Emmanuel J. Botzolakis

Enhanced macroscopic desensitization shapes the response of α4 subtype‐containing GABAA receptors to synaptic and extrasynaptic GABA

Up‐regulation of the GABAA 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 GABAA receptors in normal and abnormal physiology. We used rapid GABA application to recombinant GABAA 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 EC50 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.

A conserved Cys-loop receptor aspartate residue in the M3-M4 cytoplasmic loop is required for GABAA receptor assembly.

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 γ-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.

Microscopic kinetic determinants of macroscopic currents: insights from coupling and uncoupling of GABAA receptor desensitization and deactivation.

The time course of inhibitory postsynaptic currents (IPSCs) reflects GABAA 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 GABAA 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.

Hereditary Spastic Paraplegia-Associated Mutations in the NIPA1 Gene and Its Caenorhabditis elegans Homolog Trigger Neural Degeneration In Vitro and In Vivo through a Gain-of-Function Mechanism

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.

GABAA Receptor α1 Subunit Mutation A322D Associated with Autosomal Dominant Juvenile Myoclonic Epilepsy Reduces the Expression and Alters the Composition of Wild Type GABAA Receptors

A GABAA receptor (GABAAR) α1 subunit mutation, A322D (AD), causes an autosomal dominant form of juvenile myoclonic epilepsy (ADJME). Previous studies demonstrated that the mutation caused α1(AD) subunit misfolding and rapid degradation, reducing its total and surface expression substantially. Here, we determined the effects of the residual α1(AD) subunit expression on wild type GABAAR expression to determine whether the AD mutation conferred a dominant negative effect. We found that although the α1(AD) subunit did not substitute for wild type α1 subunits on the cell surface, it reduced the surface expression of α1β2γ2 and α3β2γ2 receptors by associating with the wild type subunits within the endoplasmic reticulum and preventing them from trafficking to the cell surface. The α1(AD) subunit reduced surface expression of α3β2γ2 receptors by a greater amount than α1β2γ2 receptors, thus altering cell surface GABAAR composition. When transfected into cultured cortical neurons, the α1(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 α1(AD) subunits, this epilepsy mutation also elicited a modest dominant negative effect that likely shapes the epilepsy phenotype.

Benzodiazepine modulation of GABAA receptor opening frequency depends on activation context: A patch clamp and simulation study

Benzodiazepines (BDZs) are GABA(A) receptor modulators with anxiolytic, hypnotic, and anticonvulsant properties. BDZs are understood to potentiate GABA(A) receptor function by increasing channel opening frequency, in contrast to barbiturates, which increase channel open duration. However, the in vitro evidence demonstrating increased opening frequency involved prolonged exposure to sub-saturating GABA concentrations, conditions most similar to those found in extrasynaptic areas. In contrast, synaptic GABA(A) receptors are transiently activated by high GABA concentrations. To determine if BDZ modulation of single-channel opening frequency would be different for BDZ-sensitive receptors activated under synaptic versus extrasynaptic conditions, a combination of patch clamp recording and kinetic modeling was used. Consistent with the original experimental findings, BDZs were found to increase receptor affinity for GABA by decreasing the unbinding rate. While this mechanism was predicted to increase opening frequency under extrasynaptic conditions, simulations predicted that the same mechanism under synaptic conditions would increase the number, but not the frequency, of single-channel openings. Thus, a single mechanism (slower GABA unbinding) can produce differential changes in opening frequency under synaptic versus extrasynaptic conditions. The functional impact of BDZs on GABA(A) receptors therefore depends upon the physiological context of receptor activation.

The effect of HSP-causing mutations in SPG3A and NIPA1 on the assembly, trafficking, and interaction between atlastin-1 and NIPA1.

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 dependent 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.

Achieving synaptically relevant pulses of neurotransmitter using PDMS microfluidics

Fast synaptic transmission is mediated by post-synaptic ligand-gated ion channels (LGICs) transiently activated by neurotransmitter released from pre-synaptic vesicles. Although disruption of synaptic transmission has been implicated in numerous neurological and psychiatric disorders, effective and practical methods for studying LGICs in vitro under synaptically relevant conditions are unavailable. Here, we describe a novel microfluidic approach to solution switching that allows for precise temporal control over the neurotransmitter transient while substantially increasing experimental throughput, flexibility, reproducibility, and cost-effectiveness. When this system was used to apply ultra-brief ( approximately 400micros) GABA pulses to recombinant GABA(A) receptors, members of the cys-loop family of LGICs, the resulting currents resembled hippocampal inhibitory post-synaptic currents (IPSCs) and differed from currents evoked by longer, conventional pulses, illustrating the importance of evaluating LGICs on a synaptic timescale. This methodology should therefore allow the effects of disease-causing mutations and allosteric modulators to be evaluated in vitro under physiologically relevant conditions.

Comparison of γ-Aminobutyric Acid, Type A (GABAA), Receptor αβγ and αβδ Expression Using Flow Cytometry and Electrophysiology: EVIDENCE FOR ALTERNATIVE SUBUNIT STOICHIOMETRIES AND ARRANGEMENTS.

The subunit stoichiometry and arrangement of synaptic αβγ GABAA receptors are generally accepted as 2α:2β:1γ with a β-α-γ-β-α counterclockwise configuration, respectively. Whether extrasynaptic αβδ receptors adopt the analogous β-α-δ-β-α subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant γ2 and δ subunits when co-transfected with α1 and/or β2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and β2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of β2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of δ subunit degradation, 10-fold less δ subunit cDNA was required to recapitulate γ2 subunit expression patterns and to eliminate the functional signature of α1β2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αβγ and αβδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αβγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical β-α-γ/δ-β-α receptors, including β-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and β-α-γ/δ-α-γ/δ receptors at higher levels of expression. These findings provide important insight into the role of GABAA receptor subunit under- or overexpression in disease states such as genetic epilepsies.

Context-dependent modulation of αβγ and αβδ GABAA receptors by penicillin : Implications for phasic and tonic inhibition

Penicillin, an open-channel blocker of GABA(A) receptors, was recently reported to inhibit phasic, but not tonic, currents in hippocampal neurons. To distinguish between isoform-specific and context-dependent modulation as possible explanations for this selectivity, the effects of penicillin were evaluated on recombinant GABA(A) receptors expressed in HEK293T cells. When co-applied with saturating GABA, penicillin decreased peak amplitude, induced rebound, and prolonged deactivation of currents evoked from both synaptic and extrasynaptic receptor isoforms. However, penicillin had isoform-specific effects on the extent of desensitization, reflecting its ability to differentially modulate peak (non-equilibrium) and residual (near-equilibrium) currents. This suggested that the context of activation could determine the apparent sensitivity of a given receptor isoform to penicillin. To test this hypothesis, we explored the ability of penicillin to modulate synaptic and extrasynaptic isoform currents that were activated under more physiologically relevant conditions. Interestingly, while currents evoked from synaptic isoforms under phasic conditions (transient activation by a saturating concentration of GABA) were substantially inhibited by penicillin, currents evoked from extrasynaptic isoforms under tonic conditions (prolonged application by a sub-saturating concentration of GABA) were minimally affected. We therefore concluded that the reported inability of penicillin to modulate tonic currents could not simply be attributed to insensitivity of extrasynaptic receptors, but rather, reflected an inability to modulate these receptors in their native context of activation.