Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is the only ligand-gated ion channel that hydrolyzes its agonist, ATP. CFTR gating has been argued to be tightly coupled to its enzymatic activity, but channels do open occasionally in the absence of ATP and are reversibly activated (albeit weakly) by nonhydrolyzable nucleotides. Why the latter only weakly activates CFTR is not understood. Here we show that CFTR activation by adenosine 5'-O-(thiotriphosphate) (ATPγS), adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP), and guanosine 5'-3-O-(thio)triphosphate (GTPγS) is enhanced substantially by gain of function (GOF) mutations in the cytosolic loops that increase unliganded activity. This enhancement correlated with the base-line nucleotide-independent activity for several GOF mutations. AMP-PNP or ATPγS activation required both nucleotide binding domains (NBDs) and was disrupted by a cystic fibrosis mutation in NBD1 (G551D). GOF mutant channels deactivated very slowly upon AMP-PNP or ATPγS removal (τdeac ∼ 100 s) implying tight binding between the two NBDs. Despite this apparently tight binding, neither AMP-PNP nor ATPγS activated even the strongest GOF mutant as strongly as ATP. ATPγS-activated wild type channels deactivated more rapidly, indicating that GOF mutations in the cytosolic loops reciprocally/allosterically affect nucleotide occupancy of the NBDs. A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPγS, indicating that these analogs interact differently with the NBDs. We conclude that poorly hydrolyzable nucleotides are less effective than ATP at opening CFTR channels even when they bind tightly to the NBDs but are converted to stronger agonists by GOF mutations.
Highlights
ATP-gated CFTR channels are weakly activated by nonhydrolyzable nucleotides
A gain of function (GOF) Mutation Increases CFTR Activation by Poorly Hydrolyzable Nucleotides—Fig. 1 shows the strong activation of a previously characterized GOF mutant (K978C-CFTR) by AMP-PNP, adenosine 5-O-(thiotriphosphate) (ATP␥S), and guanosine 5-3O-(thio)triphosphate (GTP␥S) in excised inside-out macropatches. This mutation is located in cytosolic loop 3, which lies along the axis that links the nucleotide binding domains (NBDs) to the pore in structural models of CFTR
This ligand-independent activity of K978C-CFTR is detected in excised macropatch recordings as a small current that persists after removal of bath ATP by a scavenger and subsequent bath perfusion with ATP-free solution (Fig. 1A)
Summary
Results: GOF mutations promote ATP-free CFTR activity, proportionately increase activation by nonhydrolyzable nucleotides, and rescue cystic fibrosis gating mutants. The strict coupling concept does not explain the fact that CFTR channels do open occasionally in the absence of ATP (10 –13) or can be reversibly activated (albeit weakly) by poorly hydrolyzable or nonhydrolyzable nucleotides such as ATP␥S and AMP-PNP [14, 15]. We reasoned that if these analogs are partial agonists in the nomenclature of classic allosteric activation schemes (16 –21) they should be converted to stronger agonists by GOF mutations that increase ligand-free activity Our results support this prediction, provide insights into why these analogs normally are weak CFTR activators, and show that GOF mutations can rescue the defective gating of CF channels with mutations in the NBD signature sequences
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