In Reply:
We appreciate the interest of Drs. Dilger and Steinbach in our work and are grateful for their comments on our recent publication.1To properly study the molecular mechanism behind nondepolarizing neuromuscular blocking agents' (NMBAs) inhibition of the nicotinic acetylcholine receptor (nAChR)-mediated signaling in the human neuromuscular junction, an isolated preparation of the junction is needed. For obvious reasons, such approach is not possible and in lieu of that various techniques are used, ranging from acute neuronal preparations to various heterogeneous cellular expression systems. All these methods have different shortcomings in terms of relevance. The neuronal preparations often have insufficient washing to do proper in vitro pharmacology, and the heterogeneous systems often overexpress the receptors studied. Yet, what we can do is comparative in vitro pharmacologic studies using similar methods to compare potency and efficacy in vitro using receptors from relevant species. The Xenopus oocyte two-electrode voltage clamp system is very well suited for such studies. It is a widely used and well-established technique yielding stable and comparable in vitro pharmacologic data from numerous laboratories and has been doing so for 3 decades.
We agree with Drs. Dilger and Steinbach that whole cell two-electrode voltage clamp recordings from Xenopus oocytes are not a system well suited for detailed kinetic studies of receptor and ligand interaction. However, we never claim this in our article.1What we describe is the whole cell functional pharmacology of a range of nondepolarizing NMBAs studied on the human muscle nAChR activated by acetylcholine and dimethylphenylpiperazinium. We show that acetylcholine desensitizes the receptor, whereas dimethylphenylpiperazinium does not: when using larger concentrations of ACh (10 vs. 1 μm), we increase receptor desensitization determined by a decrease in current activated by repeated applications of acetylcholine1(figs. 2C and D). At the higher concentration of agonist, a classic competitive antagonist will be less efficacious and we do not observe this. In fact, we generally observe an increased efficacy of inhibition by the nondepolarizing NMBAs1(table 2). A careful analysis of the inhibition curves1(figs. 2 and 3) shows that nondepolarizing NMBAs induce both right-shifted curves and a depression of maximum currents that are the hallmarks of competitive and noncompetitive inhibition, respectively. The noncompetitive mode of action is primarily observed at higher concentrations of acetylcholine, which also induces receptor desensitization. Further, when using the nondesensitizing antagonist dimethylphenylpiperazinium, the inhibition becomes more competitive1(fig. 5 and table 5).
In this context, we have to remember that the resolution of the nAChR family X-ray and electron microscopy structures are insufficient to determine the exact molecular interactions, neither with nondepolarizing NMBAs nor with acetylcholine itself.2The structure of an acetylcholine-binding protein is known,3but nondepolarizing NMBA binding to this crystal is not studied. Thus, we have to acknowledge the fact that it is still only a theoretical model framework describing receptor function by multiple open, closed, and desensitized states. The published structures of the nAChR do not have the resolution to dissect between multiple open, closed, or desensitized states and of course not the transition between these. To resolve the functional interaction and kinetics between the nAChRs and ligands, we agree with Drs. Dilger and Steinbach that one must use either binding studies or outside out isolated patch recordings. However, the former is unable to resolve the functional effect of the interaction (agonism or antagonism) and the latter suffers from disrupted cytoskeleton, absence of normal intracellular ion concentrations, and different membrane tension compared with the whole cell.
In short, there is no single ideal in vitro system for replacing the human neuromuscular junction. However, we believe that by using comparative in vitro pharmacology one can qualitatively describe clinically relevant pharmacological interactions on the cellular level. This is possible without resolving the exact distribution between multiple desensitized states.
*Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden. malin.jonsson.fagerlund@ki.se
