Stoichiometry and novel gating mechanism within the cystic fibrosis transmembrane conductance regulator channel

Abstract

New Findings r What is the central question of this study?We aimed to demonstrate the possibility that the cystic fibrosis transmembrane conductance regulator (CFTR) forms higher-order multimers during channel pore formation and/or channel gating using functional evidence. r What is the main finding and its importance?The majority of our electrophysiological data suggested that the CFTR channel pore is formed by transmembrane domains of only one CFTR molecule as a monomer. However, we also observed functional upregulation of CFTR activation in patches, which implies interprotein interactions among CFTR molecules. Our findings will help to resolve many contradictions among previous reports regarding whether CFTR is a monomer or a multimer.Despite its fundamental importance to the molecular mechanism underlying cystic fibrosis, many details of the structural basis for the cystic fibrosis transmembrane conductance regulator (CFTR) remain unknown. In addition, the possible interactions among the CFTR proteins have not been clearly demonstrated. In order to identify whether the CFTR channel pore is formed as a monomer or a multimer, we analysed the single-channel properties in patches of cell membrane that coexpressed selected CFTR mutants having significantly different single-channel properties. No hybrid channel opening patterns were observed. We therefore propose that the CFTR channel pore is indeed composed of a monomer. However, we also observed that coexisting CFTR monomers in the cell membrane facilitated the activation of individual CFTR channels. The functional upregulation of this CFTR channel opening probability and the different gating behaviour suggest dynamic conformational changes among the interacting CFTR proteins within the multimeric CFTR complex. Our findings regarding the CFTR monomer channel pore and the novel synergistic gating behaviour within the CFTR channel complex will help to resolve the remaining contradictions among previous studies regarding whether CFTR is a monomer or a multimer.

The Pore Architecture of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Revealed by Co-Mutation in Pore-Forming Transmembrane Regions

“…Therefore, we used T338A as the control CFTR channel variant and constructed all double mutations on this background. Our previous results regarding the stoichiometry of the CFTR channel (Qian et al 2014) and those of other researchers (Marshall et al 1994, Chen et al 2002 suggest that the CFTR channel pore is formed by TM regions from a single CFTR molecule. The CFTR channel pore is likely formed by multimers of TM regions rather than by a single TM region, as is the case for potassium channels (Doyle et al 1998); thus, we proposed that at least one other TM region in addition to TM6 must play a role in CFTR channel pore formation.…”

“…1). In contrast, T338A led to a significant increase in channel conductance, as described previously (Linsdell et al 1998, Qian et al 2014. For the double mutant (A96V/T338A), the enhancive effect of the T338A single mutation on the unitary current was mitigated by the co-mutation of A96V ( Fig.…”

“…To further investigate the CFTR mutants, we tested the blocking properties of Au(CN) 2 -, a high affinity probe for the anion binding sites in the CFTR pore (Gong et al 2002a, Qian et al 2014. In wild type CFTR Vol.…”

“…In contrast, if the two residues are located close enough to have a strong and specific collaboration with each other, a synergic outcome is expected. For example, through co-mutagenesis, we previously found that simultaneous mutations of two close residues (T338A/T339A or T338A/S341A) near the middle of TM6 significantly changed single channel conductance: the conductance was greater for the T338A/T339A double mutant than for the associated single mutants (T338A and T339A), and the conductance was less for the T338/S341A double mutant than for the associated single mutants (T338A and S341A) (Qian et al 2014). Another study showed dynamic interaction between two CFTR residues (R555 and T1246) that were expected to lie on opposite sides of the predicted NBD1-NBD2 dimer interface (Vergani et al 2005).…”

“…Based on this assumption, in the present study, we investigated whether a synergic effect exists between two CFTR residues, one in TM1 and one in TM6. The T338A-CFTR channel (Linsdell et al 1998, Ge et al 2004, Fatehi et al 2007 has outstanding functional properties because mutation of threonine to alanine causes T338A-CFTR channels to have highly characteristic functional properties of CFTR channels in terms of single channel conductance (Fatehi et al 2007, Qian et al 2014, anion permeability (Fatehi et al 2007) and channel blocker binding affinity (Gong et al 2002b, Fatehi et al 2007; therefore, the T338A mutant was selected as the background variant for the characterization of the effects of the double mutants (with an additional mutation in TM1). The site in TM1 that was most highly expected to collaborate with T338 was lysine at position 95 (K95) because K95 is located at the same layer as T338 when TM1 and TM6 are arranged antiparallel (Ge et al 2004).…”