Given the utility of GABA receptor ligands as anthelminthics and insecticides, further study of invertebrate GABA receptor modulation promises to help identify new drugs to combat emerging resistance. The C. elegans GABA receptor, UNC-49, is modulated by the neurosteroids pregnenolone sulfate and pregnanolone, identifying neurosteroids as potential lead compounds. Notably, neurosteroid sites are non-conserved between invertebrates and mammals, limiting possible cross-toxicity. This proposal is focused on better understanding neurosteroid modulation of UNC-49.
In several UNC-49 mutants, pregnanolone and sulfated neurosteroids serve as positive allosteric regulators suggesting neurosteroids may activate inhibitory and enhancing pathways simultaneously which contribute to an overall additive effect on the receptor. Our hypothesis is that there are two pathways operating separately and independently: a novel inhibitory pathway acting negatively, and the well-recognized enhancement pathway acting purely positively.
To test this hypothesis, we used data from a receptor mutation, M2 15’, designed to increase efficiency of the enhancing pathway and observed the difference to the balance between inhibition and enhancement. M2 15’ is a universal positive allosteric residue known for involvement in enhancement of GABA receptors by many modulators. We mathematically modeled the data to attribute the change in modulation caused by the mutation solely to a change in enhancement. The experiment was performed on multiple receptor chimeras to confirm wider applicability of the model.
Because M2 15’ is known as an enhancing residue, we predict that changes in modulation will be attributable solely to increased activity of the enhancement pathway, thus confirming that enhancement is operating independently of and simultaneously with inhibition.
Receptors showed decreased inhibition and/or increased enhancement when exposed to the neurosteroids consistent with multiple pathways occurring. However, unexpectedly, the mutations to the enhancement pathway also affected the activity of the inhibition pathway. These data suggest M2 15’ is not involved solely in enhancement and may be involved in neurosteroid inhibition. This reveals a new function for well-studied M2 15’ as a residue involved in both inhibition and enhancement.
In addition, modeling suggests that the neurosteroid directly affects the gating process rather than affecting the process that occurs between ligand binding and channel opening. This narrows down the set of receptor conformational changes that may be allosterically inhibited by neurosteroids. This contributes to an expanded understanding of the allosteric regulatory pathways of GABA receptors and has provided a lead-in toward drug development by adding to the characterization of a potentially useful site for the development of novel pesticides.