Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in
invertebrate nervous systems. I 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 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 [3
H]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 [3
H]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.
Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. I 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 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.