Key roles of hydrophobic rings of TM2 in gating of the alpha9alpha10 nicotinic cholinergic receptor.
We have performed a systematic mutagenesis of three hydrophobic rings (17', 13' and 9') within transmembrane region (TM) 2 of the alpha9alpha10 nicotinic cholinergic receptor (nAChR) to a hydrophilic (threonine) residue and compared the properties of mutant receptors reconstituted in Xenopus laevis oocytes. Phenotypic changes in alpha9alpha10 mutant receptors were evidenced by a decrease in the desensitization rate, an increase in both the EC(50) for ACh as well as the efficacy of partial agonists and the reduction of the allosteric modulation by extracellular Ca(2+). Mutated receptors exhibited spontaneous openings and, at the single-channel level, an increased apparent mean open time with no major changes in channel conductance, thus suggesting an increase in gating of the channel as the underlying mechanism. Overall, the degrees of the phenotypes of mutant receptors were more overt in the case of the centrally located V13'T mutant. Based on the atomic model of the pore of the electric organ of the Torpedo ray, we can propose that the interactions of side chains at positions 13' and 9' are key ones in creating an energetic barrier to ion permeation. In spite of the fact that the roles of the TM2 residues are mostly conserved in the distant alpha9alpha10 member of the nAChR family, their mechanistic contributions to channel gating show significant differences when compared to other nAChRs. These differences might be originated from slight differential intramolecular rearrangements during gating for the different receptors and might lead each nAChR to be in tune with their physiological roles.
Plazas
Paola V
PV
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, CONICET-UBA, Vuelta de Obligado 2490, Buenos Aires 1428, Argentina.
De Rosa
María J
MJ
Gomez-Casati
María E
ME
Verbitsky
Miguel
M
Weisstaub
Noelia
N
Katz
Eleonora
E
Bouzat
Cecilia
C
Elgoyhen
Ana Belén
AB
D000109:Acetylcholine / Q000494:pharmacology; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D018678:Cholinergic Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015640:Ion Channel Gating / Q000235:genetics*; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010641:Phenotype; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D014982:Xenopus laevis
D018678:Cholinergic Agents; C470699:Chrna10 protein, rat; C498541:Chrna9 protein, rat; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D000109:Acetylcholine; D002118:Calcium