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Probing ion-channel pores one proton at a time

2005; Nature Portfolio; Volume: 438; Issue: 7070 Linguagem: Inglês

10.1038/nature04293

1476-4687

Gisela D. Cymes, Ying Ni, Claudio Grosman,

Photoreceptor and optogenetics research

Although membrane proteins often rely on ionizable residues for structure and function, their ionization states under physiological conditions largely elude experimental estimation. To gain insight into the effect of the local microenvironment on the proton affinity of ionizable residues, we have engineered individual lysines, histidines and arginines along the α-helical lining of the transmembrane pore of the nicotinic acetylcholine receptor. We can detect individual proton binding–unbinding reactions electrophysiologically at the level of a single proton on a single side chain as brief blocking–unblocking events of the passing cation current. Kinetic analysis of these fluctuations yields the position-dependent rates of proton transfer, from which the corresponding pKa values and shifts in pKa can be calculated. Here we present a self-consistent, residue-by-residue description of the microenvironment around the pore-lining transmembrane α-helices (M2) in the open-channel conformation, in terms of the excess free energy that is required to keep the engineered basic side chains protonated relative to bulk water. A comparison with closed-channel data leads us to propose that the rotation of M2, which is frequently invoked as a hallmark of the gating mechanism of Cys-loop receptors, is minimal, if any. How the protonation state of ionizable residues in proteins is modulated by the microenvironment is fundamental to many processes at the interface of biology, chemistry and physics. It is has long been recognized as an experimentally elusive, if not intractable, phenomenon. Now a new approach, combining protein engineering and single-channel patch-clamp methods, makes it possible to detect individual proton binding and unbinding events with microsecond time-resolution in muscle nicotinic receptors.