Jgp_201711777 413..416

By tracking conformational transitions of individual ion channel proteins, single-channel recordings allow the observer to deduce gating mechanisms, as well as estimate rate constants of microscopic gating steps. For most ion channels, gating mechanisms are complex, comprising multiple stable open (O) and closed (C) conformations among which conformational transitions may follow a network of possible kinetic pathways. The ensemble of all states and the connectivities among them are called a gating scheme. The strictest limitation to deciphering complex gating schemes is the fact that only transitions between closed and open states can be detected, whereas transitions among closed states, or among open states, remain invisible in single-channel current traces. It is true that such silent transitions are betrayed by the shapes of the dwell-time distributions, which carry information both on the numbers of closed and open states and on the magnitudes of the rate constants of “invisible” transitions (Colquhoun and Hawkes, 1981). But in practice, extracting such information is not at all straightforward: whereas fitting a mechanism to the data by maximum likelihood is a convenient way for estimating rate constants once the underlying gating scheme is known, deciphering the mechanism itself is a much harder task. Typically, several alternative schemes provide satisfactory fits, and comparison of log-likelihood scores facilitates the choice among models only under specific circumstances (when one model is a subset of the other). Thus, clearly, rendering an invisible transition as directly observable in a single-channel current trace is extremely informative for addressing mechanisms. In the previous issue of The Journal of General Physiology, Zhang and Hwang exploit mutants in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that allow visualization of a normally invisible gating transition. In doing so, they reveal a role for the internal vestibule of the channel in gating. For the majority of ion channels, gating is an equilibrium process that does not require an external source of energy. An equilibrium implies that the frequency of transitions along each microscopic kinetic pathway is identical in both directions, so that no preferential cycling may occur around any kinetic loop. One consequence of such microscopic reversibility is that all observable features of a single-channel current recording must be time reversible: any time asymmetry signals violation of thermodynamic equilibrium and hence suggests an external source of energy input (Rothberg and Magleby, 2001). A second consequence of microscopic reversibility is that the dwell-time distributions must decay monotonically: a peaked distribution is again indicative of a nonequilibrium process (Kijima and Kijima, 1987). CFTR, the chloride channel mutated in cystic fibrosis patients, is the only ion channel member of the large family of ABC transport proteins. A large body of information confirms that opening and closing (gating) of its ion pore follows the same conserved mechanism that drives thermodynamically uphill substrate transport in most ABC proteins. In these transporters, a cycle of ATP binding and hydrolysis at two cytosolic nucleotide-binding domains (NBD1 and 2) is linked to transmembrane domain (TMD) movements that alternately expose the substrate binding site to the two sides of the membrane. The CFTR protein has also been shown to hydrolyze ATP and this activity found necessary for its channel function (Li et al., 1996). That finding raised the possibility that CFTR gating might be coupled to its enzymatic activity. Given that the free energy change (ΔG) of ATP hydrolysis is highly negative under physiological conditions (and even more so under in vitro conditions in the nominal absence of ADP and inorganic phosphate), any such coupling would imply a cyclic gating mechanism far from thermodynamic equilibrium. Several lines of experimental evidence have confirmed the aforementioned hypothesis by demonstrating violation of microscopic reversibility for CFTR gating. In heavily filtered (10 Hz) single-channel recordings of WT CFTR reconstituted in lipid bilayers, the presence of the organic buffer MOPS in the cytosolic compartment caused the appearance of a unique subconductance pattern: channels preferentially opened to a lower conductance level (termed O1) but briefly T he J o u rn al o f G e ne ra l P hy si o lo g y