title: Exon 3 splicing and mutagenesis identify residues influencing cell surface density of heterologously expressed silkworm (Bombyx mori) glutamate-gated chloride channels.
abstract: Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. Insect GluCls show alternative splicing, and, to determine its impact on channel function and pharmacology, we isolated GluCl cDNAs from larvae of the silkworm (Bombyx mori). We show that six B. mori glutamate-gated chloride channel variants are generated by splicing in exons 3 and 9 and that exons 3b and 3c are common in the brain and third thoracic ganglion. When expressed in Xenopus laevis oocytes, the three functional exon 3 variants (3a, b, c) all had similar EC50 values for l-glutamate and ivermectin (IVM); however, Imax (the maximum l-glutamate- and IVM-induced response of the channels at saturating concentrations) differed strikingly between variants, with the 3c variant showing the largest l-glutamate- and IVM-induced responses. By contrast, a partial deletion detected in exon 9 had a much smaller impact on l-glutamate and IVM actions. Binding assays using [(3)H]IVM indicate that diversity in IVM responses among the GluCl variants is mainly due to the impact on channel assembly, altering receptor cell surface numbers. GluCl variants expressed in HEK293 cells show that structural differences influenced Bmax but not Kd values of [(3)H]IVM. Domain swapping and site-directed mutagenesis identified four amino acids in exon 3c as hot spots determining the highest amplitude of the l-glutamate and IVM responses. Modeling the GluCl 3a and 3c variants suggested that three of the four amino acids contribute to intersubunit contacts, whereas the other interacts with the TM2-TM3 linker, influencing the receptor response.
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, Nakamachi Nara, Japan (S.F., M.I., K.M.); Graduate School of Life Science, University of Hyogo, Koto, Kamigori-cho, Ako-gun, Hyogo, Japan (Y.N.); Graduate School of Agriculture, Kyoto University, Sakyo-Ku, Kyoto, Japan (M.A.); Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom (A.K.J.); and Department of Medicine, Wolfson Institute for Biomedical Research, University College London, London, United Kingdom (D.B.S.).
authors.forename: Shogo
authors.initials: S
authors.lastname: Furutani
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, Nakamachi Nara, Japan (S.F., M.I., K.M.); Graduate School of Life Science, University of Hyogo, Koto, Kamigori-cho, Ako-gun, Hyogo, Japan (Y.N.); Graduate School of Agriculture, Kyoto University, Sakyo-Ku, Kyoto, Japan (M.A.); Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom (A.K.J.); and Department of Medicine, Wolfson Institute for Biomedical Research, University College London, London, United Kingdom (D.B.S.).
authors.forename: Makoto
authors.identifier: http://orcid.org/0000-0002-6403-0781
authors.initials: M
authors.lastname: Ihara
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, Nakamachi Nara, Japan (S.F., M.I., K.M.); Graduate School of Life Science, University of Hyogo, Koto, Kamigori-cho, Ako-gun, Hyogo, Japan (Y.N.); Graduate School of Agriculture, Kyoto University, Sakyo-Ku, Kyoto, Japan (M.A.); Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom (A.K.J.); and Department of Medicine, Wolfson Institute for Biomedical Research, University College London, London, United Kingdom (D.B.S.).
authors.forename: Yuri
authors.initials: Y
authors.lastname: Nishino
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, Nakamachi Nara, Japan (S.F., M.I., K.M.); Graduate School of Life Science, University of Hyogo, Koto, Kamigori-cho, Ako-gun, Hyogo, Japan (Y.N.); Graduate School of Agriculture, Kyoto University, Sakyo-Ku, Kyoto, Japan (M.A.); Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom (A.K.J.); and Department of Medicine, Wolfson Institute for Biomedical Research, University College London, London, United Kingdom (D.B.S.).
authors.forename: Miki
authors.initials: M
authors.lastname: Akamatsu
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, Nakamachi Nara, Japan (S.F., M.I., K.M.); Graduate School of Life Science, University of Hyogo, Koto, Kamigori-cho, Ako-gun, Hyogo, Japan (Y.N.); Graduate School of Agriculture, Kyoto University, Sakyo-Ku, Kyoto, Japan (M.A.); Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom (A.K.J.); and Department of Medicine, Wolfson Institute for Biomedical Research, University College London, London, United Kingdom (D.B.S.).
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Jones
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, Nakamachi Nara, Japan (S.F., M.I., K.M.); Graduate School of Life Science, University of Hyogo, Koto, Kamigori-cho, Ako-gun, Hyogo, Japan (Y.N.); Graduate School of Agriculture, Kyoto University, Sakyo-Ku, Kyoto, Japan (M.A.); Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom (A.K.J.); and Department of Medicine, Wolfson Institute for Biomedical Research, University College London, London, United Kingdom (D.B.S.).
authors.forename: David B
authors.identifier: http://orcid.org/0000-0003-0705-5242
authors.initials: DB
authors.lastname: Sattelle
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, Nakamachi Nara, Japan (S.F., M.I., K.M.); Graduate School of Life Science, University of Hyogo, Koto, Kamigori-cho, Ako-gun, Hyogo, Japan (Y.N.); Graduate School of Agriculture, Kyoto University, Sakyo-Ku, Kyoto, Japan (M.A.); Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom (A.K.J.); and Department of Medicine, Wolfson Institute for Biomedical Research, University College London, London, United Kingdom (D.B.S.) kmatsuda@nara.kindai.ac.jp.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Matsuda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D005091:Exons; D018698:Glutamic Acid / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007559:Ivermectin / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012326:RNA Splicing; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D018118:Chloride Channels; C109777:glutamate-gated chloride channels; D018698:Glutamic Acid; D007559:Ivermectin