title: Amino acid residues Gln4020 and Lys4021 of the ryanodine receptor type 1 are required for activation by 4-chloro-m-cresol.
abstract: The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.
authors.affiliation: Department of Anesthesia Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115. Electronic address: fessenden@bbri.org.
authors.forename: James D
authors.initials: JD
authors.lastname: Fessenden
authors.affiliation: Department of Veterinary Medicine-Molecular Biosciences, Davis, California 95616.
authors.forename: Wei
authors.initials: W
authors.lastname: Feng
authors.affiliation: Department of Veterinary Medicine-Molecular Biosciences, Davis, California 95616; Center for Children's Environmental Health, Davis, California 95616.
authors.forename: Isaac N
authors.initials: IN
authors.lastname: Pessah
authors.affiliation: Department of Anesthesia Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Allen
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D003408:Cresols / Q000494:pharmacology*; D005973:Glutamine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D003408:Cresols; D019837:Ryanodine Receptor Calcium Release Channel; D005973:Glutamine; C006984:chlorocresol; D008239:Lysine