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PLoS One
2013 Jan 01;84:e61733. doi: 10.1371/journal.pone.0061733.
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Partial agonism of taurine at gamma-containing native and recombinant GABAA receptors.
Kletke O
,
Gisselmann G
,
May A
,
Hatt H
,
Sergeeva OA
.
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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 GABAA-receptors (GABAAR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABAAR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α(1/2)β(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 αxβ1δ-GABAAR, we generated a chimeric γ(2) subunit carrying the δ subunit motif around F77 (MTVFLH). At α(1/2)β(1)γ2(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 GABAA receptors. Our study sheds new light on the β1 subunit conferring the widest range of taurine-efficacies modifying GABAAR function under (patho)physiological conditions.
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23637894
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Figure 2. Mutation γ2F77I reduces taurine efficacy at recombinant GABAA receptors.(A) 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) and (C). Data obtained in corresponding wild type receptors (γ2 instead of γ2F77I) are plotted in red.
Figure 3. Zinc-sensitive TMN neurons show similar efficacies for GABA and taurine.(A) 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) 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). (C) Superimposed responses to different concentrations of taurine in comparison to the maximal GABA response recorded in one zinc-sensitive neuron. (D) 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.
Figure 4. Gating of native GABAA receptors by taurine is impaired by the mutation γ2F77I.(A) Whole-cell voltage-clamp recordings (Vh = −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) γ2F77I mouse. (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. (C) GABAAR- versus GlyR-involvement in taurine-responses was tested by the co-application of taurine with gabazine (gz, GABAAR antagonist). Amplitude of the remaining response was subtracted in each neuron from the control taurine response, to construct the concentration - response curves in (B).
Figure 5. Sequence alignment of GABAA receptor subunits between amino acids 58 and 92 (γ2 mouse numbering).Underlined is a putative assembly signal conserved in different GABAA receptor subunits (36)). Note no difference between all three β- subunits in the putative assembly signal: MDYTLTMYFQQ_W with the exception for the position 81 (different residues are indicated in different colour). Interestingly, these coloured β subunit-specific residues were shown previously to affect stabilization of a homomeric assembly (45). Fat letters show δ: MTVFLH and γ2: IDIFFA motifs which were exchanged in the chimeric γ2(δ74–79) subunit. Orange field indicates location of γ2F77 site involved in zolpidem binding as well as homologous or same residues at other GABAAR subunits.
Figure 6. Chimeric αxβ1γ2(δ74–79) receptors show superagonistic properties of taurine.(A and B) Representative current traces (comparison of taurine (600 mM) - and GABA (0.3–3 mM) - evoked maximal currents) are shown for different receptor types. (A) 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 GABAAR, which renders them similarity with the α2β1δ receptors.
Figure 1. Comparison of taurine- and GABA-evoked maximal currents recorded from binary αxβx (A) or ternary αxβxγ2L (B) GABAA receptors.Note that gating by taurine of γ2 subunit containing GABAA 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 Table 1.
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