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. 2005 Jan 11;102(2):455-60.
doi: 10.1073/pnas.0408575102. Epub 2004 Dec 27.

Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain

Allan L Berger  1 , Mutsuhiro Ikuma, Michael J Welsh
Affiliations

Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain

Allan L Berger et al. Proc Natl Acad Sci U S A. .

Abstract

ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating.

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Figures

Fig. 5.
Fig. 5.
Effect of blocking ATP binding to NBD1 on current. Examples of data from excised membrane patches containing many CFTR channels are shown, along with the fraction of current recovered after NEM treatment. ATP (1 mM) and NEM (200 μM) were present during times indicated by bars (n = 3 for each channel type).
Fig. 1.
Fig. 1.
Labeling CFTR NBDs with [α-32P]8-N3-ATP. (A) CFTR was purified and blotted with antibody MM13-4 (N-terminal epitope), 13.1 (R domain), M3A7 (NBD2), and 24-1 (C terminus). Protein was uncut or treated with Genenase I. Migration of full-length CFTR (FL), NBD1-containing fragment (N1), and NBD2-containing fragment (N2) are indicated. (B) Autoradiograms of [α-32P]8-N3-ATP labeling of CFTR variants. Membranes were labeled as described in Methods. CFTR was then immunoprecipitated, digested with Genenase I, and run on SDS/PAGE. Background bands represent cellular proteins that label with [α-32P]8-N3-ATP and coimmunoprecipitate with CFTR. Because these background bands were variable between experiments, we confirmed the identity of CFTR bands by Western blotting for each experiment. Digital autoradiography of WT CFTR showed that the sum of the radioactivity in the N1 and N2 bands was 82 ± 9% of the radioactivity in the undigested (FL) CFTR (n = 5 independent labeling reactions). (C) Quantified [α-32P]8-N3-ATP labeling normalized to amount of protein measured by immunoblot. Asterisks indicate P < 0.05 (ANOVA) compared with WT labeling (n = 5).
Fig. 2.
Fig. 2.
Effect of blocking ATP binding to NBD2 on CFTR gating. (A) Examples of single-channel recordings for WT and S1248F CFTR. (B) Recording from a membrane patch containing a small number of S1248C channels before and after treatment with 200 μM NEM. (C) Single-channel gating kinetics. Asterisks indicate P < 0.05 compared with WT CFTR (n = 3–6 for each construct).
Fig. 3.
Fig. 3.
Effect of blocking ATP binding to NBD2 on the gating of CFTR-K1250A. (A) Autoradiogram of [α-32P]8-N3-ATP labeling of CFTR-K464A and K1250A; labeling of both NBDs was observed for each mutant. (B) Example of recording of an S1248C/K1250A channel before and after NEM treatment and an S1248F/K1250A channel. (C) Effect of NEM modification of CFTR-S1248C/K1250A on relative current and burst duration. Because we were not able to accurately assess the number of channels in a patch before adding NEM (K1250A has a long interburst interval), Po and the interburst interval were not determined. Asterisks indicate P < 0.05 compared with WT (n = 4).
Fig. 4.
Fig. 4.
Effect of blocking ATP binding to NBD1 on CFTR gating. (A) Examples of recordings from CFTR-A462F, and of A462C channels before and after NEM treatment. Although the A462F tracing shown has a higher Po than the mean, we chose this example to show the bursts. (B) Average data for channel gating kinetics. Asterisks indicate P < 0.05 compared with WT (n = 3–6 for each construct).
Fig. 6.
Fig. 6.
Effect of blocking ATP binding at NBD2 and hydrolysis at NBD1 on gating. (A) Examples of K464A/S1248F and K464A/S1248C channels before and after NEM treatment. (B) Relative current and burst duration from K464A/S1248C channels. Because we were not able to accurately estimate the number of channels in a patch before adding NEM, Po and the interburst interval could not be accurately determined, so we show relative current (n = 3).
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