Relationship between nucleotide binding and ion channel gating in cystic fibrosis transmembrane conductance regulator

Abstract

We have employed rate-equilibrium free energy relationship (REFER) analysis to characterize the dynamic events involved in the allosteric regulation of cystic fibrosis transmembrane conductance regulator (CFTR) function. A wide range of different hydrolysable and poorly hydrolysable nucleoside triphosphates were used to elucidate the role of ATP hydrolysis in CFTR function. The linearity of the REFER plots and Phi values near unity for all ligands tested implies that CFTR channel gating is a reversible thermally driven process with all structural reorganization in the binding site(s) completed prior to channel opening. This is consistent with the requirement for nucleotide binding for channel opening. However, the channel structural transition from the open to the closed state occurs independently of any events in the binding sites. Similar results were obtained on substitution of amino acids at coupling joints between both nucleotide binding domains (NBD) and cytoplasmic loops (CL) in opposite halves of the protein, indicating that any structural reorganization there also had occurred in the channel closed state. The fact that fractional Phi values were not observed in either of these distant sites suggests that there may not be a deterministic 'lever-arm' mechanism acting between nucleotide binding sites and the channel gate. These findings favour a stochastic coupling between binding and gating in which all structural transitions are thermally driven processes. We speculate that increase of channel open state probability is due to reduction of the number of the closed state configurations available after physical interaction between ligand bound NBDs and the channel.

Figure 1. CFTR model

CFTR structural elements are shown in scale according to a homology model (Serohijos et al. 2008). TMs, CLs and NBDs specify the areas occupied by transmembrane domains, cytoplasmic loops and nucleotide binding domains, respectively. The positions of the structural elements used for the REFER analysis are shown as a black spheres and labelled by the number of the amino acid in the sequence.

Figure 2. REFER plots for the wild-type CFTR driven by different ATP analogues as ligands

A, selective examples of single channel recordings of the wild-type CFTR driven by different ligands (second column). The top level of each trace is a channel closed state. Ligand type is shown above the traces. The values of the equilibrium constant, K, were calculated as a ratio of the areas under the peaks on the all-points histogram (left column) whereas the rate constants were calculated from the dwell-time histograms as k_o= 1/τ_c and k_c= 1/τ_o (two right columns). B, REFER plot for the wild-type CFTR ion channel openings induced by different nucleotides. The specific nucleotides are indicated opposite the experimental points. Each experimental point is a mean value ±s.e.m. of at least 3 in dependent experiments. Slope Φ is shown under the graph. The exact parameters of the best fit are: for the upper line slope is 0.965 ± 0.07 (R= 0.994, P < 0.0063). For the lower line slope is 0.974 ± 0.05 (R= 0.989, P < 0.0001). C, REFER plot for wild-type CFTR channel closing driving by the same set of different nucleotides. Each experimental point is a mean value ±s.e.m. of at least 3 in dependent experiments. Slope Φ is shown under the graph. The exact parameters of the best fit are: for the upper line slope is 0.021 ± 0.048 (R= 0.319, P= 0.681), for the lower line slope is 0.024 ± 0.058 (R= 0.226, P= 0.529).

Figure 3. REFER plots for mutants at residues 508 and 1068 driven by MgATP

A, selective examples of single channel recordings of the CFTR mutants at 508 and 1068 positions are shown. The top level of each trace is the channel closed state. The values of the equilibrium constant, K, were calculated as ratios of the areas under the peaks on the all-points histogram (left) whereas the rate constants were calculated from the dwell-time histograms as k_o= 1/τ_c and k_c= 1/τ_o. The REFER plots for the processes of channel openings for CFTR mutants at position 508 (B) and 1068 (C) are shown. The type of amino acid is shown in one-letter code near each experimental point. Experimental points shown as mean values ±s.e.m. Slope Φ is shown under the graphs. The exact parameters of the best fit are: for the left graph slope is 0.953 ± 0.06 (R= 0.998, P < 0.0001), for the right line slope is 0.968 ± 0.05 (R= 0.989, P < 0.0001).

Figure 4. REFER plots for mutants at residues 276 and 1280

A, selective examples of single channel recordings of the CFTR mutants at 276 and 1280 positions are shown. The top level of each trace is the channel closed state. The values of the equilibrium constant, K, were calculated as a ratio of the areas under the peaks on the all-points histogram (left column) whereas the rate constants were calculated from the dwell-time histograms as k_o= 1/τ_c and k_c= 1/τ_o (two right columns). B, REFER plots for the processes of openings of CFTR mutants at position 276 (A) and position 1280 (B). The type of amino acid is shown in one-letter code near each experimental point. Experimental points shown as mean value ±s.e.m. Slope Φ is shown under the graphs. The exact parameters of the best fit are: for the left graph the slope is 0.938 ± 0.04 (R= 0.997, P= 0.0002), for the right the slope is 0.967 ± 0.09 (R= 0.985, P= 0.0002).

Figure 5. A simplified model of gating energy profile transformations leading to extreme Φ values

The left energy profile in A represents ligand unbound states of the CFTR channel with N closed states and 2 open states available. It relates to the pore structure only and is assumed to be independent of the influence of unliganded NBDs. For simplicity all open (O_i) and closed (C_i) structural substates of the CFTR channel are shown as states of equal energies with identical energy barriers between them. This energy profile will result in equilibrium constant K=k_o/k_c=τ_o/τ_c= 2/N and ΔG∼ logK∼ log(2/N). The probability of the open state P_o=K/(1 +K) = 2/(2 +N) could be very low for the unbound CFTR if N≫ 2. We assume that a set of N available closed state structural configurations could be dramatically reduced to not less than 1 as a result of interaction of ligand bound NBDs with the channel structure. All open states as well as the rest of available closed states remain invariant. For simplicity we left only a single closed state configuration available after interaction of ligand bound NBDs with the channel structure and the invariant set of 2 open state configurations. The energy profile in A with a single closed state and the same set of 2 open states is a result of such an interaction and will lead to ΔG∼ logK∼ log2. The overall ΔΔG for the gating process is proportional to log N and P_o will increase. The arrest in the closed states is available after ligand dissociation only when the multiplicity of closed states resets and no additional coupling is required. Of course, the real energy profile is much more complex and we have used a simplified version to demonstrate the basic idea and distinctive properties of the mechanism suggested. The transitions to C₁ are still available in the open state on the right side of Fig. 5A and could be recorded as a brief intraburst closing if the experimental set-up has sufficient time resolution. The effective two state energy diagram available from single channel recording after strong low pass filtration is shown in B. The Φ value for the process of closed states reduction is 1 and does not impose restrictions on the backward process.