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

P. Fong, Nicholas P. Illsley, Jonathan Widdicombe, A. S. Verkman

Research output: Contribution to journalArticle

13 Scopus citations

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 (JCl) 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 in JCl from a control value of 0.37±0.03 nmol · sec-1 · mg protein-1 upon addition of 0.2 mm diphenylamine-2-carboxylate. The following did not alter JCl significantly (JCl values gives as percent change from control): 50 mmcis Na (-1±5%), 0.1 mm furosemide (-3±4%), 0.1 mm furosemide in the presence of 50 mmcis Na (-5±2%), 0.1 mm H2DIDS (-18±9%), a 1.5 pH unit inwardly directed H gradient (-7±7%), and 0.1 mm H2DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50 mm anion gradients, the initial rates of Br and I entry (JBr and J1, respectively) were not significantly different from JCl. JCl was a saturable function of Cl concentration with apparent Kd of 24 mm and apparent Vmax of 0.54 nmol · sec-1 · mg protein-1. Measurement of the temperature dependence of JCl 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.

Original languageEnglish (US)
Pages (from-to)233-239
Number of pages7
JournalThe Journal of Membrane Biology
Volume104
Issue number3
DOIs
StatePublished - Sep 1988
Externally publishedYes

Keywords

  • chloride channel
  • fluorescence
  • ion permeability
  • trachea

ASJC Scopus subject areas

  • Physiology
  • Cell Biology
  • Biophysics

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