Seeing ATP Hydrolysis in Real Time, What does it Tell Us?

Abstract

CFTR, a chloride channel evolved from the exporter member of the ATP binding cassette (ABC) Protein Superfamily, is gated by association and dissociation of its two NBDs. Single-channel data have led to a gating model with one-to-one stoichiometry between the gating cycle and the ATP hydrolysis cycle_a theme predicted based on rigid-body movements of each TMD-NBD complex for the popular alternating access model proposed for ABC transporters at large. Here, we report a CFTR mutant (R352C) that exhibits two distinguishable single-channel conductance levels (O1 and O2). The transition of these two open states follows a preferred order (C-O1-O2-C), indicating an input of free energy that drives the predominant O1-O2 transition over the opposite O2-O1 transition. This idea is further supported by the observation that only C-O1-C was seen in the presence of ATP when introducing mutations (e.g., E1371S) that abolish ATP hydrolysis. However, in the absence of ATP, R352C/E1371S channels exhibit only Ca O2a C transitions, indicating that without ATP-induced NBD dimerization, the pore conformation must be different from that opened by ATP-induced NBD dimerization. Statistical analysis of single-channel gating events also revealed a considerable amount of opening events containing more than one O1-O2 (i.e. C -(O1-O2)n-C) transition. If we accept the idea that the O1-O2 transition represents the hydrolysis of 1 ATP molecule, these surprising gating transitions would reflect openings embedded with hydrolysis of more than one ATP molecule_a violation of one-to-one stoichiometry. These new results lead us to propose a new gating model that features energetically coupled NBDs and TMDs: both NBDs and TMDs hold a certain degree of autonomy to function on their own but conformational changes in each domain are energetically coupled. Importantly, this new model offers a new target for the action of CFTR potentiators.