A cluster of negative charges at the amino terminal tail of CFTR regulates ATP‐dependent channel gating

• DOI: 10.1111/j.1469-7793.2001.0459c.xd
• Publication Date: 2004-08-05T09:02:55Z
• Authors: Jian Fu; Hong‐Long Ji; Anjaparavanda P. ...; Kevin L. Kirk
• Keywords: Chloride channel; Wild type; Adenosine triphosphate

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