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 [3H]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 [3H]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.
Footnotes
- Received September 13, 2014.
- Accepted September 26, 2014.
This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science [Grants 21310147, 26292031]; the Integrated Research Project for Plants, Insects, and Animals using Genome Technology from the Ministry of Agriculture, Forestry, and Fisheries of Japan [Grant 1302]; and the Strategic Project to Support the Formation of Research Bases at Private Universities: Matching Fund Subsidy from the Ministry of Education, Culture, Sports, Science, and Technology of Japan [Grant S1101035].
↵This article has supplemental material available at molpharm.aspetjournals.org.
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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