Regulation of CFTR Cl- channel gating by ADP and ATP analogues.

Abstract

A B S T R A C T The cystic fibrosis gene product (CFTR) isNucleotide triphosphates initiate channel activity, while nucleotide diphosphates and nonhydrolyzable ATP analogues do not. To further characterize the role of these compounds on CFTR channel activity we examined their effects on chloride channel currents in excised inside-out membrane patches from CFFR transfected mouse L cells. ADP competitively inhibited ATP-dependent CFTR channel gating with a Ki of 16 -+ 9 ~M. AMP neither initiated CVFR channel gating nor inhibited ATP-dependent CFFR channel gating. Similarly, ATP analogues with substitutions in the phosphate chain, including AMPCPP, AMPPCP, AMPPNP, and ATP~S failed to support CFTR channel activity when present at the cytoplasmic face of the membrane and none of these analogues, when present at three to 10-fold excess of ATP, detectably altered ATP-dependent CFFR channel gating. These data suggest that none of these ATP analogues interact with the ATP regulatory site of CFFR which we previously characterized and, therefore, no inference regarding a requirement for ATP hydrolysis in CFFR channel gating can be made from their failure to support channel activity. Furthermore, the data indicate that this nucleotide regulatory site is exquisitely sensitive to alterations in the phosphate chain of the nucleotide; only a nonsubstituted nucleotide di-or triphosphate interacts with this regulatory site. Alternative recording conditions, such as the presence of kinase and a reduction in temperature to 25~ result in a previously uncharacterized kinetic state of CFTR which may exhibit distinctly different nucleotide dependencies.

Ibuprofen inhibits cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion.

“…Additionally, kinetic behavior of the channels in control conditions (see Figs. 4 and 5) was indistinguishable from our previous characterization of CFTR when evaluated in this system (21,22).…”

“…Single channel amplitude (i), mean current (I), and number of channels (N) present in the patch were determined as described previously (21). For macroscopic current analysis (i.e., I in the presence of various concentrations of NSAIDs expressed as a percent of I in the absence of NSAIDs; I/I con ) and fluctuation analysis, recordings 85-170 s in length were analyzed for each control or experimental condition with Bio-Patch software (version 3.21; Molecular Kinetics, Inc., Pullman, WA) as described previously (21).…”

“…To determine the mechanism of this inhibition, the effect of ibuprofen on CFTR Cl Ϫ channels in excised, inside-out patches from mouse L cells recombinantly expressing CFTR (19) was determined. We characterized previously the kinetics of nucleotide-dependent gating (21,22) and sulfonylurea-dependent block of CFTR (23,31) in these cells in detail. The results of one experiment with ibuprofen are shown in Fig.…”

Purinergic Signaling in the Airways

“…There are some outstanding reviews concerned with purinergic signaling in CF (Schultz et al, 1995;Ramsey, 1996;Weisman et al, 1998;Yerxa, 2001;Boucher, 2002Boucher, , 2004Boucher, , 2007Kellerman et al, 2002;Bucheimer and Linden, 2004;Marcet and Boeynaems, 2006;Tarran et al, 2006;Clunes and Boucher, 2008).…”

ATP-independent CFTR channel gating and allosteric modulation by phosphorylation

“…ADP is a well-known inhibitor of CFTR channel activity by mechanisms that are incompletely understood (26,27). We were clued that ADP inhibits the ATP-independent activity of the constitutive mutants (e.g., K190C/K978C-CFTR channels) by the finding that their currents were lowest when hexokinase/glucose was added to induce current deactivation in the presence of 1.5 mM ATP (instead of perfusing ATP from the bath before adding the enzyme).…”