Pore Blocker Reports Gating-Conformational Changes of the CFTR Channel

Abstract

The CFTR chloride channel is an ABC protein which couples ATP binding and hydrolysis at two conserved intracellular nucleotide binding domains (NBDs) to gating conformational changes. In all ABC proteins ATP binding at both NBDs drives formation of a stable NBD-dimer occluding two ATP molecules at the interface. ATP hydrolysis prompts dimer dissociation, to allow initiation of a new cycle following ADP-ATP exchange. CFTR pore gating is coupled to this irreversible cycle such that dimerized NBDs correspond to an open, while dissociated NBDs to a closed pore (Nature 433:876-880): under normal conditions, most openings follow the gating sequence C→O1→O2→ C, where state C is a compound closed state, O1 is a prehydrolytic, and O2 a posthydrolytic open state (PNAS 107:1241-46). Whereas C↔O transitions involve pore opening/closure, little is known about the conformational changes associated with the hydrolytic O1→O2 step. NPPB is a voltage-dependent pore blocker which also stimulates CFTR open probability (J. Biol. Chem. 280:23622-23630). Here we have used macroscopic and single-channel recordings to dissect the mechanism of its gating effect. We found that NPPB prolongs wild-type (WT) CFTR open times by ∼4x, due to selective slowing of the O1→O2 step. In addition, NPPB appears to stabilize the C↔O1 transition state, because it increases by ∼3x both the opening rate of WT channels, and the closing rate of non-hydrolytic catalytic-site mutants. In contrast, CFTR gating is not affected by the pore blocker MOPS. Gating effects of NPPB are voltage-independent, and cannot be competed off by the presence of MOPS, suggesting that these effects involve NPPB binding to a site outside the pore; this site is likely located in the transmembrane domain, because NPPB gating effects are also insensitive to [ATP] and do not require the presence of the R domain.