@article {Furutanimol.114.095869, author = {Shogo Furutani and Makoto Ihara and Yuri Nishino and Miki Akamatsu and Andrew K. Jones and David B. Sattelle and Kazuhiko Matsuda}, title = {Exon 3 Splicing and Mutagenesis Identify Residues Influencing Cell Surface Density of Heterologously-expressed Silkworm (Bombyx mori) Glutamate-gated Chloride Channels}, elocation-id = {mol.114.095869}, year = {2014}, doi = {10.1124/mol.114.095869}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. Insect GluCls show alternative splicing but its impact on channel function and pharmacology remains to be determined. We have isolated GluCl cDNAs from larvae of the silkworm (Bombyx mori) showing that 6 BmGluCl 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 response. By contrast, a partial deletion detected in exon 9 had a much smaller impact on L-glutamate and IVM actions. Binding assays using [3H]IVM indicate that diversity in IVM responses among the GluCl variants are 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 [3H]IVM. Domain swapping and site-directed mutagenesis identified 4 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 3 of the 4 amino acids contribute to inter-subunit contacts, while the other interacts with the TM2-TM3 linker, influencing the receptor response.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/early/2014/09/30/mol.114.095869}, eprint = {https://molpharm.aspetjournals.org/content/early/2014/09/30/mol.114.095869.full.pdf}, journal = {Molecular Pharmacology} }