Timothy Angelotti

Pharmacology and mechanism of action of pregabalin: The calcium channel α2–δ (alpha2–delta) subunit as a target for antiepileptic drug discovery

Summary Pregabalin (Lyrica™) is a new antiepileptic drug that is active in animal seizure models. Pregabalin is approved in US and Europe for adjunctive therapy of partial seizures in adults, and also has been approved for the treatment of pain from diabetic neuropathy or post-herpetic neuralgia in adults. Recently, it has been approved for treatment of anxiety disorders in Europe. Pregabalin is structurally related to the antiepileptic drug gabapentin and the site of action of both drugs is similar, the alpha 2 –delta (α 2 –δ) protein, an auxiliary subunit of voltage-gated calcium channels. Pregabalin subtly reduces the synaptic release of several neurotransmitters, apparently by binding to α 2 –δ subunits, and possibly accounting for its actions in vivo to reduce neuronal excitability and seizures. Several studies indicate that the pharmacology of pregabalin requires binding to α 2 –δ subunits, including structure-activity analyses of compounds binding to α 2 –δ subunits and pharmacology in mice deficient in binding at the α 2 –δ Type 1 protein. The preclinical findings to date are consistent with a mechanism that may entail reduction of abnormal neuronal excitability through reduced neurotransmitter release. This review addresses the preclinical pharmacology of pregabalin, and also the biology of the high affinity binding site, and presumed site of action.

The Type II Isoform of cGMP-dependent Protein Kinase Is Dimeric and Possesses Regulatory and Catalytic Properties Distinct from the Type I Isoforms

The type I cGMP-dependent protein kinases (cGK Iα and Iβ) form homodimers (subunit Mr ∼ 76,000), presumably through conserved, amino-terminal leucine zipper motifs. Type II cGMP-dependent protein kinase (cGK II) has been reported to be monomeric (Mr ∼ 86,000), but recent cloning and sequencing of mouse brain cGK II cDNA revealed a leucine zipper motif near its amino terminus. In the present study, recombinant mouse brain cGK II was expressed, purified, and characterized. Sucrose gradient centrifugation and gel filtration chromatography were used to determine Mr values for holoenzymes of cGK Iα (168,000) and cGK II (152,500), which suggest that both are dimers. Native cGK Iα possessed significantly lower Ka values for cGMP (8-fold) and β-phenyl-1,N2-etheno-cGMP (300-fold) than did recombinant cGK II. Conversely, the Sp- and Rp-isomers of 8-(4-chloro-phenylthio)-guanosine-3′,5′-cyclic monophosphorothioate demonstrated selectivity toward cGK II in assays of kinase activation or inhibition, respectively. A peptide substrate derived from histone f2B had a 20-fold greater Vmax/Km ratio for cGK Iα than for cGK II, whereas a peptide based upon a cAMP response element binding protein phosphorylation site exhibited a greater Vmax/Km ratio for cGK II. Finally, gel filtration of extracts of mouse intestine partially resolved two cGK activities, one of which had properties similar to those demonstrated by recombinant cGK II. The combined results show that both cGK I and cGK II form homodimers but possess distinct cyclic nucleotide and substrate specificities.

Native and Recombinant GABAA Receptor Channels

Biochemical, pharmacological and electrophysiological techniques have been used to characterize GABAA receptor (GABAR) channels in mammalian brain and have demonstrated the existence of a p

Regulation of G-Protein Coupled Receptor Traffic by an Evolutionary Conserved Hydrophobic Signal

Plasma membrane (PM) expression of G‐protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c‐adrenergic receptors (α2c‐ARs), heterologous expression in non‐native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric α2a/α2c‐AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for α2c‐AR subtype‐specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into α2a‐ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within α2c‐ARs serves as a regulator of GPCR trafficking.

Differential targeting and function of α2A and α2C adrenergic receptor subtypes in cultured sympathetic neurons

Previous research suggested that alpha2A and alpha2C adrenergic receptor (AR) subtypes have overlapping but unique physiological roles in neuronal signaling; however, the basis for these dissimilarities is not completely known. To better understand the observed functional differences between these autoreceptors, we investigated targeting and signaling of endogenously expressed alpha2A and alpha2CARs in cultured sympathetic ganglion neurons (SGN). At Days 1 and 4, alpha2A and alpha2CARs could be readily detected in SGN from wild-type mice. By Day 8, alpha2A ARs were targeted to cell body, as well as axonal and dendritic sites, whereas alpha2C ARs were primarily localized to an intracellular vesicular pool within the cell body and proximal dendritic projections. Expression of synaptic vesicle marker protein SV2 did not differ at Day 8 nor co-localize with either subtype. By Day 16, however, alpha2C ARs had relocated to somatodendritic and axonal sites and, unlike alpha2A ARs, co-localized with SV2 at synaptic contact sites. Consistent with a functional role for alpha2 ARs, we also observed that dexmedetomidine stimulation of cultured SGN more efficiently inhibited depolarization-induced calcium entry into older, compared to younger, cultures. These results provide direct evidence of distinct developmental patterns of endogenous alpha2A and alpha2C AR targeting and function in a native cell system and that maturation of SGN in culture leads to alterations in neuronal properties required for proper targeting. More importantly, the co-localization at Day 16 of alpha2C ARs at sites of synaptic contact may partially explain the differential modulation of neurotransmitter release and responsiveness to action potential frequency observed between alpha2A and alpha2C ARs in SGN.

REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity

Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins. Therefore, some REEPs can be further described as ER membrane shaping adapter proteins.

Sex-specific modulation of spinal nociception by α2-adrenoceptors: Differential regulation by estrogen and testosterone

Sex-related differences in antinociception produced by the activation of alpha(2)-adrenoceptors (alpha(2)-ARs) have been reported, however, the precise role of gonadal steroids is still unknown. Hence, we hypothesized that estrogen and testosterone modulate antinociceptive effects of clonidine (an alpha(2)-AR agonist) on N-methyl-D-aspartate- (NMDA) and heat-induced spinal nociception. We also investigated whether estrogen or testosterone alters the expression of alpha(2A)-adrenoceptors in the spinal cord. Sprague-Dawley (SD) rats were implanted with PE10 cannulae in the intrathecal space of the lumbosacral spinal cord and divided into male, proestrous and diestrous female, ovariectomized (OVX), estradiol-treated OVX (OVX+E), castrated male (GDX), testosterone (GDX+T) and estradiol-treated castrated male (GDX+E) groups. Clonidine dose-dependently inhibited NMDA-induced scratching behavior in the male and OVX groups but to a significantly lesser extent in the OVX+E group. It also increased the tail withdrawal latency in the male, OVX, diestrous and GDX+T groups but not in the OVX+E, proestrous, GDX and GDX+E groups. Levels of alpha(2A)-AR mRNA were significantly higher in the OVX, estradiol-treated OVX, GDX and GDX+E animals. In contrast, alpha(2A)-AR protein levels were higher in estradiol-treated OVX, GDX, GDX+T and GDX+E animals as compared with the male. Indeed, no correlations were observed between changes in the mRNA or protein levels of alpha(2A)-AR and behavioral observations. These results support our hypothesis that sex-related differences in alpha(2)-AR-mediated modulation of spinal nociception are gonadal hormone-dependent: estrogen attenuates antinociceptive effects in females whereas testosterone is required for the expression of antinociception in males. In addition, results also revealed that the mechanism of action of gonadal hormones may not involve a global alternation in expression of alpha(2A)-AR in the spinal cord. Estrogen-induced attenuation of alpha(2)-AR-mediated inhibition of nociception could contribute to the higher prevalence of pain syndromes in women.

Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of α2-Adrenergic Responses Is because of Ligand-selective Internalization

Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged α2A-adrenergic receptor (AR) expression was driven by the endogenous mouse α2AAR gene locus. Exploiting this mouse line, we evaluated α2AAR trafficking and α2AAR-mediated inhibition of Ca2+ currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the α2AAR subtype. We discovered a more rapid desensitization of Ca2+ current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of α2AAR evoked by clonidine than by guanfacine. Clonidine-induced α2AAR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of α2AAR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-α2AAR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.

Nodular invasive tracheobronchitis due to Aspergillus in a patient with systemic lupus erythematosus

Nodular or pseudomembranous tracheobronchitis due to infection by Aspergillus species is an uncommon presentation of invasive aspergillosis. Most cases have been described in severely immunocompromised hosts. We describe the case of a 23-year-old woman, with recently diagnosed systemic lupus erythematosus, who developed worsening respiratory function. Bronchoscopy revealed rapid developmentand progression of multiple nodular plaques in her trachea and bronchi. Endobronchial biopsy demonstrated invasive fungal infection with tissue necrosis and the presence of hyphal elements consistent with aspergillosis. To the best of our knowledge, this is only the second report of fulminant invasive tracheobronchitis due to Aspergillus in a patient with an autoimmune disease.

Molecular and electrophysiological characterization of a allelic variant of the rat α6 GABAA receptor subunit

A 1.45 kb DNA sequence encoding the rat alpha 6 GABAA receptor subunit (nucleotides 33-1483) was cloned from a Sprague-Dawley rat brain cDNA library by PCR amplification. Dideoxy sequencing of two individual clones revealed that the nucleotide sequence differed at only one basepair (T480-->G) from that published previously. This difference altered the deduced amino acid sequence, producing a conservative amino acid substitution (His121-->Gln). A Gln residue is present at the same location in the bovine alpha 6 subunit. Restriction endonuclease analysis of the total PCR product demonstrated that this variant of the rat alpha 6 subunit was the only allele found in this particular rat brain library, the original allele was not present. These results were further verified by RNAse protection assays performed with RNA isolated from individual rat cerebella. alpha 6, beta 1, and gamma 2S subunits were transiently expressed in L929 cells for electrophysiological analysis. Whole-cell recordings obtained from the cells demonstrated that GABAA receptor channels with the expected GABA and benzodiazepine pharmacology were produced. Excised outside out single channel recordings from the same cells revealed that GABA elicited brief duration openings to a 33 pS main conductance level and to at least one smaller (approximately 21 pS) subconductance level. Thus this allelic variant of rat alpha 6 subunit could assemble with other subunits to form a functional GABAA receptor channel with similar properties to the original allelic form.