Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors

Abstract

Nicotinic acetylcholine receptors are heteropentameric ion channels that open upon activation to a single conducting state. The second transmembrane segments of each subunit were identified as channel-forming elements, but their respective contribution in the gating process remains unclear. Moreover, the detailed impact of variations of the membrane potential, such as occurring during an action potential, on the transmembrane domains, is unknown. Residues at the 12¢ position, close to the center of each second transmembrane segment, play a key role in channel gating. We examined their functional symmetry by substituting a lysine to that position of each subunit and measuring the electrical activity of single channels. For 12¢ lysines in the a, c and d subunits rapid transitions between an intermediate and large conductance appeared, which are interpreted as single lysine protonation events. From the kinetics of these transitions we calculated the pK a values of respective lysines and showed that they vary differently with membrane hyperpolarization. Respective mutations in b or subunits gave receptors with openings of either intermediate or large conductance, suggesting extreme pK a values in two open state conformations. The results demonstrate that these parts of the highly homologous transmembrane domains, as probed by the 12¢ lysines, sense unequal microenvironments and are differently affected by physiologically relevant voltage changes. Moreover, observation of various gating events for mutants of a subunits suggests that the open channel pore exists in multiple conformations, which in turn supports the notion of functional asymmetry of the channel. Abbreviations nAChRNicotinic acetylcholine receptor TM1/TM2 First/second transmembrane segment GFP Green fluorescent protein

Dynamics of heteropentameric nicotinic acetylcholine receptor: Implications of the gating mechanism

“…Furthermore, the quaternary twist motion is highly symmetric in the pore region and involves all subunits equally. This is in contradiction to some experimental data indicating a strongly nonequivalent contribution of different subunits to gating 25, 27…”

“…Previous reports already suggested that when the nature of a gate is hydrophobic instead of geometric occlusion, the required pore widening may be smaller than that commonly assumed 31, 70, 73. In addition, asymmetry of the conformational changes in our model correlates with experimental findings on the unequal contributions from different subunits to the gating movement 25, 27…”

“…The cryoelectron microscopy data collected on the Torpedo nAChRs in the presence or absence of acetylcholine24 revealed an asymmetric motion within the LBD. Several other studies25–27 on muscle‐type nAChRs provided additional evidence of asymmetry in the quaternary movement during gating.…”

Asymmetric Ligand Binding Facilitates Conformational Transitions in Pentameric Ligand-Gated Ion Channels

“…52,53 Experimental data also support the role of asymmetric motion in channel functions. 56,57 An asymmetric and independent contribution of the TM2 residues to gating of nAChR was observed in a single-channel study. 56 Thus, it is not surprising to see spontaneous asymmetric motion in our simulations.…”

Fluorescent Epibatidine Agonists for Neuronal and Muscle‐Type Nicotinic Acetylcholine Receptors

“…Details of the synthesis (Scheme 1) of fluorescent EPB ligands and all other experiments are provided in the Supporting Information and also in references [22][23][24] for radioligand competition and protection binding experiments, in references [25][26][27] for expression and electro- …”