Premium
A cluster of negative charges at the amino terminal tail of CFTR regulates ATP‐dependent channel gating
Author(s) -
Fu Jian,
Ji HongLong,
Naren Anjaparavanda P.,
Kirk Kevin L.
Publication year - 2001
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2001.0459c.xd
Subject(s) - gating , chloride channel , cystic fibrosis transmembrane conductance regulator , chemistry , xenopus , phosphorylation , biophysics , mutant , wild type , membrane potential , biochemistry , adenosine triphosphate , transmembrane domain , amino acid , biology , gene
1 The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by protein kinase A (PKA) phosphorylation of its R domain and by ATP binding at its nucleotide‐binding domains (NBDs). Here we investigated the functional role of a cluster of acidic residues in the amino terminal tail (N‐tail) that also modulate CFTR channel gating by an unknown mechanism. 2 A disease‐associated mutant that lacks one of these acidic residues (D58N 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 bursts. 4 Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation. 5 Conversely, the N‐tail triple mutant exhibited a markedly inhibited response to AMP‐PNP, a poorly hydrolysable ATP analogue that can nearly lock open the wild‐type channel. The N‐tail mutant had both a slower response to AMP‐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 mutant 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 upstream of ATP hydrolysis, probably at NBD2.