Differences in kinetics between GABAC and GABAA receptors on carp retinal bipolar cells

The present work was undertaken to characterize kinetics, including activation, desensitization and deactivation, of responses mediated by GABAA and GABAC receptors on carp retinal bipolar cells, using the whole-cell patch-clamp technique. It was revealed that the GABAC response was generally slower in kinetics than the GABAA response. Activation kinetics of both the receptors could be well fit by monoexponential functions with time constants τ, being 44.57 ms (GABAC) and 10.86 ms (GABAA) respectively. Desensitization of the GABAA response was characterized by a fast and a slow exponential component with time constants of τfast = 2.16 s and τslow = 19.78 s respectively, whereas desensitization of the GABAC response was fit by a monoexponential function of the time constant τ = 6.98 s. Deactivation at both the receptors was adequately described by biexponential functions with time constants being much higher for the GABAC response (τfast = 674.8 ms; τslow = 2 090 ms) than those for the GABAA response (τfast = 42.07 ms; τslow = 275.1 ms). These differences in kinetics suggest that GABAC and GABAA receptors may be involved in processing signals in different frequency domains.     

Differences in kinetics between GABAC and GABAA receptors on carp retinal bipolar cells

The present work was undertaken to characterize kinetics, including activation, desensitization and deactivation, of responses mediated by GABA_A and GABA_C receptors on carp retinal bipolar cells, using the whole-cell patch-clamp technique. It was revealed that the GABA_C response was generally slower in kinetics than the GABA_A response. Activation kinetics of both the receptors could be well fit by monoexponential functions with time constants t, being 44.57 ms (GABA_C) and 10.86 ms (GABA_A) respectively. Desensitization of the GABA_Aresponse was characterized by a fast and a slow exponential component with time constants of τ_fast = 2.16 s and τ_slow = 19.78 s respectively, whereas desensitization of the GABA_C response was fit by a monoexponential function of the time constant τ = 6.98 s. Deactivation at both the receptors was adequately described by biexponential functions with time constants being much higher for the GABA_C response (τ_fast= 674.8 ms; τ_slow = 2 090 ms) than those for the GABA_A response (τ_fast = 42.07 ms; τ_slow = 275.1 ms). These differences in kinetics suggest that GABA_C and GABA_A receptors may be involved in processing signals in different frequency domains.     

GABAC receptors in the vertebrate retina

In the central nervous system (CNS), the inhibitory transmitter GABA interacts with three subtypes of GABA receptors, type A, type B, and type C. Historically, GABA receptors have been classified as either the inotropic GABA_A receptors or the metabotropic GABA_B receptors. Over the past 10 yr, studies have shown that a third class, called the GABA_C receptor, also exists. GABA_C receptors are found primarily in the vertebrate retina and to some extent in other parts of the CNS. Although GABA_A and GABA_C receptors both gate chloride channels, they are pharmacologically, molecularly, and functionally distinct. The ρ subunit of the GABA_C receptor, which has about 35% amino acid homology to GABA_A receptor subunits, was cloned from the retina and, when expressed inXenopus oocytes, has properties similar to retinal GABA_C receptors. There are probably distinct roles for GABA_C receptors in the retina, because they are found on only a subset of neurons, whereas GABA_A receptors are ubiquitous. This article reviews recent electrophysiological and molecular studies that have characterized the unique properties of GABA_C receptors and describes the roles that these receptors may play in visual information processing in the retina.     

Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABAA receptor

The prevalence of aromatic residues in the ligand binding site of the GABA_A receptor, as with other cys‐loop ligand‐gated ion channels, is undoubtedly important for the ability of neurotransmitters to bind and trigger channel opening. Here, we have examined three conserved tyrosine residues at the GABA binding pocket (β₂Tyr97, β₂Tyr157, and β₂Tyr205), making mutations to alanine and phenylalanine. We fully characterized the effects each mutation had on receptor function using heterologous expression in HEK‐293 cells, which included examining surface expression, kinetics of macroscopic currents, microscopic binding and unbinding rates for an antagonist, and microscopic binding rates for an agonist. The assembly or trafficking of GABA_A receptors was disrupted when tyrosine mutants were expressed as αβ receptors, but interestingly not when expressed as αβγ receptors. Mutation of each tyrosine accelerated deactivation and slowed GABA binding. This provides strong evidence that these residues influence the binding of GABA. Qualitatively, mutation of each tyrosine has a very similar effect on receptor function; however, mutations at β₂Tyr157 and β₂Tyr205 are more detrimental than β₂Tyr97 mutations, particularly to the GABA binding rate. Overall, the results suggest that interactions involving multiple tyrosine residues are likely during the binding process.     

Trafficking and potential assembly patterns of epsilon-containing GABAA receptors

Incorporation of the epsilon subunit into the GABAA receptor has been suggested to confer unusual, but variable, biophysical and pharmacological characteristics to both recombinant and native receptors. Due to their structural similarity with the gamma subunits, epsilon subunits have been assumed to substitute at the single position of the gamma subunit in assembled receptors. However, prior work suggests that functional variability in epsilon-containing receptors may reflect alternative sites of incorporation and of not just one, but possibly multiple epsilon subunits in the pentameric receptor complex. Here we present data indicating that increased expression of epsilon, in conjunction with alpha2 and beta3 subunits, results in expression of GABAA receptors with correspondingly altered rectification, deactivation and levels of spontaneous openings, but not increased total current density. We also provide data that the epsilon subunit, like the beta3 subunit, can self-export and data from chimeric receptors suggesting that similarities between the assembly domains of the beta3 and the epsilon subunits may allow the epsilon subunit to replace the beta, as well as the gamma, subunit. The substitution of an epsilon for a beta, as well as the gamma subunit and formation of receptors with alternative patterns of assembly with respect to epsilon incorporation may underlie the observed variability in both biophysical and pharmacological properties noted not only in recombinant, but also in native receptors.     

甘氨酸对鲫鱼视网膜ERG b-波和ON型双极细胞活动的调制

本工作在分离灌流的鲫鱼视网膜上研究了甘氨酸对明,暗视视网膜电图（ＥＲＧ）ｂ－波和胞内记录的ＯＮ型双极细胞反应的作用。结果表明,甘氨酸能明显压抑ＥＲＧ ｂ－波和ＯＮ型双极细胞的反应,其作用能为士的宁所翻转;甘氨酸对用谷氨酸分离的ＥＲＧ ＰⅢ成分（光感受器电位）无明显影响。这些结果提示,甘氨酸可能直接作用于双极细胞的受体,从而调节视网膜ＯＮ通路的活动。     

On high- and low-affinity agonist sites in GABAA receptors

GABAA receptors are activated via low-affinity binding sites for the agonists GABA or muscimol. Evidence has been provided that the amino acid residue alpha 1F64 located at the beta2(+)/alpha1(-) subunit interface forms part of this binding site. In radioactive ligand binding studies the agonist [3H]muscimol has been found to interact with the receptor via a high-affinity binding site. This site has been interpreted as a conformational variant of the low-affinity site. Alternatively, the high-affinity binding site has been located to the alpha1(+)/beta2(-) interface and the homologous residue to alpha 1F64, beta 2Y62 has been proposed to constitute an important part of this site. Here we investigated the effect of the point mutation alpha 1F64L and the homologous mutation beta 2Y62L on agonist and antagonist binding and functional properties in alpha 1 beta 2 gamma 2 GABAA receptors. While the mutation in the alpha1 subunit had drastic consequences on all studied properties, including desensitization, the mutation in the beta2 subunit had little consequence. Our observations are relevant for the relative location of high- and low-affinity agonist sites in GABAA receptors.     

光学方法分析GABA及GABAA受体在脑干听觉传入通路的作用

目的旨在探讨脑干听觉传入通路中GABA能神经递质及GABAA受体对电刺激位听神经传入冲动的影响.方法使用出生后0～5 d的ddy/ddy小鼠制备脑干切片.脑片经电压敏感染料NK3041染色,电刺激与脑片相连的位听神经残端.使用16×16像素的硅光电二极管阵列测量光学信号.所采集的数据使用ARGUS50/PDA软件分析.结果多部位的光学记录方法显示了从位听神经到耳蜗核和前庭核的兴奋性传导的时间-空间分布.其中每一个光学成分由快峰电位样反应和慢反应组成.抑制性神经递质GABA可降低诱发的光学信号的快反应和慢反应,GABAA受体拮抗剂荷包牡丹碱可增强这些反应.结论16×16像素的硅光电二极管阵列可记录位听神经刺激诱发的多部位光学信号,每一个光学信号含有突触前及突触后电位成分.抑制性神经递质GABA和GJBAA受体拮抗剂可调节光学信号的兴奋性传导.     

Ischemic insult to cerebellar Purkinje cells causes diminished GABAA receptor function and allopregnanolone neuroprotection is associated with GABAA receptor stabilization

Cerebellar Purkinje cells (PC) are particularly vulnerable to ischemic injury and excitotoxicity, although the molecular basis of this sensitivity remains unclear. We tested the hypothesis that ischemia causes rapid down-regulation of GABA(A) receptors in cerebellar PC, thereby increasing susceptibility to excitotoxicity. Oxygen-glucose deprivation (OGD) caused a decline in functional GABA(A) receptors, within the first hour of re-oxygenation. Decreased amplitude of miniature inhibitory post-synaptic potentials confirmed that OGD caused a significant decrease in functional synaptic GABA(A) receptors and quantitative Western blot analysis demonstrated the loss of GABA(A) receptor current was associated with a decline in total receptor protein. Interestingly, the potent neuroprotectant allopregnanolone (ALLO) prevented the decline in GABA(A) receptor current and protein. Consistent with our in vitro data, global ischemia in mice caused a significant decline in total cerebellar GABA(A) receptor protein and PC specific immunoreactivity. Moreover, ALLO provided strong protection of PC and prevented ischemia-induced decline in GABA(A) receptor protein. Our findings indicate that ischemia causes a rapid and sustained loss of GABA(A) receptors in PC, whereas ALLO prevents the decline in GABA(A) receptors and protects against ischemia-induced damage. Thus, interventions which prevent ischemia-induced decline in GABA(A) receptors may represent a novel neuroprotective strategy.     

Relation between potassium-channel kinetics and the intrinsic dynamics in isolated retinal bipolar cells

Characterization of the intrinsic dynamics of isolated retinal bipolar cells by a whole-cell patch-clamp technique combined with estimation of effective impulse responses across a range of mean injected currents reveals strikingly adaptive behavior. At resting potential, bipolar cells' effective impulse response is slow, high gain, and low pass. Depolarization speeds up response, decreases gain, and, in most cells, induces bandpass behavior.This adaptive behavior involves two K⁺ currents. The delayed-rectifier accounts for the observed gain reduction, speed increase, and bandpass behavior. The A-channel further shortens the impulse responses but suppresses bandpass features. Computer simulations of model neurons with a delayed-rectifier and varying A-channel conductances reveal that impulse responses largely reflect the flux of electrical charge through the two K⁺ channels. The A-channel broadens the frequency response and preempts the action of the delayed-rectifier, thereby reducing the associated bandpass features. Admixtures of the two K⁺ channels produce the observed variety of dynamics of retinal bipolar cells.     

The function and the expression of forebrain GABAA receptors change with hormonal state in the adult mouse

Neurotransmission mediated by gamma‐aminobutyric acid type A (GABA_A) receptors in the mammalian medial preoptic area (mPOA) plays a pivotal role in the expression of hormone‐sensitive behaviors. Hand in hand with GABAergic control of reproduction, hormone treatments that activate gonadal steroid signaling pathways in gonadectomized rats are known to regulate the expression of specific GABA_A receptor subunit mRNAs. While the effects of exogenous hormone treatments have been well documented, little information is available as to how GABA_A receptor‐mediated transmission in the mPOA is altered by endogenous changes in hormonal state in gonadally‐intact adult animals or if those changes can be ascribed to hormone‐dependent changes in receptor subunit composition. In the present study, we found that both the peak amplitudes of GABA_A receptor‐mediated synaptic currents in the mPOA, as well as the ability of the endogenous neurosteroids to modulate those currents, varied as a function of the estrous cycle. Moreover, we found that the degree of neurosteroid modulation was also significantly different between wild‐type and the androgen‐insensitive testicular feminization (Tfm) mutant male mice. Semiquantitative RT‐PCR analysis performed to assess levels of GABA_A receptor subunit mRNAs indicated that levels of specific subunits varied over the course of the estrous cycle and between wild‐type and Tfm male mice. The variations in GABA_A receptor expression and function in the mPOA that are associated with differences in gonadal steroid signaling may contribute to the dynamic nature of GABAergic control of neuroendocrine pathways. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 137–149, 2002; DOI 10.1002/neu.10021     

Implications for treatment: GABAA receptors in aging, Down syndrome and Alzheimer's disease

In addition to progressive dementia, Alzheimer's disease (AD) is characterized by increased incidence of seizure activity. Although originally discounted as a secondary process occurring as a result of neurodegeneration, more recent data suggest that alterations in excitatory-inhibitory (E/I) balance occur in AD and may be a primary mechanism contributing AD cognitive decline. In this study, we discuss relevant research and reports on the GABA(A) receptor in developmental disorders, such as Down syndrome, in healthy aging, and highlight documented aberrations in the GABAergic system in AD. Stressing the importance of understanding the subunit composition of individual GABA(A) receptors, investigations demonstrate alterations of particular GABA(A) receptor subunits in AD, but overall sparing of the GABAergic system. In this study, we review experimental data on the GABAergic system in the pathobiology of AD and discuss relevant therapeutic implications. When developing AD therapeutics that modulate GABA it is important to consider how E/I balance impacts AD pathogenesis and the relationship between seizure activity and cognitive decline.     

Direction selectivity in retinal bipolar cell axon terminals

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Phospholipase C-related inactive protein is implicated in the constitutive internalization of GABAA receptors mediated by clathrin and AP2 adaptor complex

A mechanism for regulating the strength of synaptic inhibition is enabled by altering the number of GABA(A) receptors available at the cell surface. Clathrin and adaptor protein 2 (AP2) complex-mediated endocytosis is known to play a fundamental role in regulating cell surface GABA(A) receptor numbers. Very recently, we have elucidated that phospholipase C-related catalytically inactive protein (PRIP) molecules are involved in the phosphorylation-dependent regulation of the internalization of GABA(A) receptors through association with receptor beta subunits and protein phosphatases. In this study, we examined the implications of PRIP molecules in clathrin-mediated constitutive GABA(A) receptor endocytosis, independent of phospho-regulation. We performed a constitutive receptor internalization assay using human embryonic kidney 293 (HEK293) cells transiently expressed with GABA(A) receptor alpha/beta/gamma subunits and PRIP. PRIP was internalized together with GABA(A) receptors, and the process was inhibited by PRIP-binding peptide which blocks PRIP binding to beta subunits. The clathrin heavy chain, mu2 and beta2 subunits of AP2 and PRIP-1, were complexed with GABA(A) receptor in brain extract as analyzed by co-immunoprecipitation assay using anti-PRIP-1 and anti-beta2/3 GABA(A) receptor antibody or by pull-down assay using beta subunits of GABA(A) receptor. These results indicate that PRIP is primarily implicated in the constitutive internalization of GABA(A) receptor that requires clathrin and AP2 protein complex.     

Nonlinear spatial integration in retinal bipolar cells shapes the encoding of artificial and natural stimuli

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TRPM1: The endpoint of the mGluR6 signal transduction cascade in retinal ON‐bipolar cells

For almost 30 years the ion channel that initiates the ON visual pathway in vertebrate vision has remained elusive. Recent findings now indicate that the pathway, which begins with unbinding of glutamate from the metabotropic glutamate receptor 6 (mGluR6), ends with the opening of the transient receptor potential (TRP)M1 cation channel. As a component of the mGluR6 signal transduction pathway, mutations in TRPM1 would be expected to cause congenital stationary night blindness (CSNB), and several such mutations have already been identified in CSNB families. Furthermore, expression of TRPM1 in both the retina and skin raises the possibility that a genetic link exists between certain types of visual and skin disorders.     

Role of protein kinase A in GABAA receptor dysfunction in CA1 pyramidal cells following chronic benzodiazepine treatment

One-week treatment with the benzodiazepine (BZ) flurazepam (FZP), results in anticonvulsant tolerance, associated with reduced GABAA receptor (GABAR) subunit protein and miniature inhibitory post-synaptic current (mIPSC) amplitude in CA1 neurons of rat hippocampus. Because protein kinase A (PKA) has been shown to modulate GABAR function in CA1 pyramidal cells, the present study assessed whether GABAR dysfunction is associated with changes in PKA activity. Two days after 1-week FZP treatment, there were significant decreases in basal (- 30%) and total (- 25%) PKA activity, and a 40% reduction in PKA RIIbeta protein in the insoluble fraction of CA1 hippocampus. The soluble component of CA1 showed a significant increase in basal (100%) but not total PKA activity. Whole-cell recording in vitro showed a 50% reduction in mIPSC amplitude in CA1 pyramidal cells, with altered sensitivity to PKA modulators. Neurons from FZP-treated rats responded to 8-bromo-cAMP with a significant increase (31%) in mIPSC amplitude. Likewise, vasoactive intestinal polypeptide (VIP), an endogenous PKA activator, caused a significant 36% increase in mIPSC amplitude in FZP-treated cells. Neither agent had a significant effect on mIPSC amplitude in control cells. This study supports a role for PKA in GABAR dysfunction after chronic FZP treatment.