Chloride transport in apical membrane vesicles from bovine tracheal epithelium: Characterization using a fluorescent indicator

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Abstract
Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J Cl) was increased significantly from 0.32±0.12 nmol · sec−1 · mg protein−1 (mean±sem) to 0.50±0.07 nmol · sec−1 · mg protein−1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ Cl from a control value of 0.37±0.03 nmol · sec−1 · mg protein−1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ Cl significantly (J Cl values gives as percent change from control): 50mm cis Na (−1±5%), 0.1mm furosemide (−3±4%), 0.1mm furosemide in the presence of 50mm cis Na (−5±2%), 0.1mm H2DIDS (−18±9%), a 1.5 pH unit inwardly directed H gradient (−7±7%), and 0.1mm H2DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J Br andJ 1, respectively) were not significantly different fromJ Cl.J Cl was a saturable function of Cl concentration with apparentK d of 24mm and apparentV max of 0.54 nmol · sec−1 · mg protein−1. Measurement of the temperature dependence ofJ Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance.