Chimeric constructs endow the human CFTR Cl ⁻ channel with the gating behavior of murine CFTR

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl ؊ channel gated by ATP-driven nucleotide-binding domain (NBD) dimerization. Here we exploit species differences between human and murine CFTR to investigate CFTR channel gating. Using homologous recombination, we constructed human-murine CFTR (hmCFTR) chimeras with sequences from NBD1, NBD2, or the regulatory domain (RD) of human CFTR replaced by the equivalent regions of murine CFTR. The gating behavior of hmRD and human CFTR were indistinguishable, whereas hmNBD1 and hmNBD2 had subtle effects on channel gating, prolonging both burst duration and interburst interval. By contrast, hmNBD1؉2, containing both NBDs of murine CFTR, reproduced the gating behavior of the subconductance state of murine CFTR, which has dramatically prolonged channel openings. The CFTR potentiator pyrophosphate (PPi) enhanced human, hmRD, and hmNBD1 CFTR Cl ؊ currents, but not those of hmNBD2, hmNBD1؉2, and murine CFTR. By analyzing the rate-equilibrium free-energy relationships of chimeric channels, we obtained snapshots of the conformation of the NBDs during ATP-driven dimerization. Our data demonstrate that the conformation of NBD1 changes before that of NBD2 during channel opening. This finding suggests that NBD dimerization does not proceed by a symmetric tweezer-like motion, but instead in an asymmetric fashion led by NBD1. We conclude that the NBDs of murine CFTR determine the unique gating behavior of its subconductance state, whereas NBD2 controls channel potentiation by PPi.ATP-binding cassette transporter ͉ chloride ion channel ͉ cystic fibrosis ͉ recombinational cloning ͉ rate-equilibrium free-energy relationships M utation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl Ϫ channel causes the genetic disease cystic fibrosis (CF) (1). CFTR is composed of two membranespanning domain (MSD)-nucleotide-binding domain (NBD) motifs linked by a unique regulatory domain (RD) (1). The MSDs assemble to form a low-conductance (6-to 10-pS) anion-selective pore (2). The RD contains multiple consensus phosphorylation sites, phosphorylation of which is a prerequisite for channel opening (2). The NBDs form a head-to-tail dimer with two ATP-binding sites located at the dimer interface (3). ATP binds tightly to one ATP-binding site (site 1; formed by the Walker A and B motifs of NBD1 and the LSGGQ motif of NBD2), whereas ATP is hydrolyzed rapidly at the other ATP-binding site (site 2; formed by the Walker A and B motifs of NBD2 and the LSGGQ motif of NBD1) (4, 5). Anion flow through the CFTR pore is gated by the interaction of ATP with sites 1 and 2 powering NBD dimerization and, hence, conformational changes in the MSDs (5).A powerful strategy to investigate structure-function relationships is to exploit functional differences between homologues from divergent species. In previous work, we demonstrated that the gating behavior of murine CFTR (mCFTR) differs from that of human CFTR (hCFTR) in several important respects (6, 7). First, mCFTR opens for prolon...

Direct Sensing of Intracellular pH by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel

“…In CFTR, site 1 has no ATPase activity, but site 2 cyclically hydrolyzes ATP to drive channel gating (3,21). In this asymmetric gating model, ATP-induced dimerization at site 1 likely occurs before that at site 2, which in turn precedes CFTR opening (48).…”

Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters

“…For all three species, heterologous expression of a cDNA encoding the CFTR gene product has led to the appearance of CFTR-like channels (with characteristic chloride selectivity, activation by PKA, etc.) (5)(6)(7).…”

Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

“…Activation of CFTR anion transport requires phosphorylation of the R-D by PKA and PKC, as well as dimerization of the two NBDs (16), although the precise mechanism has not yet been described. Binding and hydrolysis of ATP at the NBD dimer interface is thought to induce a conformational change in the channel pore through interactions between the NBD and TM domains (10,33,36), allowing the passage of chloride ions and bicarbonate (9) through the plasma membrane.…”