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Robert L Macdonald

Publications and source records attributed to Robert L Macdonald.

At least 19 recordsLinked to original sources
PubMed2006

Enhanced macroscopic desensitization shapes the response of alpha4 subtype-containing GABAA receptors to synaptic and extrasynaptic GABA.

Up-regulation of the GABAA receptor alpha4 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 alpha4 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 alpha4beta3gamma2L receptors to those of the more widely distributed alpha1beta3gamma2L receptors. These receptor currents had similar peak current amplitudes and GABA EC50 values. However, alpha4beta3gamma2L currents activated more slowly when exposed to submaximal GABA concentrations, had more fast desensitization (tau = 15-100 ms), and had less residual current during long GABA applications. In addition, alpha4beta3gamma2L currents deactivated more slowly than alpha1beta3gamma2L currents. Peak currents evoked by repetitive, brief GABA applications were more strongly attenuated for alpha4beta3gamma2L currents than alpha1beta3gamma2L currents. Moreover, the time required to recover from desensitization was prolonged in alpha4beta3gamma2L currents compared to alpha1beta3gamma2L 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 alpha4beta3gamma2L currents to higher concentrations of GABA, while alpha1beta3gamma2L currents were less affected by exposure to low levels of GABA. Taken together, these data suggest that alpha4beta3gamma2L receptors have unique kinetic properties that limit the range of GABA applications to which they can respond maximally. While similar to alpha1beta3gamma2L receptors in their ability to respond to brief and low frequency synaptic inputs, alpha4beta3gamma2L receptors are less efficacious when exposed to prolonged tonic GABA or during repetitive stimulation, as may occur during learning and seizures.

Cell Line↗
PubMed2006

Why does fever trigger febrile seizures? GABAA receptor gamma2 subunit mutations associated with idiopathic generalized epilepsies have temperature-dependent trafficking deficiencies.

With a worldwide incidence as high as 6.7% of children, febrile seizures are one of the most common reasons for seeking pediatric care, but the mechanisms underlying generation of febrile seizures are poorly understood. Febrile seizures have been suspected to have a genetic basis, and recently, mutations in GABAA receptor and sodium channel genes have been identified that are associated with febrile seizures and generalized seizures with febrile seizures plus pedigrees. Pentameric GABAA receptors mediate the majority of fast synaptic inhibition in the brain and are composed of combinations of alpha(1-6), beta(1-3), and gamma(1-3) subunits. In alphabetagamma2 GABAA receptors, the gamma2 subunit is critical for receptor trafficking, clustering, and synaptic maintenance, and mutations in the gamma2 subunit have been monogenically associated with autosomal dominant transmission of febrile seizures. Here, we report that whereas trafficking of wild-type alpha1beta2gamma2 receptors was slightly temperature dependent, trafficking of mutant alpha1beta2gamma2 receptors containing gamma2 subunit mutations [gamma2(R43Q), gamma2(K289M), and gamma2(Q351X)] associated with febrile seizures was highly temperature dependent. In contrast, trafficking of mutant alpha1beta2gamma2 receptors containing an alpha1 subunit mutation [alpha1(A322D)] not associated with febrile seizures was not highly temperature dependent. Brief increases in temperature from 37 to 40 degrees C rapidly (<10 min) impaired trafficking and/or accelerated endocytosis of heterozygous mutant alpha1beta2gamma2 receptors containing gamma2 subunit mutations associated with febrile seizures but not of wild-type alpha1beta2gamma2 receptors or heterozygous mutant alpha1(A322D)beta2gamma2 receptors, suggesting that febrile seizures may be produced by a temperature-induced dynamic reduction of susceptible mutant surface GABAA receptors in response to fever.

Animals↗
PubMed2006

Delta subunit susceptibility variants E177A and R220H associated with complex epilepsy alter channel gating and surface expression of alpha4beta2delta GABAA receptors.

Most human idiopathic generalized epilepsies (IGEs) are polygenic, but virtually nothing is known of the molecular basis for any of the complex epilepsies. Recently, two GABAA receptor delta subunit variants (E177A, R220H) were proposed as susceptibility alleles for generalized epilepsy with febrile seizures plus and juvenile myoclonic epilepsy. In human embryonic kidney 293T cells, recombinant halpha1beta2delta(E177A) and halpha1beta2delta(R220H) receptor currents were reduced, but the basis for the current reduction was not determined. We examined the mechanistic basis for the current reduction produced by these variants using the halpha4beta2delta receptor, an isoform more physiologically relevant and linked to epileptogenesis, by characterizing the effects of these variants on receptor cell surface expression and single-channel gating properties. Expression of variant alpha4beta2delta(R220H) receptors resulted in a decrease in surface receptor proteins, and a smaller, but significant, reduction was observed for variant alpha4beta2delta(E177A) receptors. For both variants, no significant alterations of surface expression were observed for mixed population of wild-type and variant receptors. The mean open durations of alpha4beta2delta(E177A) and alpha4beta2delta(R220H) receptor single-channel currents were both significantly decreased compared to wild-type receptors. These data suggest that both delta(E177A) and delta(R220H) variants may result in disinhibition in IGEs by similar cellular and molecular mechanisms, and in heterozygously affected individuals, a reduction in channel open duration of delta subunit-containing GABAA receptors may be the major contributor to the epilepsy phenotypes.

Amino Acid Substitution↗
PubMed2006

Expression and function of inwardly rectifying potassium channels after experimental subarachnoid hemorrhage.

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is because of smooth muscle contraction, although the mechanism of this contraction remains unresolved. Membrane potential controls the contractile state of arterial myocytes by gating voltage-sensitive calcium channels and is in turn primarily controlled by K(+) ion conductance through several classes of K(+) channels. We characterized the role of inwardly rectifying K(+) (K(IR)) channels in vasospasm. Vasospasm was created in dogs using the double-hemorrhage model of SAH. Electrophysiological, real-time quantitative reverse-transcriptase polymerase chain reaction, Western blotting, immunohistochemistry, and isometric tension techniques were used to characterize the expression and function of K(IR) channels in normal and vasospastic basilar artery 7 days after SAH. Subarachnoid hemorrhage resulted in severe vasospasm of the basilar artery (mean of 61% +/- 5% reduction in diameter). Membrane potential of pressurized vasospastic basilar arteries was significantly depolarized compared with control arteries (-46 +/- 1.4 mV versus -29.8 +/- 1.8 mV, respectively, P < 0.01). In whole-cell patch clamp of enzymatically isolated basilar artery myocytes, average K(IR) conductance was 1.6 +/- 0.5 pS/pF in control cells and 9.2 +/- 2.2 pS/pF in SAH cells (P = 0.007). Blocking K(IR) channels with BaCl(2) (0.1 mmol/L) resulted in significantly greater membrane depolarization in vasospastic compared with normal myocytes. Expression of K(IR) 2.1 messenger ribonucleic acid (mRNA) was increased after SAH. Western blotting and immunohistochemistry also showed increased expression of K(IR) protein in vasospastic smooth muscle. Blockage of K(IR) channels in arteries under isometric tension produced a greater contraction in SAH than in control arteries. These results document increased expression of K(IR) 2.1 mRNA and protein during vasospasm after experimental SAH and suggest that this increase is a functionally significant adaptive response acting to reduce vasospasm.

Animals↗
PubMed2005

Structural determinants of benzodiazepine allosteric regulation of GABA(A) receptor currents.

Benzodiazepine enhancement of GABA(A) receptor current requires a gamma subunit, and replacement of the gamma subunit by the delta subunit abolishes benzodiazepine enhancement. Although it has been demonstrated that benzodiazepines bind to GABA(A) receptors at the junction between alpha and gamma subunits, the structural basis for the coupling of benzodiazepine binding to allosteric enhancement of the GABA(A) receptor current is unclear. To determine the structural basis for this coupling, the present study used a chimera strategy, using gamma2L-delta GABA(A) receptor subunit chimeras coexpressed with alpha1 and beta3 subunits in human embryonic kidney 293T cells. Different domains of the gamma2L subunit were replaced by delta subunit sequence, and diazepam sensitivity was determined. Chimeric subunits revealed two areas of interest: domain 1 in transmembrane domain 1 (M1) and domain 2 in the C-terminal portion of transmembrane domain 2 (M2) and the M2-M3 extracellular loop. In those domains, site-directed mutagenesis demonstrated that the following two groups of residues were involved in benzodiazepine transduction of current enhancement: residues Y235, F236, T237 in M1; and S280, T281, I282 in M2 as well as the entire M2-M3 loop. These results suggest that a pocket of residues may transduce benzodiazepine binding to increased gating. Benzodiazepine transduction involves a group of residues that connects the N terminus and M1, and another group of residues that may facilitate an interaction between the N terminus and the M2 and M2-M3 loop domains.

Allosteric Regulation↗
PubMed2005

Endoplasmic reticulum retention and associated degradation of a GABAA receptor epilepsy mutation that inserts an aspartate in the M3 transmembrane segment of the alpha1 subunit.

A GABA(A) receptor alpha1 subunit epilepsy mutation (alpha1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the alpha1 subunit. We reported previously that heterologous expression of alpha1(A322D)beta2gamma2 receptors in mammalian cells resulted in reduced total and surface alpha1 subunit protein. Here we demonstrate the mechanism of this reduction. Total alpha1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 +/- 6%) or when coexpressed with beta2 and gamma2S subunits (78 +/- 6%), indicating an expression reduction prior to subunit oligomerization. In alpha1beta2gamma2S receptors, endoglycosidase H deglycosylated only 26 +/- 5% of alpha1 subunits, consistent with substantial protein maturation, but in alpha1(A322D)beta2gamma2S receptors, endoglycosidase H deglycosylated 91 +/- 4% of alpha1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the alpha1 subunit was tagged with yellow (alpha1-YFP) or cyan (alpha1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 +/- 4% of alpha1-YFPbeta2gamma2 but only 5 +/- 1% alpha1(A322D)-YFPbeta2gamma2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 +/- 4% alpha1-YFPbeta2gamma2S, 86 +/- 3% alpha1(A322D)-YFP). Heterozygous expression of alpha1-CFPbeta2gamma2S and alpha1(A322D)-YFPbeta2gamma2S or alpha1-YFPbeta2gamma2S and alpha1(A322D)-CFPbeta2gamma2S receptors showed that membrane GABA(A) receptors contained primarily wild type alpha1 subunits. These data demonstrate that the A322D mutation reduces alpha1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane alpha1 subunits that are almost exclusively wild type subunits.

Amino Acid Substitution↗
PubMed2004

GABA(A) receptor epilepsy mutations.

Idiopathic generalized epilepsy (IGE) syndromes are diseases that are characterized by absence, myoclonic, and/or primary generalized tonic-clonic seizures in the absence of structural brain abnormalities. Although it was long hypothesized that IGE had a genetic basis, only recently have causative genes been identified. Here we review mutations in the GABA(A) receptor alpha1, gamma2, and delta subunits that have been associated with different IGE syndromes. These mutations affect GABA(A) receptor gating, expression, and/or trafficking of the receptor to the cell surface, all pathophysiological mechanisms that result in neuronal disinhibition and thus predispose affected patients to seizures.

Epilepsy↗
PubMed2004

The GABAA receptor gamma2 subunit R43Q mutation linked to childhood absence epilepsy and febrile seizures causes retention of alpha1beta2gamma2S receptors in the endoplasmic reticulum.

The GABA(A) receptor gamma2 subunit mutation R43Q is an autosomal dominant mutation associated with childhood absence epilepsy and febrile seizures. Previously, we demonstrated that homozygous alpha1beta3gamma2L(R43Q) receptor whole-cell currents had reduced amplitude with unaltered time course, suggesting reduced cell surface expression of functional receptors. In human embryonic kidney 293-T cells, we demonstrate that both heterozygous and homozygous alpha1beta2gamma2S(R43Q) GABA(A) receptor current amplitudes were reduced when receptors were assembled from coexpressed alpha1, beta2, and gamma2S subunits and from beta2-alpha1 tandem subunits coexpressed with the gamma2L subunit. Using fluorescence confocal microscopy, we demonstrated that mutant receptors containing enhanced yellow fluorescent protein-tagged gamma2S subunits had reduced surface expression and were retained in the endoplasmic reticulum. In addition, using biotinylation of surface receptors and immunoblotting, we confirmed that alpha1beta2gamma2S(R43Q) receptors had reduced surface expression. These results provide evidence that the gamma2S(R43Q) mutation impaired GABA(A) receptor function by compromising receptor trafficking and reducing surface expression.

Animals↗
PubMed2004

Multiple actions of propofol on alphabetagamma and alphabetadelta GABAA receptors.

GABAA receptors are predominantly composed of alphabetagamma and alphabetadelta isoforms in the brain. It has been proposed that alphabetagamma receptors mediate phasic inhibition, whereas alphabetadelta receptors mediate tonic inhibition. Propofol (2,6-di-isopropylphenol), a widely used anesthetic drug, exerts its effect primarily by modulating GABAA receptors; however, the effects of propofol on the kinetic properties of alphabetagamma and alphabetadelta receptors are uncertain. We transfected human embryonic kidney (HEK293T) cells with cDNAs encoding rat alpha1, alpha6, beta3, gamma2L, or delta subunits and performed whole-cell patch-clamp recordings to explore this issue. Propofol (3 microM) increased GABA concentration-response curve maximal currents similarly for both alpha1beta3gamma2L and alpha6beta3gamma2L receptors, but propofol increased those for alpha1beta3delta and alpha6beta3delta receptors differently, the increase being greater for alpha1beta3delta than for alpha6beta3delta receptors. Propofol (10 microM) produced similar alterations in alpha1beta3gamma2L and alpha6beta3gamma2L receptor currents when using a preapplication protocol; peak currents were not altered, desensitization was reduced, and deactivation was prolonged. Propofol enhanced peak currents for both alpha1beta3delta and alpha6beta3delta receptors, but the enhancement was greater for alpha1beta3delta receptors. Desensitization of these two isoforms was not modified by propofol. Propofol did not alter the deactivation rate of alpha1beta3delta receptor currents but did slow deactivation of alpha6beta3delta receptor currents. The findings that propofol reduced desensitization and prolonged deactivation of gamma2L subunit-containing receptors and enhanced peak currents or prolonged deactivation of delta subunit-containing receptors suggest that propofol enhancement of both phasic and tonic inhibition may contribute to its anesthetic effect in the brain.

Cell Line↗
PubMed2004

Pentobarbital differentially modulates alpha1beta3delta and alpha1beta3gamma2L GABAA receptor currents.

GABAA receptors are modulated by a variety of compounds, including the neurosteroids and barbiturates. Although the effects of barbiturates on alphabetagamma isoforms, thought to dominate phasic (synaptic) GABAergic inhibition, have been extensively studied, the effects of pentobarbital on kinetic properties of alphabetadelta GABAA receptors, thought to mediate tonic (extra- or perisynaptic) inhibition, are unknown. Using ultrafast drug delivery and single channel recording techniques, we demonstrate isoform-specific pentobarbital modulation of low-efficacy, minimally desensitizing alpha1beta3 currents and high-efficacy, rapidly desensitizing alpha1beta3gamma2L currents. Specifically, with saturating concentrations of GABA, pentobarbital substantially potentiated peak alpha1beta3delta receptor currents but failed to potentiate peak alpha1beta3gamma2L receptor currents. Also, pentobarbital had opposite effects on the desensitization of alpha1beta3delta (increased) and alpha1beta3gamma2L (decreased) receptor currents evoked by saturating GABA. Pentobarbital increased steady-state alpha1beta3delta receptor single channel open duration primarily by introducing a longer duration open state, whereas for alpha1beta3gamma2L receptor channels, pentobarbital increased mean open duration by increasing the proportion and duration of the longest open state. The data support previous suggestions that GABA may be a partial agonist at alphabetadelta isoforms, which may render them particularly sensitive to allosteric modulation. The remarkable increase in gating efficacy of alpha1beta3delta receptors suggests that alphabetadelta isoforms, and by inference tonic forms of inhibition, may be important targets for barbiturates.

Cells, Cultured↗
PubMed2004

The juvenile myoclonic epilepsy GABA(A) receptor alpha1 subunit mutation A322D produces asymmetrical, subunit position-dependent reduction of heterozygous receptor currents and alpha1 subunit protein expression.

Individuals with autosomal dominant juvenile myoclonic epilepsy are heterozygous for a GABA(A) receptor alpha1 subunit mutation (alpha1A322D). GABA(A) receptor alphabetagamma subunits are arranged around the pore in a beta-alpha-beta-alpha-gamma sequence (counterclockwise from the synaptic cleft). Therefore, each alpha1 subunit has different adjacent subunits, and heterozygous expression of alpha1(A322D), beta, and gamma subunits could produce receptors with four different subunit arrangements: beta-alpha1-beta-alpha1-gamma (wild type); beta-alpha1(A322D)-beta-alpha1-gamma (Het(betaalphabeta)); beta-alpha1-beta-alpha1(A322D)-gamma (Het(betaalphagamma));beta-alpha1(A322D)-beta-alpha1(A322D)-gamma (homozygous). Expression of a 1:1 mixture of wild-type andalpha1(A322D) subunits with beta2S and gamma2S subunits (heterozygous transfection) produced smaller currents than wild type and much larger currents than homozygous mutant transfections. Western blot and biotinylation assays demonstrated that the amount of total and surface alpha1 subunit from heterozygous transfections was also intermediate between those of wild-type and homozygous mutant transfections. alpha1(A322D) mutations were then made in covalently tethered triplet (gamma2S-beta2S-alpha1) and tandem (beta2S-alpha1) concatamers to target selectively alpha1(A322D) to each of the asymmetric alpha1 subunits. Coexpression of mutant and wild-type concatamers resulted in expression of either Het(betaalphabeta) or Het(betaalphagamma) receptors. Het(betaalphabeta) currents were smaller than wild type and much larger than Het(betaalphagamma) and homozygous currents. Furthermore, Het(betaalphabeta) transfections contained less beta-alpha concatamer than wild type but more than both Het(betaalphagamma) and homozygous mutant transfections. Thus, whole-cell currents and protein expression of heterozygous alpha1(A322D)beta2Sgamma2S receptors depended on the position of the mutant alpha1 subunit, and GABA(A) receptor currents in heterozygous individuals likely result primarily from wild-type and Het(betaalphabeta) receptors with little contribution from Het(betaalphagamma) and homozygous receptors.

Cell Line↗
PubMed2004

Proton modulation of alpha 1 beta 3 delta GABAA receptor channel gating and desensitization.

Alphabetagamma GABA(A) receptor currents are phasic and desensitizing, whereas alphabetadelta GABA(A) receptor currents are tonic and have no fast desensitization. alphabetagamma receptors are subsynaptic and mediate phasic inhibition, whereas alphabetadelta receptors are extra- or perisynaptic and mediate tonic inhibition. Given the different roles of these GABA(A) receptor isoforms and the fact that GABA(A) receptors are allosterically regulated by extracellular pH in a subunit-dependent manner, we compared the effects of changing pH on rat delta or gamma2L subunit-containing GABA(A) receptor currents. Human embryonic kidney cells (HEK293T) were transfected with cDNAs encoding rat alpha1, beta3, gamma2L, or delta GABA(A) receptor subunits in several binary and ternary combinations, and whole cell and single channel patch-clamp recordings were obtained. Lowering pH substantially enhanced alpha1beta3 receptor currents. This effect was significantly more pronounced for ternary alpha1beta3delta receptors, whereas ternary alpha1beta3gamma2L receptors were relatively insensitive to lowered pH. Lowering pH did not affect the extent of desensitization of alpha1beta3 and alpha1beta3gamma2L receptor currents, but significantly increased the extent of desensitization of alpha1beta3delta receptor currents. Lowering pH prolonged deactivation of alpha1beta3 and alpha1beta3delta receptor currents and enhanced the "steady-state" currents of alpha1beta3delta receptors evoked by long-duration (28 s) GABA applications. Lowering pH significantly increased mean open duration of alpha1beta3delta steady-state single channel currents due to introduction of a longer-duration open state, suggesting that low pH enhances alpha1beta3delta receptor steady-state currents by modifying GABA(A) receptor gating properties.

Cell Line↗
PubMed2004

GABRD encoding a protein for extra- or peri-synaptic GABAA receptors is a susceptibility locus for generalized epilepsies.

A major challenge in understanding complex idiopathic generalized epilepsies has been the characterization of their underlying molecular genetic basis. Here, we report that genetic variation within the GABRD gene, which encodes the GABAA receptor delta subunit, affects GABA current amplitude consistent with a model of polygenic susceptibility to epilepsy in humans. We have found a GABRD Glu177Ala variant which is heterozygously associated with generalized epilepsy with febrile seizures plus. We also report an Arg220His allele in GABRD which is present in the general population. Compared with wild-type receptors, alpha1beta2Sdelta GABAA receptors containing delta Glu177Ala or Arg220His have decreased GABAA receptor current amplitudes. As GABAA receptors mediate neuronal inhibition, the reduced receptor current associated with both variants is likely to be associated with increased neuronal excitability. Since delta subunit-containing receptors localize to extra- or peri-synaptic membranes and are thought to be involved in tonic inhibition, our results suggest that alteration of this process may contribute to the common generalized epilepsies.

Amino Acid Substitution↗
PubMed2004

Functional characterization of mouse alpha4beta2 nicotinic acetylcholine receptors stably expressed in HEK293T cells.

Mouse alpha4beta2 nicotinic acetylcholine receptors (nAchRs) were stably expressed in HEK293T cells. The function of this stable cell line, termed mmalpha4beta2, was assessed using an aequorin-based luminescence method that measures agonist-evoked changes in intracellular calcium. Agonist-elicited changes in intracellular calcium were due primarily to direct entry of calcium through the alpha4beta2 channel, although release of calcium from intracellular stores contributed approximately 28% of the agonist-evoked response. Agonist pharmacologies were very similar between the mmalpha4beta2 cells and most cell lines that stably express human alpha4beta2 nAchRs. Based on agonist profiles and sensitivity to the antagonist dihydro-beta-erythroidine (DHbetaE), the predominant alpha4beta2 nAchR expressed in the mmalpha4beta2 cells exhibits a pharmacology that most resembles the DHbetaE-sensitive component of 86Rb+ efflux from mouse brain synaptosomes. However, when evaluated with the aequorin assay, the mmalpha4beta2 nAchR was found to be atypically sensitive to blockade by the presumed alpha7-selective antagonist methyllycaconitine (MLA), exhibiting an IC50 value of 31 +/- 0.1 nm. Similar IC50 values have been reported for the MLA inhibition of nicotine-stimulated dopamine release, a response that is mediated by beta2-subunit-containing nAchRs and not alpha7-subunit-containing nAchRs. Consequently, at low nanomolar concentrations, MLA may not be as selective for alpha7-containing nAchRs as previously thought.

Acetylcholine↗
PubMed2003

Beta subunit phosphorylation selectively increases fast desensitization and prolongs deactivation of alpha1beta1gamma2L and alpha1beta3gamma2L GABA(A) receptor currents.

We studied the effects of phosphorylation by protein kinase A (PKA) on GABA(A) receptors (alpha1beta1gamma2L andalpha1beta3gamma2L) transiently expressed in HEK 293T cells. Under conditions favorable for PKA activation, currents obtained using whole-cell patch clamp of lifted cells displayed increased rate and extent of the fast phases of desensitization, decreased rate of current deactivation after GABA removal, and prolongation of brief IPSC-like currents. Mutation of serine residues (beta1 S409, beta3 S407, beta3 S408) revealed that only beta1 S409 and beta3 S408 were critical for the modulatory effect of PKA on GABA(A) receptor currents. Additionally, repeated pulse inhibition was increased in receptors after mutation of the critical serine to glutamate and decreased when the serine was mutated to alanine. These data demonstrate that PKA phosphorylation modulated GABA(A) receptor currents by increasing fast phases of macroscopic desensitization and suggest a role for PKA in regulating GABAergic IPSC duration.

Cell Line↗
PubMed2003

Neurosteroids shift partial agonist activation of GABA(A) receptor channels from low- to high-efficacy gating patterns.

Although GABA activates synaptic (alphabetagamma) GABA(A) receptors with high efficacy, partial agonist activation of alphabetagamma isoforms and GABA activation of the primary extrasynaptic (alphabetadelta) GABA(A) receptors are limited to low-efficacy activity, characterized by minimal desensitization and brief openings. The unusual sensitivity of alphabetadelta receptor channels to neurosteroid modulation prompted investigation of whether this high sensitivity was dependent on the delta subunit or the low-efficacy channel function that it confers. We show that the isoform specificity (alphabetadelta > alphabetagamma) of neurosteroid modulation could be reversed by conditions that reversed isoform-specific activity modes, including the use of beta-alanine to achieve increased efficacy with alphabetadelta receptors and taurine to render alphabetagamma receptors low efficacy. We suggest that neurosteroids preferentially enhance low-efficacy GABA(A) receptor activity independent of subunit composition. Allosteric conversion of partial to full agonism may be a general mechanism for reversibly scaling the efficacy of GABA(A) receptors to endogenous partial agonists.

Allosteric Regulation↗
PubMed2003

Mutations linked to generalized epilepsy in humans reduce GABA(A) receptor current.

The first mutations of the GABA(A) receptor channel linked to familial epilepsy in humans were reported in the gamma2L subunit. When expressed in Xenopus oocytes, the gamma2 subunit R43Q mutation abolished current enhancement by the benzodiazepine, diazepam, and the gamma2 subunit K289M mutation decreased current amplitudes. In this study, single channel recording and concentration jump techniques were used to evaluate the effects of these mutations on GABA(A) receptor currents from receptors expressed in human embryonic kidney cells. When co-expressed with alpha1 and beta3 subunits, the mutated gamma2 subunits did not alter current activation rates or rates or extent of desensitization during prolonged (400 ms) GABA application (1 mM). Deactivation following brief (5 ms) or prolonged (400 ms) GABA application was accelerated for the K289M, but not the R43Q mutation. Single channel analysis showed shorter mean open duration, suggesting that the faster deactivation was likely caused by altered gating efficacy. Interestingly, the R43Q mutation did not alter diazepam potentiation. However, significantly smaller current amplitudes were observed that were not explained by decreased single channel conductance or open time, suggesting this mutation resulted in reduced surface expression of functional receptors. These two gamma mutations likely produce disinhibition and familial epilepsy by distinct mechanisms, suggesting that maintenance of normal neuronal inhibition depends on both the peak amplitude of IPSCs and their time course.

Amino Acids, Basic↗