ subunits which modify channel trafficking and function. We previously showed that α(2)δ-1 (and likely the other mammalian α(2)δ isoforms--α(2)δ-2, 3 and 4) is required for targeting Ca(v)s to lipid rafts, although the mechanism remains unclear. Whilst originally understood to have a classical type I transmembrane (TM) topology, recent evidence suggests the α(2)δ subunit contains a glycosylphosphatidylinositol (GPI)-anchor that mediates its association with lipid rafts. To test this notion, we have used a strategy based on the expression of chimera, where the reported GPI-anchoring sequences in the gabapentinoid-sensitive α(2)δ-1 subunit have been substituted with those of a functionally inert Type I TM-spanning protein--PIN-G. Using imaging, electrophysiology and biochemistry, we find that lipid raft association of PIN-α(2)δ is unaffected by substitution of the GPI motif with the TM domain of PIN-G. Moreover, the presence of the GPI motif alone is not sufficient for raft localisation, suggesting that upstream residues are required. GPI-anchoring is susceptible to phosphatidylinositol-phospholipase C (PI-PLC) cleavage. However, whilst raft localisation of PIN-α(2)δ is disrupted by PI-PLC treatment, this is assay-dependent and non-specific effects of PI-PLC are observed on the distribution of the endogenous raft marker, caveolin, but not flotillin. Taken together, these data are most consistent with a model where α(2)δ-1 retains its type I transmembrane topology and its targeting to lipid rafts is governed by sequences upstream of the putative GPI anchor, that promote protein-protein, rather than lipid-lipid interactions.

Formation of N-type (Cav2.2) voltage-gated calcium channel membrane microdomains: Lipid raft association and clustering.

Voltage-gated calcium channels (Ca(v)s) comprise a pore-forming α1 with auxiliary α2δ and ß subunits which modulate Ca(v) function and surface expression. Ca(v)α1 and α2δ are present in signalling complexes termed lipid rafts but it is unclear whether α2δ is obligatory for targeting Ca(v)s to rafts or to what extent this influences cell surface organisation of Ca(v)s. Here, we have used imaging, biochemistry and electrophysiology to determine localisation and raft-partitioning of WT and functionally active HA-epitope tagged α2δ-1 and Ca(v)2.2 subunits expressed in COS-7 cells. We show that α2δ-1 not only partitions into lipid rafts itself but also mediates raft-partitioning of Ca(v)2.2/ß(1b) complexes. Ca(v)α2δ-1, Ca(v)2.2/ß(1b) and Ca(v)2.2/ß(1b)/α2δ-1 complexes are all organised into cell surface clusters although only in the presence of α2δ-1 do they co-localise with raft markers, caveolin and flotillin. Such clusters persist in the presence of 3-methyl-ß-cyclodextrin even though the raft markers disperse. However, clustering is profoundly sensitive to disruption of the actin-based cytoskeleton by cytochalasin-D. We conclude that α2δ-1, and likely other α2δ subunits, is necessary and sufficient for targeting Ca(v)s to lipid rafts. However, formation of clusters supporting "hotspots" of Ca(v) activity requires aggregation of macromolecular complexes containing raft components, stabilised by interactions with the cytoskeleton.

Locomotor response to L-DOPA in reserpine-treated rats following central inhibition of aromatic L-amino acid decarboxylase: further evidence for non-dopaminergic actions of L-DOPA and its metabolites.

L-DOPA is the most widely used treatment for Parkinson's disease. The anti-parkinsonian and pro-dyskinetic actions of L-DOPA are widely attributed to its conversion, by the enzyme aromatic L-amino acid decarboxylase (AADC), to dopamine. We investigated the hypothesis that exogenous L-DOPA can induce behavioural effects without being converted to dopamine in the reserpine-treated rat-model of Parkinson's disease. A parkinsonian state was induced with reserpine (3 mg/kg s.c.). Eighteen hours later, the rats were administered L-DOPA plus the peripherally acting AADC inhibitor benserazide (25 mg/kg), with or without the centrally acting AADC inhibitor NSD1015 (100 mg/kg). L-DOPA/benserazide alone reversed reserpine-induced akinesia (4158+/-1125 activity counts/6 h, cf vehicle 1327+/-227). Addition of NSD1015 elicited hyperactive behaviour that was approximately 7-fold higher than L-DOPA/benserazide (35755+/-5226, P<0.001). The hyperactivity induced by L-DOPA and NSD1015 was reduced by the alpha(2C) antagonist rauwolscine (1 mg/kg) and the 5-HT(2C) agonist MK212 (5 mg/kg), but not by the D2 dopamine receptor antagonist remoxipride (3 mg/kg) or the D1 dopamine receptor antagonist SCH23390 (1 mg/kg). These data suggest that L-DOPA, or metabolites produced via routes not involving AADC, might be responsible for the generation of at least some L-DOPA actions in reserpine-treated rats.

Binding of dopamine and 3-methoxytyramine as l-DOPA metabolites to human alpha(2)-adrenergic and dopaminergic receptors.

The ability of l-3,4-dihydroxyphenylalanine (l-DOPA), l-DOPA-methyl ester and their major metabolites, dopamine, dihydroxyphenylacetic acid (DOPAC), homovanillic (HVA), 3-O-methyldopa and 3-methoxytyramine (3-MT) to bind to alpha(2) adrenergic and D1 and D2 dopamine receptors was assessed by radioligand binding to cloned human receptors expressed in cell lines. As anticipated, dopamine bound with high affinity to D1 (IC(50) 1.1 + or - 0.16 microM) and D2 (IC(50) 0.7 + or - 0.3 microM) dopamine receptors. However, dopamine also bound with high affinity to alpha(2A) (IC(50) was 2.6 + or - 0.5 microM), alpha(2C) (IC(50) 3.2 + or - 0.7 microM). 3-MT bound to alpha(2A) with high affinity (IC(50), 3.6 + or - 0.2 microM) though moderate affinity to alpha(2)c, D1 and D2 receptors (values of IC(50) were 55 + or - 14, 121 + or - 43, 36 + or - 14 microM, respectively). l-DOPA-methyl ester bound with high affinity to alpha(2) (IC(50) 17-36 microM) but not dopamine receptors (IC(50) 0.9-2.5 mM). l-DOPA, 3-O-methyldopa and DOPAC had no observable effect on binding to any of the receptors tested. These data suggest that the effects of l-DOPA in Parkinson's disease may result from actions of its metabolites dopamine and 3-MT on both dopaminergic and non-dopaminergic receptors. These findings may provide explanations for the differences between l-DOPA and dopamine receptor agonists in mediating anti-parkinsonian effects and propensity to be associated with dyskinesia and motor complications such as wearing-off and on-off.

PIN-G reporter for imaging and defining trafficking signals in membrane proteins.

The identification of motifs that control the intracellular trafficking of proteins is a fundamental objective of cell biology. Once identified, such regions should, in principle, be both necessary and sufficient to direct any randomly distributed protein, acting as a reporter, to the subcellular compartment in question. However, most reporter proteins have limited versatility owing to their endogenous expression and limited modes of detection--especially in live cells. To surmount such limitations, we engineered a plasmid--pIN-G--encoding an entirely artificial, type I transmembrane reporter protein (PIN-G), containing HA, cMyc and GFP epitope, and fluorescence tags. Although originally designed for trafficking studies, pIN technology is a powerful tool applicable to almost every area of biology. Here we describe the methodologies used routinely in analyzing pIN constructs and some of their derivatives.

A lentivirally delivered photoactivatable GFP to assess continuity in the endoplasmic reticulum of neurones and glia.

The endoplasmic reticulum (ER) is the largest intracellular membranous organelle. Functions of the ER are many and diverse, which include various biosynthetic, transport and signalling roles, central to cellular physiology, such as the biosynthesis of membrane and secretory proteins and the regulation of intracellular calcium. Its continuous lumen also serves as a highway for the distribution of proteins and ions to different regions of the cell, independent of the cytosol. The ER is an excitable organelle, capable of generating a regenerative wave of calcium release, which can propagate along the endomembrane throughout the entire cell, serving as a system of intracelluar communication in polarised cells. Nowhere is this feature of ER function more crucial than in neurones. The extremely polarised nature of nerve cells presents a unique challenge for the global co-ordination of localised physiological events such as growth cone guidance and synaptic plasticity. Clearly, the physical continuity of the neuronal ER lumen is central to its functionality as a conduit for communication. To further probe the continuity of ER in neurones and glia, we developed LV-PA-pIN-KDEL, a photoactivatable analogue of our recently described vector LV-pIN-KDEL, a lentivirally delivered ER-targeting soluble GFP. We demonstrate the ability of this vector to transduce astrocytes and neurones in culture and in cortical explants. Furthermore, we exploit the photoactivatable attributes of the vector together with a focal laser photoactivation protocol to reveal the continuous nature of the ER lumen in these cell types, presenting the first direct evidence of an astrocytic ER luminal continuum and providing more data to support the existence of a single ER lumen in neurones.

Identification of an evolutionarily conserved extracellular threonine residue critical for surface expression and its potential coupling of adjacent voltage-sensing and gating domains in voltage-gated potassium channels.

The dynamic expression of voltage-gated potassium channels (Kvs) at the cell surface is a fundamental factor controlling membrane excitability. In exploring possible mechanisms controlling Kv surface expression, we identified a region in the extracellular linker between the first and second of the six (S1-S6) transmembrane-spanning domains of the Kv1.4 channel, which we hypothesized to be critical for its biogenesis. Using immunofluorescence microscopy, flow cytometry, patch clamp electrophysiology, and mutagenesis, we identified a single threonine residue at position 330 within the Kv1.4 S1-S2 linker that is absolutely required for cell surface expression. Mutation of Thr-330 to an alanine, aspartate, or lysine prevented surface expression. However, surface expression occurred upon co-expression of mutant and wild type Kv1.4 subunits or mutation of Thr-330 to a serine. Mutation of the corresponding residue (Thr-211) in Kv3.1 to alanine also caused intracellular retention, suggesting that the conserved threonine plays a generalized role in surface expression. In support of this idea, sequence comparisons showed conservation of the critical threonine in all Kv families and in organisms across the evolutionary spectrum. Based upon the Kv1.2 crystal structure, further mutagenesis, and the partial restoration of surface expression in an electrostatic T330K bridging mutant, we suggest that Thr-330 hydrogen bonds to equally conserved outer pore residues, which may include a glutamate at position 502 that is also critical for surface expression. We propose that Thr-330 serves to interlock the voltage-sensing and gating domains of adjacent monomers, thereby yielding a structure competent for the surface expression of functional tetramers.

Surface expression and distribution of voltage-gated potassium channels in neurons (Review).

The last decade has witnessed an exponential increase in interest in one of the great mysteries of nerve cell biology: Specifically, how do neurons know where to place the ion channels that control their excitability? Many of the most important insights have been gleaned from studies on the voltage-gated potassium channels (Kvs) which underlie the shape, duration and frequency of action potentials. In this review, we gather recent evidence on the expression, trafficking and maintenance mechanisms which control the surface density of Kvs in different subcellular compartments of neurons and how these may be regulated to control cell excitability.

LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology, dynamics and continuity of the endoplasmic reticulum in live neurones.

BACKGROUND: The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca2+ pumps, and Ca2+ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones. RESULTS: Following its expression in HEK293 (or COS-7) cells, LV-pIN-KDEL yielded a pattern of fluorescence that co-localised exclusively with the ER marker sec61beta but with no other major organelle. We found no evidence for cytotoxicity and only rarely inclusion body formation. To explore the utility of the probe in resolving the ER in live cells, HEK293 or COS-7 cells were transduced with LV-pIN-KDEL and, after 48 h, imaged directly at intervals from 1 min to several hours. LV-pIN-KDEL fluorescence revealed the endoplasmic reticulum as a tubular lattice structure whose morphology can change markedly within seconds. Although GFP can be phototoxic, the integrity of the cells and ER was retained for several weeks and even after light exposure for periods up to 24 h. Using LV-pIN-KDEL we have imaged the ER in diverse fixed neuronal cultures and, using real-time imaging, found evidence for extensive, dynamic remodelling of the neuronal ER in live hippocampal cultures, brain slices, explants and glia. Finally, through a Fluorescence Loss in Photobleaching (FLIP) approach, continuous irradiation at a single region of interest removed all the fluorescence of LV-pIN-KDEL-transduced nerve cells in explant cultures, thus, providing compelling evidence that in neurons the endoplasmic reticulum is not only dynamic but also continuous. CONCLUSION: The lentiviral-based ER-targeted reporter, LV-pIN-KDEL, offers considerable advantages over present systems for defining the architecture of the ER, especially in primary cells such as neurones that are notoriously difficult to transfect. Images and continuous photobleaching experiments of LV-pIN-KDEL-transduced neurones demonstrate that the endoplasmic reticulum is a dynamic structure with a single continuous lumen. The introduction of LV-pIN-KDEL is anticipated to greatly facilitate a real-time visualisation of the structural plasticity and continuous nature of the neuronal ER in healthy and diseased brain tissue.

Kinetics of internalization and degradation of N-type voltage-gated calcium channels: role of the alpha2/delta subunit.

The contribution of voltage-gated calcium channels to excitable cell function depends, critically, upon the mechanisms that control their expression at the cell surface. While co-assembly of the pore forming alpha(1) and auxiliary beta subunits enhances channel surface expression, the levels are still only 30-40% of those seen with the core alpha(1B)/beta(1b)/alpha(2)delta calcium channel complex. To rationalize this observation, it has been suggested that the alpha(2)/delta subunit might stabilize calcium channel expression at the cell surface. To test this notion, we have resolved the effect of the alpha(2)/delta subunit on the rates of binding, internalization and degradation of defined N-type calcium channel surface complexes expressed in HEK293 cells, through pulse-labeling with the selective, cell impermeable, radioligand [(125)I]-omega-CgTx. Through detailed kinetic and sensitivity analysis we show that alpha(1B)/beta(1b)/alpha(2)delta complexes are internalized slowly (k(int) 0.4/h), whereupon, most become degraded (k(deg) 0.02/h). In contrast, alpha(1B)/beta(1b) complexes are internalized more rapidly (k(int) 0.8/h), following which they are either quickly degraded (k(deg) 0.1/h) or are sequestered slowly (k(tra) 0.1/h) to a pool that is metabolically stable within the time-frame of our experiments (24h). In neither case did we find evidence for recycling via the cell surface. Thus, our data argue for a novel mechanism where complexes lacking an alpha(2)/delta subunit are cleared from the cell surface and are rapidly degraded or stored, possibly for further attempts at complexation as new alpha(2)/delta subunits become available. The slower rate of internalization of complexes containing the alpha(2)/delta subunit rationalizes the stabilizing effect this subunit has upon calcium channel surface expression and suggests a mechanism by which alpha(2)delta mutations may cause severe neurological deficits.

Biosynthesis of the dystonia-associated AAA+ ATPase torsinA at the endoplasmic reticulum.

TorsinA is a widely expressed AAA(+) (ATPases associated with various cellular activities) ATPase of unknown function. Previous studies have described torsinA as a type II protein with a cleavable signal sequence, a single membrane spanning domain, and its C-terminus located in the ER (endoplasmic reticulum) lumen. However, in the present study we show that torsinA is not in fact an integral membrane protein. Instead we find that the mature protein associates peripherally with the ER membrane, most likely through an interaction with an integral membrane protein. Consistent with this model, we provide evidence that the signal peptidase complex cleaves the signal sequence of torsinA, and we show that the region previously suggested to form a transmembrane domain is translocated into the lumen of the ER. The finding that torsinA is a peripheral, and not an integral membrane protein as previously thought, has important implications for understanding the function of this novel ATPase.

Development of a pheromone trap monitoring system for orange wheat blossom midge, Sitodiplosis mosellana, in the UK.

Field-trapping experiments with synthetic 2,7-nonadiyl dibutyrate, the female-produced sex pheromone of the orange wheat blossom midge, Sitodiplosis mosellana (Géhin), demonstrated that pheromone traps were highly attractive to males and caught very few non-target organisms. Different formulations of pheromone were tested to identify the optimum release rate and dispenser type for use in pheromone traps in the UK. Key findings were that racemic pheromone was as effective as enantiomerically pure (2S,7R)-2,7-nonadiyl dibutyrate, that release rates higher than 0.5 microg day(-1) were not necessary and that the optimal formulation was a 1 mg pheromone loading in a rubber septum. Pheromone traps gave a reliable indication of peak midge emergence, onset of flight and abundance of midges throughout the season. A strong correlation between maximum trap catch and crop infestation levels was obtained.

Molecular basis of inherited calcium channelopathies: role of mutations in pore-forming subunits.

The pore-forming alpha subunits of voltage-gated calcium channels contain the essential biophysical machinery that underlies calcium influx in response to cell depolarization. In combination with requisite auxiliary subunits, these pore subunits form calcium channel complexes that are pivotal to the physiology and pharmacology of diverse cells ranging from sperm to neurons. Not surprisingly, mutations in the pore subunits generate diverse pathologies, termed channelopathies, that range from failures in excitation-contraction coupling to night blindness. Over the last decade, major insights into the mechanisms of pathogenesis have been derived from animals showing spontaneous or induced mutations. In parallel, there has been considerable growth in our understanding of the workings of voltage-gated ion channels from a structure-function, regulation and cell biology perspective. Here we document our current understanding of the mutations underlying channelopathies involving the voltage-gated calcium channel alpha subunits in humans and other species.

PIN-G--a novel reporter for imaging and defining the effects of trafficking signals in membrane proteins.

BACKGROUND: The identification of protein trafficking signals, and their interacting mechanisms, is a fundamental objective of modern biology. Unfortunately, the analysis of trafficking signals is complicated by their topography, hierarchical nature and regulation. Powerful strategies to test candidate motifs include their ability to direct simpler reporter proteins, to which they are fused, to the appropriate cellular compartment. However, present reporters are limited by their endogenous expression, paucity of cloning sites, and difficult detection in live cells. RESULTS: Consequently, we have engineered a mammalian expression vector encoding a novel trafficking reporter--pIN-G--consisting of a simple, type I integral protein bearing permissive intra/extracellular cloning sites, green fluorescent protein (GFP), cMyc and HA epitope tags. Fluorescence imaging, flow cytometry and biochemical assays of transfected HEK293 cells, confirm the size, topology and surface expression of PIN-G. Moreover, a pIN-G fusion construct, containing a Trans-Golgi Network (TGN) targeting determinant, internalises rapidly from the cell surface and localises to the TGN. Additionally, another PIN-G fusion protein and its mutants reveal trafficking determinants in the cytoplasmic carboxy terminus of Kv1.4 voltage-gated potassium channels. CONCLUSION: Together, these data indicate that pIN-G is a versatile, powerful, new reporter for analysing signals controlling membrane protein trafficking, surface expression and dynamics.

alpha2-Adrenoceptor-mediated modulation of the release of GABA and noradrenaline in the rat substantia nigra pars reticulata.

The control of movement by the basal ganglia is influenced by inputs from diverse brain structures. Unfortunately, the mechanisms of modulation are poorly defined. Based on neuroanatomical evidence for alpha2A and alpha2C subtypes of alpha2 adrenergic receptors within this region, we hypothesize that noradrenergic alpha2-receptors can influence transmitter release in the SNr. To test this hypothesis we examined the effect of the alpha 2 adrenergic agonist, clonidine, and antagonist, rauwolscine, on the efflux of [3H]-GABA and [3H]-noradrenaline from brain slices of the rat substantia nigra pars reticulata. At low concentrations (10 nM), rauwolscine caused an 84.2 +/- 18.51% (p < 0.01) increase in KCl-evoked GABA release. At higher concentrations, rauwolscine caused a dose-dependent return to basal levels. Rauwolscine also enhanced basal GABA efflux after KCl washout with a similar biphasic concentration-dependence. Surprisingly, clonidine also enhanced [3H]-GABA release but had no effect on KCl-evoked [3H]-GABA release at concentrations which inhibited [3H]-NA efflux. These effects were potentiated by the GABA re-uptake inhibitor nipecotic acid. Together, our data indicate an important role for noradrenergic modulation in the SNr. The enhancing effect of both the alpha2 adrenoceptor agonist and antagonist on GABA release, while appearing paradoxical, can be rationalised by actions at distinct subsets of alpha2 adrenoceptors, using a simple model where alpha2A adrenoceptors are localized on the terminals of noradrenergic afferents impinging upon alpha2C adrenoceptor-containing GABAergic striato-nigral neurones.

Ca2+ channels and epilepsy.

The epilepsies encompass diverse seizure disorders afflicting as many as 50 million people worldwide. Many forms of epilepsy are intractable to current therapies and there is a pressing need to develop agents and strategies to not only suppress seizures, but also cure epilepsy. Recent insights from molecular genetics and pharmacology now point to an important role for voltage-dependent calcium channels in epilepsy. In this article, I first provide an introduction to the classification of the epilepsies and an overview of neuronal Ca(2+) channels. Next, I attempt to review the evidence for a role of Ca(2+) channels in epilepsy and the insights gained from genetics and pharmacology. Lastly, I describe new avenues for how such information might be exploited in the development of therapeutic reagents.

Cell surface targeting and clustering interactions between heterologously expressed PSD-95 and the Shal voltage-gated potassium channel, Kv4.2.

Kv4.2 is a voltage-gated potassium channel that is critical in controlling the excitability of myocytes and neurons. Processes that influence trafficking and surface distribution patterns of Kv4.2 will affect its ability to contribute to cellular functions. The scaffolding/clustering protein PSD-95 regulates trafficking and distribution of several receptors and Shaker family Kv channels. We therefore investigated whether the C-terminal valine-serine-alanine-leucine (VSAL) of Kv4.2 is a novel binding motif for PSD-95. By using co-immunoprecipitation assays, we determined that full-length Kv4.2 and PSD-95 interact when co-expressed in mammalian cell lines. Mutation analysis in this heterologous expression system showed that the VSAL motif of Kv4.2 is necessary for PSD-95 binding. PSD-95 increased the surface expression of Kv4.2 protein and caused it to cluster, as shown by deconvolution microscopy and biotinylation assays. Deleting the C-terminal VSAL motif of Kv4.2 eliminated these effects, as did substituting a palmitoylation-deficient PSD-95 mutant. In addition to these effects of PSD-95 on Kv4.2 distribution, the channel itself promoted redistribution of PSD-95 to the cell surface in the heterologous expression system. This work represents the first evidence that a member of the Shal subfamily of Kv channels can bind to PSD-95, with functional consequences.