Mutational Analysis of the Charge Selectivity Filter of the a 7 Nicotinic Acetylcholine Receptor receptor from Torpedo electric organ with channel blockers

receptor from Torpedo electric organ with channel blockers (Giraudat et al., 1986; Hucho et al., 1986; Pedersen and Cohen, 1990; White and Cohen, 1992), together with mutagenesis of the affinity-labeled amino acids or their homologs in muscle-type 2a1b1gd receptor Pierre-Jean Corringer,* Sonia Bertrand,† Jean-Luc Galzi,*# Anne Devillers-Thiéry,* Jean-Pierre Changeux,*‡ and Daniel Bertrand† *Neurobiologie Moléculaire Unité de Recherche Associée au Centre National (Leonard et al., 1988; Charnet et al., 1990), pointed to de la Recherche Scientifique D1284 the M2 membrane-spanning segment of all subunits as Institut Pasteur contributing to the pharmacological site for ion channel 25 rue du Docteur Roux blockers. The pattern of labeling supports a model ac75724 Paris Cedex 15 cording to which: (1) the ion channel blocker–binding France site is located along the axis of pseudosymmetry of the †Département de Physiologie five M2 segments organized in a barrel-like structure; Faculté de Médecine (2) the binding site is formed by the superimposition of Centre Médical Universitaire chemically defined rings of homologous amino acids; 1211 Geneva 4 and (3) the M2 segment is coiled into an a helix (reviewed Switzerland in Galzi and Changeux, 1995). These conclusions were further supported by the substituted cysteine accessibility method (SCAM) developed to map the residues acSummary cessible to charged organic reagents (Akabas et al., 1994). Moreover, site-directed mutagenesis experiIn the a7 nicotinic acetylcholine receptors, we analyze ments carried out within or in the vicinity of M2 unambigthe contribution of mutations E237A and V251T, touously implicated this segment in ion transport propergether with the proline insertion P2369, in the converties (Imoto et al., 1986). Mutations of muscle-type and/ sion of the charge selectivity from cationic to anionic. or neuronal a7 receptors at the level of the rings of We show that the triple mutant exhibits spontaneous negatively charged residues located at both ends of M2 openings displaying anionic selectivity. Furthermore, and referred to as “inner,” “intermediate,” and “outer” at position 251, hydrophilic or even negatively charged negative rings (corresponding to a7 D234, E237, and residues are compatible with an anionic channel. In E258) (Imoto et al., 1988; Kienker et al., 1994) or affecting contrast, the additional proline yields an anionic chanpolar (corresponding to a7 S240 and T244) (Leonard et nel only when inserted between positions 234 and 237; al., 1988; Villarroel et al., 1991, 1992) or even hydrophoinsertion before 234 yields a cationic channel and after bic amino acids (corresponding to a7 L247 and V251) 238 alters the receptor surface expression. The coiled (Revah et al., 1991; Bertrand et al., 1993) within M2 234–238 loop thus directly contributes to the charge altered channel conductance and, in some cases, the selectivity filter of the a7 channel. selectivity among monovalent (Konno et al., 1991; Cohen et al., 1992) or divalent cations (Bertrand et al., Introduction 1993). Also, electron microscopy of Torpedo receptor revealed structural features at 9 Å resolution within Ligand-gated ion channels are allosteric membrane prothe transmembrane domains (Unwin, 1993) in both the teins (Changeux and Edelstein, 1998) that transduce the closed and open conformations (Unwin, 1995), and the binding of specific agonists into the opening of selective five rods arranged around the axis of rotational symmechannels through which ions passively diffuse across try of the receptor molecule were tentatively assigned the cell membrane. From one neurotransmitter receptor to the M2 segments. These data are summarized in to another, a wide range of ionic selectivities are Figure 1. observed that determine their individual physiological Comparison of the M2 sequences and their flanking actions. Among these channel-linked receptors, the suregions within the superfamily of ligand-gated ion chanperfamily of phylogenetically related receptors for acenels revealed minor though striking differences between tylcholine/nicotine (nAChR), glycine (GlyR), g-aminobuthe homooligomeric cationic chick a7 nAChR and antyric acid (GABAAR), and serotonin (5-HT3R) have been ionic glycine a1 receptor (Galzi et al., 1992), thus paving intensively investigated for several decades, but fundathe way for the identification of the charge selectivity mental questions concerning gating and selectivity refilter of the ion channel. In a first step, the amino acids main unresolved (Karlin and Akabas, 1995; Le Novère assumed to face the lumen of the ion channel were and Changeux, 1995; Lindstrom, 1996; Role and Berg, transferred from the glycine a1 receptor into the a7 1996). receptor (Table 1). The resulting construct, a7-1, was Affinity labeling of the membrane-bound muscle-type found to possess an anion-selective channel gated by acetylcholine. Then, all the amino acids that did not contribute to the conversion of ionic selectivity were ‡ To whom correspondence should be addressed (e-mail: changeux@ eliminated. The minimal set of mutations sufficient to pasteur.fr). confer anionic selectivity was found located by Galzi et # Present address: Unité Propre de Recherche CNRS 9050, Ecole al. (1992) at the level of (1) one ring of hydrophobic Supérieure de Biologie de Strasbourg, boulevard Sébastien Brant,