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    • We observed the IVM potentiation of currents induced by the

    2020-06-19

    We observed the IVM potentiation of currents induced by the EC5 of Glu or GABA and the IVM inhibition of currents induced by the EC90 of Glu or GABA in wild-type GluCls and GABACls. The EC50 of IVM for the potentiation in GluCls was 3.5-fold smaller than that in GABACls (Fig. 4, Fig. 8B, Table 2), and the IC50 of IVM for the inhibition in GluCls is 8.5-fold smaller than that in GABACls (Fig. 5, Fig. 9B, Table 2). Thus, GluCls are also more sensitive to IVM than GABACls in these actions. When IVM potentiates agonist-induced currents in both receptors, it is worth noting that the potentiation seems to be accompanied by the elicitation of slow, sustained currents by IVM itself and the diminishment of Glu- or GABA-induced currents at a later stage as exemplified by currents traces in Fig. 4, Fig. 8A. This suggests that IVM might change the interacting amino ProteOrange residues by moving inward in the crevice during channel activation. The amino acids at the 36′ position in TM3, G312 in GluCls and G333 in GABACls (Fig. 1), are likely among the most important residues for the action of IVM as the equivalent amino acid residues were reported to be essential for IVM actions in Cys-loop receptors (Lynagh and Lynch, 2010). We therefore examined how the substitution of G36′ affects the triple action of IVM. The Met mutants, G312M GluCl and G333M GABACl, did not undergo activation provoked by IVM. However, diminished activation was observed in G333A GABACls, resulting in a decrease in the potency of IVM. As the interaction of IVM with the 15′ amino acid in TM2 is required for the activation of channels (Hibbs and Gouaux, 2011), bulky side-chain amino acids at the entrance of the IVM binding crevice might destabilize or inhibit the binding of IVM to the position of the 15′ amino acid. Currents induced by the EC5 of Glu were potentiated in G312M GluCls, albeit by high concentrations of IVM. G333M GABACls showed little potentiation of EC5 GABA-induced currents, even with the perfusion of high concentrations of IVM. GABACls are more affected by the steric hindrance at the 36′ position during potentiation compared with GluCls. However, G333A GABACls showed greater potentiation but a decrease in the potency of IVM compared with wild-type GABACls during the perfusion of high concentrations of IVM. The maximum amplitude of the potentiation of GABA-induced currents in G333A GABACls was 6.7-fold greater compared with that of wild-type GABACls. However, this is most likely because these mutant channels do not undergo the diminishment or antagonism of GABA-induced currents by IVM, which occurs in wild-type GABACls at a later stage of response (Fig. 8, Fig. 9B and C). Although G333M and G333A GABACls did not undergo IVM antagonism of currents induced by high concentrations of GABA, G312M GluCls exhibited the IVM potentiation of currents induced by high concentrations of Glu rather than antagonism. It remains to be investigated whether this peculiar action of G312M GluCls is associated with the agonist profile, such as a small Hill coefficient, in this mutant. Together, these findings indicate that G36′ at the entrance of the IVM binding crevice plays crucial roles in IVM activation, potentiation, and antagonism in GluCls and GABACls. Of these effects, the antagonism seems to be most critically affected by the 36’ amino acid, given that it was abolished by mutations. In α1β2γ2L GABACls, IVM binding to non-equivalent intersubunit sites with different interacting amino acids has been shown to induce different conformational states leading to the activation, potentiation, and inhibition of currents (Estrada-Mondragon and Lynch, 2015). In the case of homo-pentameric channels such as Musca GluCls and GABACls, there are five equivalent binding sites for IVM, given that IVM binds to intersubunit crevices. We show in the present study that whether IVM potentiates or inhibits agonist-induced currents depends on agonist concentrations. In homo-pentamers, which have five orthosteric binding sites, global structural changes to produce an open state of a channel differ by how many agonists bind to the orthosteric sites. (Rayes et al., 2009). Our findings may indicate that the binding of IVM to its binding sites with different conformations, which are induced by different concentrations of agonists, leads to potentiation or antagonism of agonist-induced currents.