Extended Data Fig. 7: Correlating CFTR pore opening and closure with NBD conformation.

a. Example recordings of current responses to ATP application. 3 mM ATP was rapidly applied to an inside-out excised patch containing C-terminally GFP-fused wild-type CFTR (at the dashed lined). PKA-phosphorylation was performed prior to the recording. Time-courses were fitted with monoexponential functions (red lines). b. Example measurements of FRET responses to ATP application. 3 mM ATP was rapidly applied to PKA-phosphorylated CFTR_FRET (at the dashed line). Time-courses were fitted with monoexponential functions (red lines). c. Representative inside-out excised patch showing the rate of solute exchange with local perfusion. CFTR was phosphorylated prior to the recording. CFTR current was then elicited by application of 3 mM ATP. The chloride Nernst potential was switched by exchanging from a chloride-containing perfusion solution (blue bar) to a sulfate-containing perfusion solution (red bar). d. Rate of current relaxation after solute exchange. Data represent means (solid black line) and standard errors (grey shaded area) for 7 patches. The time-course was fit with a monoexponential function (red line). e. Rate of relaxation in fluorescence intensity after injection (at the dashed line) of a DNA-conjugated Cy2 fluorophore into the imaging chamber. The experimental time-course (black points) was fitted with a monoexponential function (red line). f–h. Representative recordings of CFTR current relaxation upon ATP-withdrawal in inside-out excised patches: ATP withdrawal from wild-type CFTR, BeF₃⁻-trapped wild-type CFTR, and E1371Q CFTR (f); ATP withdrawal from wild-type CFTR in the absence of Mg²⁺, and upon reapplication of Mg²⁺ (g); ATP withdrawal from W401A CFTR (h). 2 mM Mg²⁺ was present throughout the recordings in f and h. 3 mM ATP, 0.5 mM BeF₃⁻, 2 mM Mg²⁺, and 10 mM EDTA were perfused onto the patches where indicated. CFTR was activated by application of 300 nM PKA and 3 mM ATP prior to the displayed recordings. i–k. Contour plots of FRET responses after ATP-withdrawal from phosphorylated CFTR_FRET: ATP withdrawal from wild-type CFTR_FRET in absence and presence of BeF₃⁻ or AlF₄⁻ and E1371Q CFTR_FRET (i); ATP withdrawal from wild-type CFTR_FRET in the absence of Mg²⁺ and upon reapplication of Mg²⁺ (j); ATP withdrawal from W401A CFTR_FRET (k). 2 mM Mg²⁺ was present throughout the experiments in i and k. 3 mM ATP, 0.5 mM BeF₃⁻, 1 mM AlF₄⁻, 2 mM Mg²⁺, and 10 mM EDTA were present where indicated. l. Schematic of events underlying CFTR pore closure and NBD separation. CFTR bound to two ATP molecules is dimerized, open, and competent for hydrolysis. ATP hydrolysis at the consensus site is followed by release of ADP and inorganic phosphate (P_i). The degenerate site remains occupied by ATP and this intermediate dynamically transitions between dimerized and separated conformations. The dimerized state has low open probability without ATP in the consensus site. ATP release from the degenerate site leads to stable NBD separation and channel closure. ATP is shown as blue circles.