No abstract

Up-regulation of the GABA A receptor α4 subunit subtype has been consistently shown in multiple animal models of chronic epilepsy. This isoform is expressed in both thalamus and hippocampus and is likely to play a significant role in regulating corticothalamic and hippocampal rhythms. However, little is known about its physiological properties, thus limiting understanding of the role of α4 subtype-containing GABA A receptors in normal and abnormal physiology. We used rapid GABA application to recombinant GABA A receptors expressed in HEK293T cells to compare the macroscopic kinetic properties of α4β3γ2L receptors to those of the more widely distributed α1β3γ2L receptors. These receptor currents had similar peak current amplitudes and GABA EC 50 values. However, α4β3γ2L currents activated more slowly when exposed to submaximal GABA concentrations, had more fast desensitization (τ = 15-100 ms), and had less residual current during long GABA applications. In addition, α4β3γ2L currents deactivated more slowly than α1β3γ2L currents. Peak currents evoked by repetitive, brief GABA applications were more strongly attenuated for α4β3γ2L currents than α1β3γ2L currents. Moreover, the time required to recover from desensitization was prolonged in α4β3γ2L currents compared to α1β3γ2L currents. We also found that exposure to prolonged low levels of GABA, similar to those that might be present in the extrasynaptic space, greatly suppressed the response of α4β3γ2L currents to higher concentrations of GABA, while α1β3γ2L currents were less affected by exposure to low levels of GABA. Taken together, these data suggest that α4β3γ2L receptors have unique kinetic properties that limit the range of GABA applications to which they can respond maximally. While similar to α1β3γ2L receptors in their ability to respond to brief and low frequency synaptic inputs, α4β3γ2L receptors are less efficacious when exposed to prolonged tonic GABA or during repetitive stimulation, as may occur during learning and seizures. GABA A receptors are pentameric cys-loop receptors composed primarily of two α subunits, two β subunits, and either a δ or a γ subunit selected from six α, three β, one δ, and three γ subunit subtypes. The distribution of specific subtypes is highly brain region and cell type specific, and varies during development and in certain disease states. The presence of a specific subunit subtype confers different pharmacological and physiological properties to receptor isoforms. For example, α subtypes strongly influence GABA A receptor pharmacology. When assembled with β and γ subunits, GABA A receptors containing α1, 2, 3 or 5 subtypes are highly diazepam sensitive. However, α1 subtype-containing receptors are much more sensitive to zolpidem than receptors containing α2 or α3 subtypes, and those containing α5 subtypes are completely insensitive to this drug. In contrast, GABA A receptors containing α4 or α6 subtypes are insensitive to both diazepam and zolpidem. Furthermore, the imidazobenzodiazepine Ro 15-4513, which is an inverse benz...

We studied the consequences of expression of wild-type (WT) human NIPA1 and two mutant forms of NIPA1 with known HSP-associated mutations (T45R and G106R) on cultured rat cortical neurons and using equivalent substitutions in the Caenorhabditis elegans NIPA1 homolog CeNIPA. WT NIPA1 localized in transfected neuronal and non-neuronal cells to the Golgi complex, a subset of synaptic vesicles, to a subset of early endosomes, and plasma cell membrane. Mutant NIPA1 accumulated in the endoplasmic reticulum (ER) triggering ER stress and features of apoptotic cell death. Flow cytometric analysis of NIPA1 surface expression demonstrated relatively intact trafficking of mutant forms and only the T45R mutant exhibited modestly reduced patterns of surface expression without evidence for a dominant-negative effect. In vivo pan-neuronal expression of the WT C. elegans NIPA1 homolog (CeNIPA) was well tolerated, with no obvious impact on neuronal morphology or behavior. In striking contrast, expression of CeNIPA bearing HSP-associated mutations caused a progressive neural degeneration and a clear motor phenotype. Neuronal loss in these animals began at day 7 and by day 9 animals were completely paralyzed. These effects appeared to arise from activation of the apoptotic program triggered by unfolded protein response (UPR), as we observed marked modifications of motor and cellular phenotype when mutant NIPA1 was expressed in caspase (ced-3)-and UPR (xbp-1)-deficient backgrounds. We propose that HSP-associated mutations in NIPA1 lead to cellular and functional deficits through a gain-of-function mechanism supporting the ER accumulation of toxic NIPA1 proteins.

The time course of inhibitory postsynaptic currents (IPSCs) reflects GABA A receptor deactivation, the process of current relaxation following transient activation. Fast desensitization has been demonstrated to prolong deactivation, and these processes have been described as being 'coupled'. However, the relationship between desensitization and deactivation remains poorly understood. We investigated the 'uncoupling' of GABA A receptor macroscopic desensitization and deactivation using experimental conditions that affected these two processes differently. Changing agonist affinity preferentially altered deactivation, changing agonist concentration preferentially altered macroscopic desensitization, and a pore domain mutation prolonged deactivation despite blocking fast desensitization. To gain insight into the mechanistic basis for coupling and uncoupling, simulations were used to systematically evaluate the interplay between agonist affinity, gating efficacy, and desensitized state stability in shaping macroscopic desensitization and deactivation. We found that the influence of individual kinetic transitions on macroscopic currents depended not only on model connectivity, but also on the relationship among transitions within a given model. In addition, changing single rate constants differentially affected macroscopic desensitization and deactivation, thus providing parsimonious kinetic explanations for experimentally observed uncoupling. Finally, these findings permitted development of an algorithmic framework for kinetic interpretation of experimental manipulations that alter macroscopic current properties. Transient synaptic release of GABA onto clusters of postsynaptic αβγ GABA A receptors represents a major mechanism of inhibition in the brain. The time course of GABA A receptor synaptic currents influences the complex behaviour of inhibitory circuits, and many clinically useful drugs that potentiate GABA A receptor function act by prolonging IPSCs. Because IPSC duration depends primarily on postsynaptic GABA A receptor properties, the kinetic principles governing channel behaviour have been the focus of active experimental investigation (Twyman et

Despite its genetic heterogeneity, hereditary spastic paraplegia (HSP) is characterized by similar clinical phenotypes, suggesting that a common biochemical pathway underlies its pathogenesis. In support of this hypothesis, we used a combination of immunoprecipitation, confocal microscopy, and flow cytometry to demonstrate that two HSP-associated proteins, atlastin-1 and NIPA1, are direct binding partners, and interestingly, that the endogenous expression and trafficking of these proteins is highly dependant upon their coexpression. In addition, we demonstrated that the cellular distribution of atlastin-1:NIPA1 complexes was dramatically altered by HSP-causing mutations, as missense mutations in atlastin-1 (R239C and R495W) and NIPA1 (T45R and G106R) caused protein sequestration in the Golgi complex (GC) and endoplasmic reticulum (ER), respectively. Moreover, we demonstrated that HSP-causing mutations in both atlastin-1 and NIPA1 reduced axonal and dendritic sprouting in cultured rat cortical neurons. Together, these findings support the hypothesis that NIPA1 and atlastin-1 are members of a common biochemical pathway that supports axonal maintenance, which may explain in part the characteristic degeneration of long spinal pathways observed in patients with HSP.

No abstract

Members of the Cys-loop superfamily of ligand-gated ion channels, which mediate fast synaptic transmission in the nervous system, are assembled as heteropentamers from a large repertoire of neuronal subunits. Although several motifs in subunit N-terminal domains are known to be important for subunit assembly, increasing evidence points toward a role for C-terminal domains. Using a combination of flow cytometry, patch clamp recording, endoglycosidase H digestion, brefeldin A treatment, and analytic centrifugation, we identified a highly conserved aspartate residue at the boundary of the M3-M4 loop and the M4 domain that was required for binary and ternary ␥-aminobutyric acid type A receptor surface expression. Mutation of this residue caused mutant and partnering subunits to be retained in the endoplasmic reticulum, reflecting impaired forward trafficking. Interestingly although mutant and partnering wild type subunits could be coimmunoprecipitated, analytic centrifugation studies demonstrated decreased formation of pentameric receptors, suggesting that this residue played an important role in later steps of subunit oligomerization. We thus conclude that C-terminal motifs are also important determinants of Cys-loop receptor assembly.The Cys-loop superfamily of ligand-gated ion channels, which includes ␥-aminobutyric acid type A (GABA A ) 2 and type C (GABA C ), nicotinic acetylcholine, glycine, and 5-hydroxytryptamine type 3 receptors, mediates fast synaptic transmission in the nervous system. Mutations that alter Cys-loop receptor surface density by affecting receptor biogenesis have been associated with idiopathic generalized epilepsies (1-4), congenital myasthenic syndromes (5), and psychiatric disorders (6). Unfortunately because the structural and cellular determinants of receptor biogenesis are poorly understood, development of effective treatment strategies remains a significant challenge.A wealth of evidence suggests that Cys-loop receptors are assembled as heteropentamers from a large repertoire of neuronal subunits (7-10). Subunits share a similar topology that includes an extracellular N-terminal domain, four transmembrane domains, three loops including a large cytoplasmic loop, and a variable length extracellular C-terminal tail (7, 11). Receptor assembly is thought to occur in the endoplasmic reticulum (ER) following glycosylation and folding of de novo synthesized subunits (12-15). Assembly is closely monitored by ER quality control machinery, and consequently subunits that fail to assemble properly are retained and degraded (15)(16)(17). Although N-terminal motifs are known to be important for subunit assembly (18,19), recent studies in nicotinic acetylcholine receptors (nAChRs) suggest that C-terminal motifs may also play a role (20).GABA A receptors are the most abundant Cys-loop receptor in the mammalian brain and are responsible for the majority of fast inhibitory neurotransmission. Like other Cys-loop superfamily receptors, they are pentamers assembled from combinations of 16 subunit subtypes ...

A GABA A receptor (GABA A R) ␣1 subunit mutation, A322D (AD), causes an autosomal dominant form of juvenile myoclonic epilepsy (ADJME). Previous studies demonstrated that the mutation caused ␣1(AD) subunit misfolding and rapid degradation, reducing its total and surface expression substantially. Here, we determined the effects of the residual ␣1(AD) subunit expression on wild type GABA A R expression to determine whether the AD mutation conferred a dominant negative effect. We found that although the ␣1(AD) subunit did not substitute for wild type ␣1 subunits on the cell surface, it reduced the surface expression of ␣1␤2␥2 and ␣3␤2␥2 receptors by associating with the wild type subunits within the endoplasmic reticulum and preventing them from trafficking to the cell surface. The ␣1(AD) subunit reduced surface expression of ␣3␤2␥2 receptors by a greater amount than ␣1␤2␥2 receptors, thus altering cell surface GABA A R composition. When transfected into cultured cortical neurons, the ␣1(AD) subunit altered the time course of miniature inhibitory postsynaptic current kinetics and reduced miniature inhibitory postsynaptic current amplitudes. These findings demonstrated that, in addition to causing a heterozygous loss of function of ␣1(AD) subunits, this epilepsy mutation also elicited a modest dominant negative effect that likely shapes the epilepsy phenotype. GABA A Rs2 are ligand-gated ion channels that provide the major source of inhibitory control to the mammalian central nervous system. Each GABA A R is a pentamer whose five subunits arise from seven subunit families that contain multiple subtype isoforms. Neurons preferentially express GABA A Rs composed of distinct combinations of subunit isoforms in different brain regions at well defined times in development (1-3).At maturity, the most prevalent GABA A R throughout the brain consists of two ␣1 subunits, two ␤2 subunits, and one ␥2 subunit in a ␤2-␣1-␤2-␣1-␥2 assembly (4 -6). To date, 13 autosomal dominant mutations in GABA A R subunit genes have been associated with different epilepsy syndromes (7).The missense AD mutation in the GABA A R ␣1 subunit gene (GABRA1) causes ADJME (8), a monogenic form of a common epilepsy syndrome that begins at a distinct developmental time point (adolescence) and confers myoclonic, generalized tonicclonic, and absence seizures as well as neuropsychiatric comorbidities (9). We demonstrated previously that the AD mutation, which substitutes a negatively charged aspartate for a neutral alanine within the M3 transmembrane domain, causes the ␣1(AD) subunit to misfold with altered topology (10). Cells rapidly degrade the misfolded ␣1(AD) subunit through both proteasome-and lysosome-mediated processes (10, 11). Therefore, the ␣1(AD) subunit is expressed at substantially lower levels than the wild type ␣1 subunit. GABA A Rs that do incorporate the residual, nondegraded ␣1(AD) subunits exhibit substantially altered electrophysiological properties (8,12,13). Therefore, a major consequence of heterozygous expression of the AD mutation i...