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  • It was reported that IVM modulates various ion channels


    It was reported that IVM modulates various ion channels, such as type-A GABACls, α7 acetylcholine-gated cation channels, P2X4 ATP-gated cation channels, and glycine-gated chloride Iohexol synthesis in vertebrates (Adelsberger et al., 2000, Khakh et al., 1999, Krause et al., 1998, Krůšek and Zemková, 1994, Shan et al., 2001, Sigel and Baur, 1987). IVMPO4, a water soluble IVM analog, acts on C. elegans GluCls to elicit currents by itself (activation) at high nanomolar concentrations and to enhance Glu-induced currents (potentiation) at low nanomolar concentrations (Cully et al., 1994). IVMPO4 activates currents in Drosophila GluCls, whereas it only slightly potentiates Glu-induced currents (Cully et al., 1996). Aside from the actions that IVM exerts on GluCls, it was recently reported that IVM acts on Drosophila GABACls as an allosteric agonist or an antagonist (Lees et al., 2014, Nakao et al., 2015). In the present study, we performed electrophysiological analyses to clarify what types of IVM actions exerted on insect GluCls or GABACls play major roles in the manifestation of IVM's insecticidal effects. We here show that IVM exerts a unique triple action (activation, potentiation, and antagonism) on both GluCls and GABACls cloned from houseflies (Musca domestica), depending on application conditions. For all three actions, GluCls are shown to be more sensitive to IVM than GABACls.
    Materials and methods
    Discussion In the present study, we show that IVM, which possesses high insecticidal activity against the housefly M. domestica, exerts a unique triple action (activation, potentiation, and antagonism) on both Musca GluCls and GABACls expressed in Xenopus oocytes. We have previously reported that Musca GluCls were activated by a 10-s application of 500 nM IVM, whereas Musca GABACls were not activated (Eguchi et al., 2006). However, when IVM was perfused for a longer time on oocytes expressing wild-type Musca GABACls, IVM elicited slow, sustained currents (Fig. 6A). The EC50 of IVM required to activate Musca GABACls was similar to that (2.3 μM) for rat α1β2γ2S GABACls (Adelsberger et al., 2000). With respect to activation by IVM, wild-type GluCls were 184-fold more sensitive than wild-type GABACls (Fig. 3, Fig. 6B, Table 2). X-ray crystallographic analysis revealed that when IVM binds to the C. elegans GluCl-α channel, it forms hydrogen bonds with S260 (15′) in TM2 and T285 (40’) in TM3 in the subunit interface (Hibbs and Gouaux, 2011). These polar amino acids are conserved or conservatively substituted by hydrophilic amino acids in the Musca GluCl subunit but are substituted with Met and Val, respectively, in the Musca GABACl Rdl subunit (Fig. 1C). These changes in amino acids might result in the lower potency of IVM in Musca GABACls. Aside from activation, we show that IVM potentiates currents induced by concentrations of GABA below its EC50 in wild-type GABACls, whereas it inhibits currents induced by concentrations of GABA above its EC50 (Fig. 7). IVM demonstrates the same effects for GluCls as for GABACls in this respect (Fig. 4A and B, Fig. 5A and B). IVM was reported to act as an antagonist in GABACls containing Drosophila Rdlbd subunits when co-applied with 100 μM GABA (above EC50) (Lees et al., 2014), whereas IVM alone acted as an allosteric agonist in Drosophila GABACls composed of the same Rdlbd subunits (Nakao et al., 2015). In native GABACls in mouse hippocampal embryonic neurons, IVM induced small currents and potentiated currents induced by low concentrations of GABA with an EC50 of 17.8 nM, whereas it inhibited currents induced by high concentrations of GABA (Krůšek and Zemková, 1994). In Drosophila GluCls, currents induced by 10 μM Glu were slightly (only 24%) potentiated after pretreatments with 1 nM IVMPO4, whereas currents elicited by 300 μM Glu were not significantly altered (Cully et al., 1996). The Glu response was 49% reduced by 10 nM IVMPO4. The IVM potentiation and antagonism are less prominent compared with the activation in Drosophila GluCls. Our data clearly indicate that whether IVM induces potentiation or antagonism in GluCls and GABACls depends on agonist concentrations (Fig. 7).