The pre‐M1 segment of the α1 subunit is a transduction element in the activation of the GABAA receptor

CV Starr Laboratory for Molecular Pharmacology, Department of Anesthesiology, Weill Medical College, Cornell University, A-1040, 1300 York Avenue, New York, NY 10021, USA.
The Journal of Physiology (Impact Factor: 5.04). 09/2006; 575(Pt 1):11-22. DOI: 10.1113/jphysiol.2005.102756
Source: PubMed


The binding of the neurotransmitter GABA induces conformational changes in the GABAA receptor (GABAAR), leading to the opening of a gate that controls ion permeation through an integral transmembrane pore. A number of structural elements within each subunit, located near the membrane interface, are believed to undergo relative movements during this activation process. In this study, we explored the functional role of the beta-10 strand (pre-M1 segment), which connects the extracellular domain to the transmembrane domain. In alpha1beta2gamma2s GABAARs, analysis of the 12 residues of the beta-10 strand in the alpha1 subunit proximal to the first transmembrane domain identified two residues, alpha1V212 and alpha1K220, in which mutations produced rightward shifts in the GABA concentration-response relationship and also reduced the relative efficacy of the partial agonist, piperidine-4-sulphonic acid. Ultra-fast agonist techniques were applied to mutant alpha1(K220A)beta2gamma2s GABAARs and revealed that the macroscopic functional deficit in this mutant could be attributed to a slowing of the opening rate constant, from approximately 1500 s(-1) in wild-type (WT) channels to approximately 730 s(-1) in the mutant channels, and a reduction in the time spent in the active state for the mutant. These changes were accompanied by a decrease in agonist affinity, with half-maximal activation rates achieved at 0.77 mM GABA in WT and 1.4 mM GABA in the alpha1(K220A)beta2gamma2s channels. The beta-10 strand (pre-M1 segment) emerges, from this and other studies, as a key functional component in the activation of the GABAAR.

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    • "Its importance in function has been extensively studied because of the naturally occurring hyperekplexia mutation of G221 (Rajendra et al. 1997). The 'Pre-TM1' segment preceding G221 also has been studied extensively (Castaldo et al. 2004; Keramidas et al. 2006). We previously used the C-terminal segment of the AChBP (Brejc et al. 2001) to orient the ligand-binding domain of the GlyR and GABA A R with respect to the center of the TM1 alpha helix (Trudell and Bertaccini 2004). "
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    ABSTRACT: J. Neurochem. (2008) 104, 1649–1662. The glycine receptor is a member of the Cys-loop, ligand-gated ion channel family and is responsible for inhibition in the CNS. We examined the orientation of amino acids I229 in transmembrane 1 (TM1) and A288 in TM3, which are both critical for alcohol and volatile anesthetic action. We mutated these two amino acids to cysteines either singly or in double mutants and expressed the receptors in Xenopus laevis oocytes. We tested whether disulfide bonds could form between A288C in TM3 paired with M227C, Y228C, I229C, or S231C in TM1. Application of cross-linking (mercuric chloride) or oxidizing (iodine) agents had no significant effect on the glycine response of wild-type receptors or the single mutants. In contrast, the glycine response of the I229C/A288C double mutant was diminished after application of either mercuric chloride or iodine only in the presence of glycine, indicating that channel gating causes I229C and A288C to fluctuate to be within 6 Å apart and form a disulfide bond. Molecular modeling was used to thread the glycine receptor sequence onto a nicotinic acetylcholine receptor template, further demonstrating that I229 and A288 are near-neighbors that can cross-link and providing evidence that these residues contribute to a single binding cavity.
    Journal of Neurochemistry 04/2008; 104(6):1649-62. DOI:10.1111/j.1471-4159.2007.05090.x · 4.28 Impact Factor
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    • "The activation of the Cys-loop ligand-gated ion channels involves the binding of the agonist to the receptor, the transduction of the binding signal to the channel gate, and the opening of the channel. The structural determinants in portions of loops 2, 7, and 9 and ␤8-␤9 linker of the extracellular N terminus (Absalom et al., 2003; Lee and Sine, 2005; Xiu et al., 2005; Sine and Engel, 2006; Gay and Yakel, 2007), preTM1 (Hu et al., 2003; Kash et al., 2003; Lee and Sine, 2005; Xiu et al., 2005; Keramidas et al., 2006; Mercado and Czajkowski, 2006), and transmembrane domain 2-3 loop (Grosman et al., 2000; Kash et al., 2003; Lummis et al., 2005; Xiu et al., 2005) have been implicated in relaying agonist binding to channel gating. It is interesting to note that those elements in the transduction pathway are also critical for allosteric modulation (Mihic et al., 1997; Krasowski et al., 1998b; Boileau and Czajkowski, 1999; Carlson et al., 2000; Chang et al., 2003; Hu and Lovinger, 2005; Jones-Davis et al., 2005). "
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    ABSTRACT: Ligand-gated ion channels participate in synaptic transmission, and they are involved in neurotransmitter release. The functions of the channels are regulated by a variety of modulators. The interaction of 2,2,2-trichloroethanol, the active hypnotic metabolite of chloral hydrate, with the 5-hydroxytryptamine (5-HT) (serotonin) type 3 receptor results in a positive allosteric modulation. We have demonstrated previously that arginine 246 (R246) located in the pretransmembrane domain 1 is critical for coupling agonist binding to gating. In this study, we examined the role of R246 in the action of trichloroethanol with a combination of mutagenesis and whole-cell patch-clamp techniques. The R246A mutation converted the partial agonist dopamine into a full agonist at the 5-HT(3A) receptor, and it facilitated activation of the mutant receptor by dopamine, suggesting an enhanced gating process due to the mutation. The positive modulation of the 5-HT(3A) receptor by trichloroethanol was dramatically reduced by the R246A mutation. Trichloroethanol had little agonist activity in the wild-type receptor (<1% of maximal 5-HT response). However, the R246A mutation significantly increased the direct activation of the receptor by trichloroethanol in the absence of agonist ( approximately 10% of maximal 5-HT response). The current activated by trichloroethanol could be blocked by the competitive 5-HT(3) receptor antagonist tropanyl 3,5-dichlorobenzoate (MDL 72222), and it had a similar reversal potential to those of current activated by 5-HT. In addition, predesensitization of the mutant receptor by trichloroethanol prevented 5-HT from activating the receptor. These data suggest that R246 is a crucial site for mediating the actions of both agonists and modulators.
    Journal of Pharmacology and Experimental Therapeutics 03/2008; 324(3):1011-8. DOI:10.1124/jpet.107.131011 · 3.97 Impact Factor
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    • "Given these results and the location of these closely apposed residues (Unwin, 2005; Dellisanti et al., 2007), about halfway between the transmitter binding sites and an equatorial gate and in the center of the α-subunit, they proposed that the " principle pathway " for the propagation of the gating conformational wave is as follows, in brief: agonist binding perturbs loop C, which perturbs the R209–E45 salt bridge, which perturbs other nearby elements that are somehow linked to the porelining transmembrane M2 helix and the gate (Lee and Sine, 2005). Several groups have also noted that charged residues located in the pre-M1 segment in AChRs and other pentameric, ligand-gated ion channels play a signifi cant role in expression and gating, for example in 5- HT 3A receptors (Hu et al., 2003), Gly receptors (Castaldo et al., 2004), GABA A receptors (Kash et al., 2004; Keramidas et al., 2006; Mercado and Czajkowski, 2006), and ACh receptors (Tamamizu et al., 1995; Lee and Sine, 2005; Xiu et al., 2005). Recently, evidence for a salt bridge between residues in pre-M1 and loop 2 was Correspondence to Anthony Auerbach: "
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    ABSTRACT: Charged residues in the beta10-M1 linker region ("pre-M1") are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational "wave." We examined the effects of mutations to all five residues in pre-M1 (positions M207-P211) plus E45 in loop 2 in the mouse alpha(1)-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (K(eq)), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in K(eq) (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Phi values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Phi values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only -0.33 kcal/mol (for both alpha subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.
    The Journal of General Physiology 01/2008; 130(6):559-68. DOI:10.1085/jgp.200709857 · 4.79 Impact Factor
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