Pharmacological properties of vertebrate GABAAGABA_{A} and glycine receptors

Gamma-Aminobuttersäure GABA und Glycin sind die beiden wichtigsten Mediatoren neuronaler Inhibition. Die Wirkung dieser Neurotransmitter wird vor allem durch den Gamma-Aminobuttersäure Rezeptor Typ A ($GABA_{A}$) und den Strychnin-sensitiven Glycin-Rezeptor vermittelt, welche zu der Superfamilie der "Cys-loop" Liganden-gesteuerten Ionenkanäle gehören. Viele verschiedene chemische Substanzklassen sind dafür bekannt, dass sie $GABA_{A}$- und Glycin-Rezeptoren modulieren, aber nur wenige endogene Substanzen wurden identifiziert, die mit diesen Rezeptoren interagieren. Im Rahmen dieser Arbeit haben wir den Wirkmechanismus der endogenen Signalmoleküle Histamin und Taurin auf $GABA_{A}$ und Glycin-Rezeptoren elektrophysiologisch untersucht. Des Weiteren haben wir das beta-Carbolin DMCM und die Substanzen PI24513 und Vertacetal$\circledR$ coer, welche zur Klasse der "fragrant (1,3)-dioxane derivatives" gehören, als Untereinheiten-spezifische Modulatoren von $GABA_{A}$-Rezeptoren identifiziert

Partial Agonism of Taurine at Gamma-Containing Native and Recombinant GABA_A Receptors

<div><p>Taurine is a semi-essential sulfonic acid found at high concentrations in plasma and mammalian tissues which regulates osmolarity, ion channel activity and glucose homeostasis. The structural requirements of GABA_A-receptors (GABA_AR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABA_AR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α_1/2β_1/3 receptors taurine was as efficient as GABA, whereas incorporation of the γ_1/2 subunit reduced taurine efficacy to 60–90% of GABA. The mutation γ_2F77I, which abolishes zolpidem potentiation, significantly reduced taurine efficacy at recombinant and native receptors compared to the wild type controls. As taurine was a full- or super- agonist at recombinant α_xβ₁δ-GABA_AR, we generated a chimeric γ₂ subunit carrying the δ subunit motif around F77 (MTVFLH). At α_1/2β₁γ_2(MTVFLH) receptors taurine became a super-agonist, similar to δ-containing ternary receptors, but remained a partial agonist at β₃-containing receptors. In conclusion, using site-directed mutagenesis we found structural determinants of taurine’s partial agonism at γ-containing GABA_A receptors. Our study sheds new light on the β₁ subunit conferring the widest range of taurine-efficacies modifying GABA_AR function under (patho)physiological conditions.</p></div

Mutation γ_2F77I reduces taurine efficacy at recombinant GABA_A receptors.

<p>(<b>A</b>) Representative current traces show responses to the maximal GABA and taurine concentrations at different receptor types. For two representative receptor types (marked with symbols) concentration-response plots for GABA (filled symbols) and taurine (open symbols) are shown in (<b>B</b>) and (<b>C</b>). Data obtained in corresponding wild type receptors (γ₂ instead of γ_2F77I) are plotted in red.</p

Chimeric α_xβ₁γ_2(δ74–79) receptors show superagonistic properties of taurine.

<p>(<b>A and B)</b> Representative current traces (comparison of taurine (600 mM) - and GABA (0.3–3 mM) - evoked maximal currents) are shown for different receptor types. (<b>A</b>) Concentration - response curves for the β₁ -containing receptors. Concentrations of agonist are plotted versus current amplitudes normalized on maximal GABA response (filled symbols for GABA -, open symbols for taurine - responses). Red curves are given for comparison with γ₂ (WT) - containing receptors. Note the dramatic increase in taurine efficacy over GABA in chimeric β₁ - containing GABA_AR, which renders them similarity with the α₂β₁δ receptors.</p

Comparison of taurine- and GABA-evoked maximal currents recorded from binary α_xβ_x (A) or ternary α_xβ_xγ_2L (B) GABA_A receptors.

<p>Note that gating by taurine of γ₂ subunit containing GABA_A receptors is significantly less efficacious compared to the corresponding binary receptors. Representative current traces (comparison of taurine (600 mM) - and GABA (0.1–1 mM) -evoked maximal currents at different receptor subtypes) are shown at the left. Scale markers represent 0.1 µA vertically and 20 s horizontally for all figures with oocyte recordings. Right: averaged concentration - response curves. Concentration of agonist (filled symbols for GABA -, open symbols for taurine - responses) is plotted versus normalized response amplitudes. Each individual measurement was first normalized to the observed maximal GABA - current amplitude and subsequently averaged. Number of investigated oocytes, Hill coefficients (nHill) and concentrations evoking a half - maximal response (EC₅₀) are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061733#pone-0061733-t001" target="_blank">Table 1</a>.</p

GABA- and taurine- gating of different GABA_A receptor types recombinantly expressed in Xenopus oocytes.

<p>GABA I _max = 1; Responses to different taurine concentrations relative to I GABA max were fitted with a logistic Hill equation as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061733#pone-0061733-g001" target="_blank">Fig. 1:</a> EC₅₀, nHill and maximal efficacy (relative to GABA), obtained from this curve-fit analysis are provided for each receptor type. Exceptions are marked with ∼ (in these two cases, the curve-fit failed due to the large deviation from mean of experimental values. Mean relative response amplitude (to 0.6 M taurine) is given). Values represent mean ± SEM. n.p. = not predictable. All values were compared to those seen in the corresponding binary (α_xβ_x) receptor type (fat); significant difference is indicated (*p<0.05, **p<0.01, non - parametrical Mann - Whitney U-test).</p

Zinc-sensitive TMN neurons show similar efficacies for GABA and taurine.

<p>(<b>A</b>) Zinc-inhibition of GABA-evoked currents in two representative neurons. Note that these neurons respond differently to ZnCl₂ 10 µM. Block of the GABA-response by this concentration served as a criterion for the selection of “zinc-sensitive” neurons. (<b>B</b>) Photographs of two neurons and gels illustrating single-cell RT-PCR analysis of γ-subunit (γ1–γ3) expression. Note the lack of a detectable amount of γ-subunit transcripts in zinc-sensitive cell (#2). (<b>C</b>) Superimposed responses to different concentrations of taurine in comparison to the maximal GABA response recorded in one zinc-sensitive neuron. (<b>D</b>) Averaged concentration - response plots for the two neuronal groups. Significant difference between individual data points is indicated: * = p<0.05. The maximal taurine-evoked currents represented 100±5% (filled squares, EC₅₀ = 12.6±0.6 mM, n = 5) vs 74±2% (open squares, EC₅₀ = 14.9±0.9 mM, n = 6) of maximal GABA-evoked currents.</p

Gating of native GABA_A receptors by taurine is impaired by the mutation γ_2F77I.

<p>(<b>A</b>) Whole-cell voltage-clamp recordings (V_h = −50 mV) from adult WT or KI mouse TMN neurons isolated from hypothalamic slices. Taurine evokes maximal responses (at 50 and 100 mM) which are comparable in amplitude to the maximal GABA (0.5 mM)-evoked currents in wild-type (WT) mouse but represents only half of the GABA-response in the knock-in (KI) γ_2F77I mouse. (<b>B</b>) Averaged concentration - response curves obtained from 10 WT and 14 KI neurons. Significant difference between individual data points is indicated: * p<0.05; *** p<0.005. (<b>C</b>) GABA_AR- versus GlyR-involvement in taurine-responses was tested by the co-application of taurine with gabazine (gz, GABA_AR antagonist). Amplitude of the remaining response was subtracted in each neuron from the control taurine response, to construct the concentration - response curves in (B).</p

Disease-associated HCN4 V759I variant is not sufficient to impair cardiac pacemaking

The hyperpolarization-activated cation current