title: Loop diuretics are open-channel blockers of the cystic fibrosis transmembrane conductance regulator with distinct kinetics.
abstract: OBJECTIVE
  Loop diuretics are widely used to inhibit the Na(+), K(+), 2Cl(-) co-transporter, but they also inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Here, we investigated the mechanism of CFTR inhibition by loop diuretics and explored the effects of chemical structure on channel blockade.
  
  
  METHODS
  Using the patch-clamp technique, we tested the effects of bumetanide, furosemide, piretanide and xipamide on recombinant wild-type human CFTR.
  
  
  RESULTS
  When added to the intracellular solution, loop diuretics inhibited CFTR Cl(-) currents with potency approaching that of glibenclamide, a widely used CFTR blocker with some structural similarity to loop diuretics. To begin to study the kinetics of channel blockade, we examined the time dependence of macroscopic current inhibition following a hyperpolarizing voltage step. Like glibenclamide, piretanide blockade of CFTR was time and voltage dependent. By contrast, furosemide blockade was voltage dependent, but time independent. Consistent with these data, furosemide blocked individual CFTR Cl(-) channels with 'very fast' speed and drug-induced blocking events overlapped brief channel closures, whereas piretanide inhibited individual channels with 'intermediate' speed and drug-induced blocking events were distinct from channel closures.
  
  
  CONCLUSIONS
  Structure-activity analysis of the loop diuretics suggests that the phenoxy group present in bumetanide and piretanide, but absent in furosemide and xipamide, might account for the different kinetics of channel block by locking loop diuretics within the intracellular vestibule of the CFTR pore. We conclude that loop diuretics are open-channel blockers of CFTR with distinct kinetics, affected by molecular dimensions and lipophilicity.
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, UK.
authors.forename: Min
authors.initials: M
authors.lastname: Ju
authors.forename: Toby S
authors.initials: TS
authors.lastname: Scott-Ward
authors.forename: Jia
authors.initials: J
authors.lastname: Liu
authors.forename: Pissared
authors.initials: P
authors.lastname: Khuituan
authors.forename: Hongyu
authors.initials: H
authors.lastname: Li
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Cai
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Husbands
authors.forename: David N
authors.initials: DN
authors.lastname: Sheppard
mesh_terms: D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism*; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005665:Furosemide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D015394:Molecular Structure; D051381:Rats; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000494:pharmacology; D014988:Xipamide / Q000494:pharmacology
chemical_list: C505032:CFTR protein, human; D002712:Chlorides; D049994:Sodium Potassium Chloride Symporter Inhibitors; D013449:Sulfonamides; D002034:Bumetanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D014988:Xipamide; D005665:Furosemide; C015451:piretanide
keywords: ATP-binding cassette transporter; CFTR; NKCC; bumetanide; chloride ion channel; epithelial ion transport; furosemide; glibenclamide; loop diuretics; open-channel blockade