Complementary regulation of anaesthetic activation of human (α₆β₃γ(2L)) and Drosophila (RDL) GABA receptors by a single amino acid residue

1. The influence of a transmembrane (TM2) amino acid located at a homologous position in human beta1 (S290) and beta3 (N289) GABAA receptor subunits and the RDL GABA receptor of Drosophila (M314) upon allosteric regulation by general anaesthetics has been investigated. 2. GABA-evoked currents mediated by human wild-type (WT) alpha6beta3gamma2L or WT RDL GABA receptors expressed in Xenopus laevis oocytes were augmented by propofol or pentobarbitone. High concentrations of either anaesthetic directly activated alpha6beta3gamma2L, but not RDL, receptors. 3. GABA-evoked currents mediated by human mutant GABAA receptors expressing the RDL methionine residue (i.e. alpha6beta3N289Mgamma2L) were potentiated by propofol or pentobarbitone with approximately 2-fold reduced potency and, in the case of propofol, reduced maximal effect. Conspicuously, the mutant receptor was refractory to activation by either propofol or pentobarbitone. 4. Incorporation of the homologous GABAA beta1-subunit residue in the RDL receptor (i.e. RDLM314S) increased the potency, but not the maximal effect, of GABA potentiation by either propofol or pentobarbitone. Strikingly, either anaesthetic now activated the receptor, an effect confirmed for propofol utilizing expression of WT or mutant RDL subunits in Schnieder S2 cells. At RDL receptors expressing the homologous beta3-subunit residue (i.e. RDLM314N) the actions of propofol were similarly affected, whereas those of pentobarbitone were unaltered. 5. The results indicate that the identity of a homologous amino acid affects, in a complementary manner, the direct activation of human (alpha6beta3gamma2L) and RDL GABA receptors by structurally distinct general anaesthetics. Whether the crucial residue acts as a regulator of signal transduction or as a component of an anaesthetic binding site per se is discussed

Mutation at the putative GABA(A) ion-channel gate reveals changes in allosteric modulation

1. We have mutated a conserved leucine in the putative membrane-spanning domain to serine in human GABA(A) β2 and investigated the actions of a number of GABA(A) agonists, antagonists and modulators on human α1β2ΔL259Sγ2s compared to wild type α1β2γ2s GABA(A) receptors, expressed in Xenopus oocytes. 2. The mutation resulted in smaller maximum currents to γ-aminobutyric acid (GABA) compared to α1β2γ2s receptors, and large leak currents resulting from spontaneous channel opening. As reported, this mutation significantly decreased the GABA EC(50) (110 fold), and reduced desensitization. Muscimol and the partial agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and piperidine-4-sulphonic acid (P4S) also displayed a decrease in EC(50). 3. In addition to competitively shifting GABA concentration response curves, the antagonists bicuculline and SR95531 both inhibited the spontaneous channel activity on α1β2ΔL259Sγ2s receptors, with different degrees of maximum inhibition. 4. The effects of a range of allosteric modulators, including benzodiazepines and anaesthetics were examined on a submaximal GABA concentration (EC(20)). Compared to wild type, none of these modulators potentiated the EC(20) response of α1β2ΔL259Sγ2s receptors, however they all directly activated the receptor in the absence of GABA. 5. To conclude, the above mutation resulted in receptors which exhibit a degree of spontaneous activity, and are more sensitive to agonists. Benzodiazepines and other agents modulate constitutive activity, but positive modulation of GABA is lost. The competitive antagonists bicuculline and SR95531 can also act as allosteric channel modulators through the same GABA binding site

Effects of extracellular pH on agonism and antagonism at a recombinant P2X(2) receptor

1. Under voltage-clamp conditions, the activity of agonists and antagonists at a recombinant P2X(2) receptor expressed in Xenopus oocytes was examined at different levels of extracellular pH (pH(e)). 2. In normal Ringer (Mg(2+) ions absent), the amplitude of submaximal inward currents to ATP was increased by progressively lowering pH(e) (8.0–5.5). ATP-responses reached a maximum at pH 6.5 with a 5 fold increase in ATP-affinity; the apparent pK(a) was 7.05±0.05. 3. Receptor affinity for ATP was lowered when extracellular Ca(2+) ions were replaced with equimolar Mg(2+) ions. However, the amplitude of the ATP-responses was still enhanced under acidic conditions, reaching maximal activity at pH 6.5 with a 5 fold increase in ATP-affinity; the apparent pK(a) was 7.35±0.05. 4. ATP species present in the superfusate (for the above ionic conditions and pH levels) were calculated to determine the forms of ATP which activate P2X(2) receptors: possible candidates include HATP, CaHATP and MgHATP. However, levels of these protonated species increase below pH 6.5, suggesting that receptor protonation rather than agonist protonation is more important. 5. The potency order for agonists of P2X(2) receptors was: ATP>2-MeS-ATP⩾ATPγS> ATPαS>>CTP⩾BzATP, while other nucleotides were inactive. EC(50) and n(H) values for full agonists were determined at pH 7.4 and re-examined at pH 6.5. Extracellular acidification increased the affinity by approximately 5 fold for full agonists (ATP, 2-MeSATP, ATPγS and ATPαS), without altering the potency order. 6. The potency order for antagonists at P2X(2) receptors was: Reactive blue-2>trinitrophenol-ATP⩾Palatine fast black⩾Coomassie brilliant blue⩾PPADS>suramin (at pH 7.4). IC(50) values and slopes of the inhibition curves were re-examined at different pH levels. Only blockade by suramin was affected significantly by extracellular acidification (IC(50) values: 10.4±2 μM, at pH 7.4; 78±5 nM, at pH 6.5; 30±6 nM, at pH 5.5). 7. In summary, a lowered pH(e) enhanced the activity of all agonists at P2X(2) receptors but, with the exception of suramin, not antagonists. Since a lowered pH(e) is also known to enhance agonist activity at P(2X) receptors on sensory neurones containing P2X(2) transcripts, the sensitization by metabolic acidosis of native P(2X) receptors containing P2X(2) subunits may have a significant effect on purinergic cell-to-cell signalling