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 alpha-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 pK(a) values and shifts in pK(a) can be calculated. Here we present a self-consistent, residue-by-residue description of the microenvironment around the pore-lining transmembrane alpha-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.

Gap junctions represent a ubiquitous and integral part of multicellular organisms, providing the only conduit for direct exchange of nutrients, messengers and ions between neighboring cells. However, at the molecular level we have limited knowledge of their endogenous permeants and selectivity features. By probing the accessibility of systematically substituted cysteine residues to thiol blockers (a technique called SCAM), we have identified the pore-lining residues of a gap junction channel composed of Cx32. Analysis of 45 sites in perfused Xenopus oocyte pairs defined M3 as the major pore-lining helix, with M2 (open state) or M1 (closed state) also contributing to the wider cytoplasmic opening of the channel. Additional mapping of a close association between M3 and M4 allowed the helices of the low resolution map (Unger et al., 1999. Science. 283:1176–1180) to be tentatively assigned to the connexin transmembrane domains. Contrary to previous conceptions of the gap junction channel, the residues lining the pore are largely hydrophobic. This indicates that the selective permeabilities of this unique channel class may result from novel mechanisms, including complex van der Waals interactions of permeants with the pore wall, rather than mechanisms involving fixed charges or chelation chemistry as reported for other ion channels.

The gating mechanism of the acetylcholine receptor channel (AChR) was investigated by using rate equilibrium linear free energy relationships (LFERs) to probe the transition state between the closed and open conformations. The properties of the transition state of gating in the second transmembrane segment (M2) of the delta subunit, one of the five homologous pore-lining segments, was measured on a residue-by-residue basis. Series of point mutations were engineered at individual positions of this domain, and the corresponding constructs were characterized electrophysiologically, at the single-channel level. Fully liganded AChR opening and closing rate constants were estimated, and Phi-values (which are a measure of the extent of the conformational change realized at the transition state) were calculated for each reaction series as the slope of the Brønsted relationship (log rate constant versus log equilibrium constant). Our results indicate that, at the transition state of gating, the extracellular half of deltaM2 partly resembles the open state (Phi-values between 0.24 and 0.38) while the intracellular half completely resembles the closed state (Phi-values between -0.18 and 0.03), with a break point near the middle of the M2 segment. This suggests that during gating the two halves of deltaM2 move asynchronously, with the rearrangement of the extracellular portion preceding (following) that of the intracellular part of deltaM2 during opening (closing). This particular sequence of molecular events indicates that the gating conformational change, which starts at the extracellular acetylcholine-binding sites (when opening), does not propagate exclusively along the primary sequence of the protein. In addition, our data are consistent with the deltaM2 segment bending or swiveling around its central residues during gating. We also elaborate on unsettled aspects of the analysis such as the accuracy of two-point LFERs, the physical interpretation of fractional Phi-values, and the existence of single versus parallel transition states for the gating reaction.

The conformational changes underlying cysteine-loop receptor channel gating remain elusive and controversial. We previously developed a single-channel electrophysiological method that allows structural inferences about the transient open-channel conformation to be made from the effect and properties of introduced charges on systematically engineered ionizable amino acids. Here we have applied this methodology to the entire M1 and M3 segments of the muscle nicotinic acetylcholine receptor, two transmembrane alpha-helices that pack against the pore-lining M2 alpha-helix. Together with our previous results on M2, these data suggest that the pore dilation that underlies channel opening involves only a subtle rearrangement of these three transmembrane helices. Such a limited conformational change seems optimal to allow rapid closed-open interconversion rates, and hence a fast postsynaptic response upon neurotransmitter binding. Thus, this receptor-channel seems to have evolved to take full advantage of the steep dependence of ion- and water-conduction rates on pore diameter that is characteristic of model hydrophobic nanopores.

Neuromuscular acetylcholine receptors (AChRs) are ion channels that alternatively adopt stable conformations that either allow (open) or prohibit (closed) ionic conduction. We probed the dynamics of pore (M2) residues at the diliganded gating transition state by using single-channel kinetic and rate-equilibrium free energy relationship (⌽-value) analyses of mutant AChRs. The mutations were at the equatorial (9) position of the ␣, ␤, and subunits (n ‫؍‬ 15) or at sites between the equator and the extracellular domain in the ␣-subunit (n ‫؍‬ 8). We also studied AChRs having only one of the two ␣-subunits mutated. The results indicate that the ␣-subunit, like the ␦-subunit, has a region of flexure near the middle of M2, that the two ␣-subunits experience distinct energy barriers to gating at the equator (but not elsewhere), and that the collective subunit motions at the equator are asymmetric during the AChR gating isomerization.ion channels ͉ phi-value ͉ rate-equilibrium free energy relationship (REFER) analysis T he acetylcholine receptor (AChR) is an allosteric protein that couples a change in affinity for ligands at the two transmitter binding sites with a change in conductance of its pore (1-3). To understand the mechanism of AChR gating, it is important to illuminate the complex molecular motions that link the stable closed (C) and open (O) conformations. The pore-lining, M2 segment has long been recognized as a critical structure in AChR gating (4). In particular, the equatorial 9Ј position (see Fig. 1 A) is a highly conserved Leu (in all five subunits) that forms a flexible region (a ''kink'') in the otherwise helical segment (5-7). It has been suggested that the equatorial region of M2 serves as the main barrier to ion permeation in the closed-channel conformation (5, 8), whereas substituted-Cys-accessibility studies place the ''gate'' closer to the cytoplasmic limit of M2 (9). Mutations at the M2 9Ј position have been shown to left-shift the whole-cell dose-response curve and cause a substantial increase in open channel lifetimes (10-13) and also affect desensitization (11,14). However, the effects of these 9Ј mutations on gating have not been analyzed at the level of equilibrium and rate constants.There is a spatial gradient in the extent to which AChR mutations that change the gating equilibrium constant alter the opening vs. the closing rate constant (15, 16). The closing rate constant is increasingly affected as one proceeds down the length of the protein, from the transmitter binding sites to the cytoplasmic limit of the transmembrane domain. Further, residues appear to be organized into contiguous domains within which all members have a similar behavior with regard to the effect of mutations on the opening vs. the closing rate constant (17). In some cases, these domains overlap secondary structural elements (e.g., helices), whereas in other cases such a direct correlation between function and structure is not apparent. The boundaries between these domains appear to be discrete (18).For a two-state r...