A cluster of negative charges at the amino terminal tail of CFTR regulatesATP-dependent channel gating

1. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by protein kinase A (PXA) phosphorylation of its R dom ain and by ATP binding at its nucleotide-binding domains (NBDs). Here we in vestigated the functional role of a cluster of acidic residues in the amino terminal tail (N-tail) that also modulate CFTR channel gating by an unknow n mechanism. 2. A disease-associated mutant that lacks one of these acidic residues (D58 N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP-activating cocktail than wild-type CFTR. 3. In excised membrane patches D58N CFTR exhibited a two-fold reduction in single channel open probability due primarily to shortened open channel bur sts. 4. Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PK A phosphorylation or on the ATP dependence of channel activation. 5. Conversely, the N-tail triple mutant exhibited a markedly inhibited resp onse to AMP-PNP, a poorly hydrolysable ATP analogue that can nearly lock op en the wild-type channel. The N-tail mutant had both a slower response to A MP-PNP (activation half-time of 140 +/- 20 s vs. 21 +/- 4 s for wild type) and a lower steady-state open probability following AMP-PNP addition (0.68 +/- 0.08 vs. 0.92 +/- 0.03 for wild type). 6. Introducing the N-tail mutations into K1250A CFTR, an NBD2 hydrolysis mu tant that normally exhibits very long open channel bursts, destabilized the activity of this mutant as evidenced by decreased macroscopic currents and shortened open channel bursts. 7. We propose that this cluster of acidic residues modulates the stability of CFTR channel openings at a step that is downstream of ATP binding and up stream of ATP hydrolysis, probably at NBD2.