title: Engineered Ionizable Side Chains.
abstract: One of the great challenges of mechanistic ion-channel biology is to obtain structural information from well-defined functional states. In the case of neurotransmitter-gated ion channels, the open-channel conformation is particularly elusive owing to its transient nature and brief mean lifetime. In this Chapter, we show how the analysis of single-channel currents recorded from mutants engineered to contain single ionizable side chains in the transmembrane region can provide specific information about the open-channel conformation without any interference from the closed or desensitized conformations. The method takes advantage of the fact that the alternate binding and unbinding of protons to and from an ionizable side chain causes the charge of the protein to fluctuate by 1 unit. We show that, in mutant muscle acetylcholine nicotinic receptors (AChRs), this fluctuating charge affects the rate of ion conduction in such a way that individual proton-transfer events can be identified in a most straightforward manner. From the extent to which the single-channel current amplitude is reduced every time a proton binds, we can learn about the proximity of the engineered side chain to the lumen of the pore. And from the kinetics of proton binding and unbinding, we can calculate the side-chain's affinity for protons (pK a), and hence, we can learn about the electrostatic properties of the microenvironment around the introduced ionizable group. The application of this method to systematically mutated AChRs allowed us to identify unambiguously the stripes of the M1, M2 and M3 transmembrane α-helices that face the pore's lumen in the open-channel conformation in the context of a native membrane.
authors.affiliation: Department of Molecular and Integrative Physiology, Center for Biophysics and Computational Biology, Program in Neuroscience, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: Gisela D
authors.initials: GD
authors.lastname: Cymes
authors.affiliation: Department of Molecular and Integrative Physiology, Center for Biophysics and Computational Biology, Program in Neuroscience, University of Illinois at Urbana-Champaign, Urbana, IL, USA. grosman@illinois.edu.|, 524 Burrill Hall, 407 S. Goodwin Ave, 61801, Urbana, IL, USA. grosman@illinois.edu.
authors.forename: Claudio
authors.initials: C
authors.lastname: Grosman
mesh_terms: D000818:Animals; D001665:Binding Sites; D005838:Genotype; D006801:Humans; D015640:Ion Channel Gating; D017136:Ion Transport; D007700:Kinetics; D008564:Membrane Potentials; D008958:Models, Molecular; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011522:Protons; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013499:Surface Properties
chemical_list: D011522:Protons; D011976:Receptors, Muscarinic; D011978:Receptors, Nicotinic
keywords: Acetylcholine receptor; Acid-base chemistry; Ion-channel electrostatics; Nicotinic receptor; Proton transfer; Single-molecule electrophysiology