Modulation of nicotinic receptors by an allosteric ligand assessed using fast jumps in acetylcholine

Abstract

Properties of ligand-gated ion channels such as α4β2 nicotinic acetylcholine receptors (nAChRs) and their interactions with various pharmacologic compounds have been studied using voltage clamping techniques for decades. The peak current amplitude, measured in whole-cell experiments, gives us an idea of how receptors will respond to a ligand in situ. Some ligands have the potential to potentiate the peak amplitude, by various mechanisms such as destabilization of receptor desensitized states. The ability of a ligand to increase the peak amplitude of α4β2 nAChRs has the potential to treat a variety of neuronal disorders; however unique properties of these receptors such as fast activation and long-lived desensitized states create significant challenges in determining the extent of modulation of the peak current using these techniques. To correctly assess the peak amplitude, the experiment must achieve synchronous activation of all surface receptors by optimizing solution exchange rates. Failure to do so leads to blunted peak-amplitude measurements in acetylcholine (ACh). This study found that previous reports of the modulating effects of desformylflustrabromine (dFBr), a positive allosteric modulator (PAM) of α4β2 nAChRs, neglected to account for the large surface area of Xenopus oocytes and slow solution exchange rates, leading to an artificially large potentiation of the peak current in dFBr. This study utilized cell lines with a relatively small surface area and a high-speed perfusion system to achieve fast solution exchange rates, and found the potentiation of the peak current by dFBr to be ~1.5-fold. Further studies involving PAMs of α4β2 nAChRs should take necessary steps to optimize solution exchange rates to improve accuracy and reproducibility of their results. In addition, analysis of the whole-cell responses of α4β2 nAChRs to dFBr and Ach have lead to new insights on their effect on not only the peak amplitude, but also on the time-to peak, and the steady-state current. On average, we found that dFBr decreased the time-to peak by 38% and increased the steady-state current ~1.5-fold. Further studies should also consider modulation of the steady-state current to be just as, if not more important than the peak amplitude, as this feature may be a better predictor of the therapeutic benefit of PAMs of α4β2 nAChRs.