Splice Variants of pH-Sensitive Chloride Channel Identify a Key Determinant of Ivermectin Sensitivity in the Larvae of the Silkworm Bombyx mori

Abstract

The pH-sensitive chloride channels (pHCls) are broadly expressed in insects, but little is known about their physiologic role, diversity, and sensitivity to insecticides acting on relevant chloride channels. Here we have sequenced 50 transcripts of the pHCl-1 gene from the brain, third thoracic ganglion (T3G), and midgut of larvae of silkworm Bombyx mori It was found that >50 variants were expressed with distinct splicing in the T3G compared with the brain and midgut. Of the variants detected, variant 9, which was expressed most abundantly in the larvae, was reconstituted in Xenopus laevis oocytes to characterize its pH and ivermectin sensitivity. Variant 9 formed a functional pHCl with half-maximal activation at a pH of 7.87, and was activated by ivermectin irrespective of the extracellular pH. This was in contrast to variant 1, which was activated more profoundly at acidic rather than basic pH. To identify a key determinant for such differential ivermectin sensitivity, different amino acids in variants 1 and 9 were swapped, and the effects of the mutations on ivermectin sensitivity were investigated. The V275S mutation of variant 1 enhanced ivermectin sensitivity, whereas the S275V mutation of variant 9 caused a reduction in sensitivity. In homology models of the Bombyx pHCls, Val275 of variant 1 interacted more strongly with Ala273 than Ser275 of variant 9 at the channel gate. This interaction is likely to prevent ivermectin-induced opening of the channel, accounting, at least partially, for the differential macrolide action on the two variants.