title: Involvement of the left-flipper-to-dorsal-fin interface of the zebrafish P2X4 receptor in ATP binding and structural rearrangement.
abstract: P2X receptors are trimeric ATP-activated non-selective cation channels. The ATP binding pocket is positioned between two neighboring subunits. Accompanying ligand binding, subunit-subunit contacts are most likely involved in receptor function and drive a conformational change to open the ion permeation pathway. In this way, we sought to determine the function of side chains of the zebrafish P2X4 receptor ectodomain left-flipper-to-dorsal-fin interface residues in ligand binding. By combining site-directed mutagenesis and electrophysiology methods, we showed that cysteine substitutions of I212, S215, Y216 and L217 resulted in decreased sensitivity to ATP. In addition, the ATP induced current at L217C was completely inhibited by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)), indicating a role for this residue in ATP action. Deletion of residues 285-293 from the zebrafish P2X4 receptor abolished channel function. However, insertion of the same sequence frame into a homologous position of the rat P2X6 receptor did not rescue channel function, suggesting that these residues are necessary but not sufficient for achieving the correct ATP-induced conformation.
authors.affiliation: Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Jie
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei 230027, China.
authors.forename: Longmei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Xu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei 230027, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Gao
authors.affiliation: Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors.forename: Wenbo
authors.initials: W
authors.lastname: Ma
authors.affiliation: Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China. Electronic address: li_zhiyuan@gibh.ac.cn.
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Li
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D058500:Animal Fins; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000235:genetics / Q000502:physiology; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics; D058848:Receptors, Purinergic P2X4 / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D015027:Zebrafish
chemical_list: D021122:Protein Subunits; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; C110590:purinoceptor P2X6; D000255:Adenosine Triphosphate
keywords: ATP-binding; Conformational changes; Left-flipper-to-dorsal-fin interface; Mutagenesis; P2X receptor