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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells

¹Cell Biophysics Group, ²Department of Pathology, and ³Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio

Submitted 22 August 2007 ; accepted in final form 3 January 2008

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 (K_c) 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 90% of the total Rb influx occurred through the Na-K pump and NKCC and 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 K_c and water loss. Rb influx equaled K_c loss, both blocked by clotrimazole (IC₅₀ 25 µM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel K_Ca3.1 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 K_c loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of K_Ca3.1 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.

K-Rb fluxes; K_Ca3.1 channels; volume regulation; Na-K-2Cl and K-Cl cotransport isoforms; reverse transcriptase polymerase chain reaction; immunochemistry

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