A Targeted Bioinformatics Assessment of Adrenocortical Carcinoma Reveals Prognostic Implications of GABA System Gene Expression.

Adrenocortical carcinoma (ACC) is a rare but deadly cancer for which few treatments exist. Here, we have undertaken a targeted bioinformatics study of The Cancer Genome Atlas (TCGA) ACC dataset focusing on the 30 genes encoding the γ-aminobutyric acid (GABA) system-an under-studied, evolutionarily-conserved system that is an emerging potential player in cancer progression. Our analysis identified a subset of ACC patients whose tumors expressed a distinct GABA system transcriptome. Transcript levels of ABAT (encoding a key GABA shunt enzyme), were upregulated in over 40% of tumors, and this correlated with several favorable clinical outcomes including patient survival; while enrichment and ontology analysis implicated two cancer-related biological pathways involved in metastasis and immune response. The phenotype associated with ABAT upregulation revealed a potential metabolic heterogeneity among ACC tumors associated with enhanced mitochondrial metabolism. Furthermore, many GABAA receptor subunit-encoding transcripts were expressed, including two (GABRB2 and GABRD) prognostic for patient survival. Transcripts encoding GABAB receptor subunits and GABA transporters were also ubiquitously expressed. The GABA system transcriptome of ACC tumors is largely mirrored in the ACC NCI-H295R cell line, suggesting that this cell line may be appropriate for future functional studies investigating the role of the GABA system in ACC cell growth phenotypes and metabolism.

A genetically encoded fluorescent sensor for in vivo imaging of GABA.

Current techniques for monitoring GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in vertebrates, cannot follow transients in intact neural circuits. To develop a GABA sensor, we applied the design principles used to create the fluorescent glutamate receptor iGluSnFR. We used a protein derived from a previously unsequenced Pseudomonas fluorescens strain and performed structure-guided mutagenesis and library screening to obtain intensity-based GABA sensing fluorescence reporter (iGABASnFR) variants. iGABASnFR is genetically encoded, detects GABA release evoked by electric stimulation of afferent fibers in acute brain slices and produces readily detectable fluorescence increases in vivo in mice and zebrafish. We applied iGABASnFR to track mitochondrial GABA content and its modulation by an anticonvulsant, swimming-evoked, GABA-mediated transmission in zebrafish cerebellum, GABA release events during interictal spikes and seizures in awake mice, and found that GABA-mediated tone decreases during isoflurane anesthesia.

Pharmacological Chaperones: Beyond Conformational Disorders.

Pharmacological chaperones (PCs) are small molecules that bind to nascent protein targets to facilitate their biogenesis. The ability of PCs to assist in the folding and subsequent forward trafficking of disease-causative protein misfolding mutants has opened new avenues for the treatment of conformational diseases such as cystic fibrosis and lysosomal storage disorders. In this chapter, an overview of the use of PCs for the treatment of conformational disorders is provided. Beyond the therapeutic application of PCs for the treatment of these disorders, pharmacological chaperoning of wild-type integral membrane proteins is discussed. Central to this discussion is the notion that the endoplasmic reticulum is a reservoir of viable but inefficiently processed wild-type protein folding intermediates whose biogenesis can be facilitated by PCs to increase functional pools. To date, the potential therapeutic use of PCs to enhance the biogenesis of wild-type proteins has received little attention. Here the rationale for the development of PCs that target WT proteins is discussed. Also considered is the likelihood that some commonly used therapeutic agents may exert unrecognized pharmacological chaperoning activity on wild-type targets in patient populations.

Cognate Ligand Chaperoning: a Novel Mechanism for the Post-translational Regulation of Neurotransmitter Receptor Biogenesis.

The functional unit for inter-neuronal communication in the central nervous system is the neuronal synapse. The number of postsynaptic neurotransmitter receptors at the cell surface is an important determinant of synaptic efficacy and plasticity. A diverse array of post-translational processes regulate postsynaptic receptor number, including receptor exocytosis, lateral diffusion, surface stabilization, endocytosis, and recycling, thus highlighting the importance of mechanisms that control postsynaptic receptor levels. Another putative post-translational mechanism for regulating receptor surface expression is cognate ligand chaperoning. It has been proposed that neurotransmitters function as cognate ligand chaperones by binding, within the endoplasmic reticulum (ER) lumen, to their nascent neurotransmitter receptors and facilitating receptor biogenesis. Here we discuss proof-of-concept evidence that small molecules can selectively facilitate the biogenesis of their targets and examine the specific evidence in support of cognate ligand chaperoning of neurotransmitter receptor biogenesis.

Neuronal gamma-aminobutyric acid (GABA) type A receptors undergo cognate ligand chaperoning in the endoplasmic reticulum by endogenous GABA.

GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endoplasmic reticulum (ER). We have previously shown that exogenous GABA can act as a ligand chaperone of recombinant GABAA receptors in the early secretory pathway leading us to now investigate whether endogenous GABA facilitates the biogenesis of GABAA receptors in primary cerebral cortical cultures. In immunofluorescence labeling experiments, we have determined that neurons expressing surface GABAA receptors contain both GABA and its degradative enzyme GABA transaminase (GABA-T). Treatment of neurons with GABA-T inhibitors, a treatment known to increase intracellular GABA levels, decreases the interaction of the receptor with the ER quality control protein calnexin, concomittantly increasing receptor forward-trafficking and plasma membrane insertion. The effect of GABA-T inhibition on the receptor/calnexin interaction is not due to the activation of surface GABAA or GABAB receptors. Consistent with our hypothesis that GABA acts as a cognate ligand chaperone in the ER, immunogold-labeling of rodent brain slices reveals the presence of GABA within the rough ER. The density of this labeling is similar to that present in mitochondria, the organelle in which GABA is degraded. Lastly, the effect of GABA-T inhibition on the receptor/calnexin interaction was prevented by pretreatment with a GABA transporter inhibitor. Together, these data indicate that endogenous GABA acts in the rough ER as a cognate ligand chaperone to facilitate the biogenesis of neuronal GABAA receptors.

Pharmacological chaperoning: a primer on mechanism and pharmacology.

Approximately forty percent of diseases are attributable to protein misfolding, including those for which genetic mutation produces misfolding mutants. Intriguingly, many of these mutants are not terminally misfolded since native-like folding, and subsequent trafficking to functional locations, can be induced by target-specific, small molecules variably termed pharmacological chaperones, pharmacoperones, or pharmacochaperones (PCs). PC targets include enzymes, receptors, transporters, and ion channels, revealing the breadth of proteins that can be engaged by ligand-assisted folding. The purpose of this review is to provide an integrated primer of the diverse mechanisms and pharmacology of PCs. In this regard, we examine the structural mechanisms that underlie PC rescue of misfolding mutants, including the ability of PCs to act as surrogates for defective intramolecular interactions and, at the intermolecular level, overcome oligomerization deficiencies and dominant negative effects, as well as influence the subunit stoichiometry of heteropentameric receptors. Not surprisingly, PC-mediated structural correction of misfolding mutants normalizes interactions with molecular chaperones that participate in protein quality control and forward-trafficking. A variety of small molecules have proven to be efficacious PCs and the advantages and disadvantages of employing orthostatic antagonists, active-site inhibitors, orthostatic agonists, and allosteric modulator PCs are considered. Also examined is the possibility that several therapeutic agents may have unrecognized activity as PCs, and this chaperoning activity may mediate/contribute to therapeutic action and/or account for adverse effects. Lastly, we explore evidence that pharmacological chaperoning exploits intrinsic ligand-assisted folding mechanisms. Given the widespread applicability of PC rescue of mutants associated with protein folding disorders, both in vitro and in vivo, the therapeutic potential of PCs is vast. This is most evident in the treatment of lysosomal storage disorders, cystic fibrosis, and nephrogenic diabetes insipidus, for which proof of principle in humans has been demonstrated.

GABA acts as a ligand chaperone in the early secretory pathway to promote cell surface expression of GABAA receptors.

GABA (gamma-aminobutyric acid) is the primary inhibitory neurotransmitter in brain. The fast inhibitory effect of GABA is mediated through the GABA(A) receptor, a postsynaptic ligand-gated chloride channel. We propose that GABA can act as a ligand chaperone in the early secretory pathway to facilitate GABA(A) receptor cell surface expression. Forty-two hours of GABA treatment increased the surface expression of recombinant receptors expressed in HEK 293 cells, an effect accompanied by an increase in GABA-gated chloride currents. In time-course experiments, a 1h GABA exposure, followed by a 5h incubation in GABA-free medium, was sufficient to increase receptor surface expression. A shorter GABA exposure could be used in HEK 293 cells stably transfected with the GABA transporter GAT-1. In rGAT-1HEK 293 cells, the GABA effect was blocked by the GAT-1 inhibitor NO-711, indicating that GABA was acting intracellularly. The effect of GABA was prevented by brefeldin A (BFA), an inhibitor of early secretory pathway trafficking. Coexpression of GABA(A) receptors with the GABA synthetic enzyme glutamic acid decarboxylase 67 (GAD67) also resulted in an increase in receptor surface levels. GABA treatment failed to promote the surface expression of GABA binding site mutant receptors, which themselves were poorly expressed at the surface. Consistent with an intracellular action of GABA, we show that GABA does not act by stabilizing surface receptors. Furthermore, GABA treatment rescued the surface expression of a receptor construct that was retained within the secretory pathway. Lastly, the lipophilic competitive antagonist (+)bicuculline promoted receptor surface expression, including the rescue of a secretory pathway-retained receptor. Our results indicate that a neurotransmitter can act as a ligand chaperone in the early secretory pathway to regulate the surface expression of its receptor. This effect appears to rely on binding site occupancy, rather than agonist-induced structural changes, since chaperoning is observed with both an agonist and a competitive antagonist.

Regulation of excitation by GABA(A) receptor internalization.

Neuronal inhibition is of paramount importance in maintaining the delicate and dynamic balance between excitatory and inhibitory influences in the central nervous system. GABA (gamma-aminobutyric acid), the primary inhibitory neurotransmitter in brain, exerts its fast inhibitory effects through ubiquitously expressed GABA(A) receptors. Activation of these heteropentameric receptors by GABA results in the gating of an integral chloride channel leading to membrane hyperpolarization and neuronal inhibition. To participate in neurotransmission, the receptor must reside on the cell surface. The trafficking of nascent receptors to the cell surface involves posttranslational modification and the interaction of the receptor with proteins that reside within the secretory pathway. The subsequent insertion of the receptor into specialized regions of the plasma membrane is dictated by receptor composition and other factors that guide insertion at synaptic or perisynaptic/extrasynaptic sites, where phasic and tonic inhibition are mediated, respectively. Once at the cell surface, the receptor is laterally mobile and subject to both constitutive and regulated endocytosis. Following endocytosis the receptor undergoes either recycling to the plasma membrane or degradation. These dynamic processes profoundly affect the strength of GABAergic signaling, neuronal inhibition, and presumably synaptic plasticity. Heritable channelopathies that affect receptor trafficking have been recently recognized and compelling evidence exists that mechanisms underlying acquired epilepsy involve GABA(A) receptor internalization. Additionally, GABA(A) receptor endocytosis has been identified as an early event in the ischemic response that leads to excitotoxicity and cell death. This chapter summarizes what is known regarding the regulation of receptor trafficking and cell surface expression and its impact on nervous system function from both cell biology and disease perspectives.

Mechanisms of homomeric alpha1 glycine receptor endocytosis.

Little is known regarding the mechanism(s) by which glycine receptors are endocytosed. Here we examined the endocytosis of homomeric alpha1 glycine receptors expressed in HEK 293 cells using immunofluorescence/confocal microscopy and whole-cell patch-clamp recordings. Our studies demonstrate that constitutive endocytosis of glycine receptors is blocked by the dominant negative dynamin construct K44A and that intracellular dialysis with peptide P4, a dynamin/amphiphysin-disrupting peptide, increased whole-cell glycine-gated chloride currents. To examine whether receptor endocytosis could be regulated by PKC, experiments with the PKC activator PMA (phorbol 12-myristate 13-acetate) were performed. PMA, but not its inactive analogue PMM (phorbol 12-monomyristate), stimulated receptor endocytosis and inhibited glycine-gated chloride currents. Similar to constitutive endocytosis, PKC-stimulated endocytosis was blocked by dynamin K44A. Mutation of a putative AP2 adaptin dileucine motif (L314A, L315A) present in the receptor cytoplasmic loop blocked PMA-stimulated receptor endocytosis and also prevented PMA inhibition of glycine receptor currents. In patch-clamp experiments, intracellular dialysis of a 12-amino acid peptide corresponding to the region of the receptor containing the dileucine motif prevented PKC modulation of wild-type glycine receptors. Unlike PKC modulation of the receptor, constitutive endocytosis was not affected by mutation of this dileucine motif. These results demonstrate that PKC activation stimulates glycine receptor endocytosis, that both constitutive endocytosis and PKC-stimulated endocytosis are dynamin-dependent, and that PKC-stimulated endocytosis, but not constitutive endocytosis, occurs via the dileucine motif (L314A, L315A) within the cytoplasmic loop of the receptor.

The role of loop F residues in determining differential d-tubocurarine potencies in mouse and human 5-hydroxytryptamine 3A receptors.

The competitive antagonist d-tubocurarine (curare) has greater potency at mouse than at human 5-hydroxytryptamine 3A (5-HT3A) receptors, despite 84% amino acid sequence identity between the receptors. Within the ligand binding domain of this receptor are six loops (A-F). A previous report demonstrated that loop C of the 5-HT3A receptor contributed to differential potency between the receptors [Hope, A. G. et al. (1999) Mol. Pharmacol. 55, 1037-1043]. The present study tested the hypothesis that loop F plays a significant role in conferring interspecies curare potency differences. Wild-type, chimeric, and point mutant 5-HT3A receptors were expressed in Xenopus oocytes, and two-electrode voltage clamp electrophysiological recordings were performed. Our data suggest that loops C and F contribute to curare potency, given that the curare IC50's (concentration of drug that produces 50% inhibition of the response) for chimeric human receptors with substitutions of mouse residues in loop C (40.07 +/- 2.52 nM) or loop F (131.8 +/- 5.95 nM) were intermediate between those for the mouse (12.99 +/- 0.77 nM) and human (1817 +/- 92.36 nM) wild-type receptors. Two human point mutant receptors containing mouse receptor substitutions in loop F (H-K195E or H-V202I) had significantly lower curare IC50's than that of the human receptor. The human double mutant receptor, H-K195E,V202I, had the same curare IC50 (133.8 +/- 6.38 nM) as that of the human receptor containing all six loop F mouse substitutions. These results demonstrate that two loop F residues make a significant contribution in determining curare potency at the 5-HT3A receptor.

Temperature: an important experimental variable in studying PKC modulation of ligand-gated ion channels.

Amphibian oocyte and mammalian heterologous expression systems are often used to investigate the function of recombinant ion channels using electrophysiological techniques. Although both systems have yielded important information, the results obtained in these systems are sometimes conflicting. Oocytes and mammalian cells differ in their physiological temperature requirements. While room temperature is within the physiological temperature range for oocytes, this temperature is far below that required by mammalian cells. Since electrophysiological studies are often performed in both oocytes and mammalian cells at room temperature, we sought to determine if recording temperature could be a factor in some disparate results obtained in these cell types. For these studies, we examined phorbol ester modulation of GABA(A) and glycine receptors. Consistent with the literature, at room temperature, PMA (phorbol 12-myristate 13-acetate) produced a large reproducible decrease in the peak amplitude of GABA and glycine-gated currents in Xenopus oocytes. In contrast, PMA was ineffective in modulating these heterologously expressed receptors at room temperature in human embryonic kidney (HEK) 293 cells. However, when electrophysiological experiments were performed at 35 degrees C in HEK 293 cells, PMA decreased the function of these receptors. Our results indicate that the temperature at which electrophysiological studies are conducted is an important experimental variable. To determine the extent to which electrophysiological recordings are performed at physiological temperatures in HEK 293 cells, a PubMed search was conducted using the search terms "patch clamp" and "HEK" for the years 2003-2004. This search revealed that only 15% of the patch clamp studies were reported to have been conducted in the temperature range of 32-37 degrees C. The results of our study indicate that temperature is an important experimental variable that requires rational consideration in the design of electrophysiological experiments.

Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins.

Adeno-associated virus (AAV) serotype 8 appears to be the strongest of the natural serotypes reported to date for gene transfer in liver and muscle. In this study, we evaluated AAV8 in the brain by several methods, including biophotonic imaging of green fluorescent protein (GFP). In the adult rat hippocampus, levels of GFP expressed were clearly greater with AAV8 than with AAV2 or AAV5 by Western blot and biophotonic imaging and slightly but significantly greater than AAV1 by Western blot. In the substantia nigra, the GFP expression conferred by AAV8 was toxic to dopamine neurons, although toxicity could be avoided with dose titration. At the low dose at which there was no GFP toxicity from the GFP vector, another AAV8 vector for a disease-related (P301L) form of the microtubule-associated protein tau caused a 78% loss of dopamine neurons and significant amphetamine-stimulated rotational behavior. The AAV8 tau vector-induced cell loss was greater than that from AAV2 or AAV5 tau vectors, demonstrating that the increased gene transfer was functional. While the toxicity observed with GFP expression warrants great caution, the efficient AAV8 is promising for animal models of neurodegenerative diseases and potentially as well for gene therapy of brain diseases.

PKC modulation of GABAA receptor endocytosis and function is inhibited by mutation of a dileucine motif within the receptor beta 2 subunit.

The modulation of GABAA receptors by protein kinase C is complex and involves effects on both ion channel function and receptor trafficking. Although PKC regulates receptor cell surface expression the mechanism is not well understood. Using immunofluorescence studies in HEK 293 cells, we demonstrate that activation of PKC by the phorbol ester PMA promotes receptor endocytosis and is dependent on the presence of a gamma subunit. This endocytosis is blocked by the dominant negative dynamin mutant K44A indicating that PKC-induced receptor endocytosis involves the dynamin endocytic pathway. Mutation of a dileucine motif within the receptor beta2 subunit inhibits the effect of PKC activation on receptor endocytosis. Using patch clamp analysis, we show that PKC activation produces a robust inhibition of GABA-gated chloride currents in cells expressing wildtype GABAA receptors, but it is ineffective in modulating receptors lacking the dileucine motif. Furthermore, the introduction into the patch pipette of a 10-amino acid peptide corresponding to the dileucine motif present in the receptor beta2 subunit prevents PKC modulation of wildtype recombinant receptors. Furthermore, in cerebral cortical neuronal slices inclusion of this peptide in the patch pipette prevents PKC modulation of native GABAA receptors. Using limited chymotrypsin digestion assays, we also show that PKC increases receptor internalization in primary cultures of cerebral cortical neurons. Lastly, PKC inhibitors do not block constitutive receptor endocytosis or affect GABA-gated chloride currents suggesting that PKC-dependent phosphorylation is not required for GABAA receptor endocytosis but plays a modulatory role in the process.

Inhibition of the activity of the native gamma-aminobutyric acid A receptor by metabolites of thyroid hormones: correlations with molecular modeling studies.

To characterize the direct effects of thyroid hormones on native gamma-aminobutyric acid(A) (GABA(A)) receptors, rapid (5 s) actions of a series of iodothyronines on muscimol-stimulated uptake of (36)Cl(-) were investigated in synaptoneurosomes prepared from rat brain. The results were correlated with molecular modeling of the active compounds. Dose-response curves for muscimol in the presence of 3,3', 5-L-triiodothyronine (L-T3) indicated a noncompetitive inhibition of muscimol-stimulated (36)Cl(-) uptake by the thyroid hormone. Synaptoneurosomes prepared from cerebellum were less sensitive to L-T3 than those from cerebral cortex, in terms of the potency of the hormone. The overall efficacy approached complete inhibition for both brain regions. Muscimol-stimulated (36)Cl(-) uptake was inhibited differentially by iodothyronine derivatives. One group of compounds with IC(50) values of 18-30 microM included L-thyroxine (L-T4), D-thyroxine (D-T4), 3,3', 5,5'-tetraiodothyroacetic acid (Tetrac), and 3,3', 5-triiodothyroacetic acid (Triac). A second group with values of 75-100 microM included 3,3', 5'-l-triiodothyronine (reverse T3; r-T3), 3,3'-diiodo-L-thyronine (3,3'-l-T2) and 3,5-diiodo-L-thyronine (3,5-D-T2). A final group of inactive compounds with IC(50) values greater than 100 microM included 3',5'-diiodo-L-thyronine (3',5'-l-T2), 3-iodo-L-thyronine (L-T1), 3'-iodo-L-thyronine (3'-L-T1), and L-thyronine (L-T0). Molecular modeling of the active iodothyronines using the Gaussian03 series of programs indicated close correspondences with models of the GABA-inhibitory neurosteroid pregnenolone sulfate (PREGS), suggesting common mechanisms of action at the GABA(A) receptor.

Constitutive GABAA receptor endocytosis is dynamin-mediated and dependent on a dileucine AP2 adaptin-binding motif within the beta 2 subunit of the receptor.

Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA(A) receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA(A) receptors composed of either alpha1beta2gamma2Lor alpha1beta2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor beta2 subunit that is critical for endocytosis. Internalization of GABAA receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor beta2 subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA(A) receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.