Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells.
This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.
Lauf
Peter K
PK
Cell Biophysics Group, 054 Biological Sciences Bldg., Wright State Univ. Boonshoft School of Medicine, Dayton, OH 45435, USA. Peter.Lauf@wright.edu
Misri
Sandeep
S
Chimote
Ameet A
AA
Adragna
Norma C
NC
D000085:Acetates / Q000494:pharmacology; D015153:Blotting, Western; D002034:Bumetanide / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability; D048429:Cell Size / Q000187:drug effects; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism; D003022:Clotrimazole / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007189:Indans / Q000494:pharmacology; D007192:Indenes / Q000494:pharmacology; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating; D007700:Kinetics; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009544:Niflumic Acid / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology; D009995:Osmosis; D010042:Ouabain / Q000494:pharmacology; D010693:Phloretin / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012413:Rubidium; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D027981:Symporters / Q000378:metabolism; D050994:Voltage-Dependent Anion Channels / Q000187:drug effects / Q000378:metabolism; D000096922:K Cl- Cotransporters
D000085:Acetates; D002034:Bumetanide; D018118:Chloride Channels; D003022:Clotrimazole; D003517:Cyclopentanes; D007189:Indans; D007192:Indenes; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D009544:Niflumic Acid; D009579:Nitrobenzoates; D010042:Ouabain; D010693:Phloretin; D011188:Potassium; D026902:Potassium Channel Blockers; D011720:Pyrazoles; D012413:Rubidium; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase; D027981:Symporters; D050994:Voltage-Dependent Anion Channels; D000096922:K Cl- Cotransporters; C443139:4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-one-5-yl)oxybutyric acid; C496923:KCNN4 protein, human; C411671:TRAM 34; C095651:((2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy)acetic acid; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid