• Investigation Of The Allosteric Modulators Desformylflustrabromine And 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid (Hepes) Interactions On Nicotinic Acetylcholine Receptors

      Daniello-Weltzen, Maegan M.; Schulte, Marvin K. (2011)
      Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop super family of ligand gated ion channels. Dysregulation of nAChRs can lead to pathologies such as Alzheimer's disease, Parkinson's disease, Autism and nicotine addiction. Possible new therapeutic avenues are positive allosteric modulators (PAMs). The natural product desformylflustrabromine (dFBr), a tryptophan metabolite of the marine bryozoan Flustra foliacea, was found to be PAM of alpha4beta2 nAChR. Evaluation of our synthetic water soluble dFBr salt by two-electrode voltage clamp of Xenopus laevis oocytes expressing human nAChR confirmed that synthetic dFBr displayed similar properties as the natural product. Low concentrations of the synthetic dFBr enhanced ACh's efficacy on alpha4beta2 receptors. At higher dFBr concentrations, dFBr inhibited ACh potentiated responses. On alpha7 receptors, dFBr inhibited ACh induced currents. Further pharmacological characterization of dFBr revealed that dFBr was able to enhance partial agonist potencies and efficacies. Evaluation of dFBr on antagonists showed no effect on antagonist inhibition. The mechanisms of biphasic modulation (potentiation and inhibition) of dFBr on alpha4beta2 nAChR were also investigated. Enhanced efficacy of ACh induced currents by dFBr appeared to be accomplished by dFBr stabilization of the open receptor conformation by destabilization of the desensitized state. The inhibition of ACh potentiated currents by dFBr appeared to involve open-channel block. To better understand dFBr mechanisms, its putative binding site was examined. Alanine mutations were made in non-orthosteric clefts on the beta2+ and alpha4- faces. Results revealed residues located on these faces are involved in ACh induced conformational change of the receptor. In addition our data supports our hypothesis that allosteric modulation by dFBr interacts with residues located on the beta2+ and alpha4- faces. The new novel actions of (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) as a alpha4beta2 stoichiometric PAM was discovered and characterized. We showed that HEPES, a common buffering agent, potentiated the high ACh sensitivity alpha4beta2 receptor while only inhibiting the low ACh sensitivity alpha4beta2 receptor. Mutagenesis results suggested that residue beta2D217 is a critical residue in the HEPES binding site. Results from these studies will aid in the development of therapeutic ligands that will assist in the treatment of diseases where nAChRs are dysregulated.