Candidate proteins for conductive chloride transport in porcine ileal brush-border membrane

Conductive transport of chloride ion is important in controlling ion and fluid secretion by exocrine tissues. The current study was directed at identifying proteins in the intestinal brush-border membrane that may be involved with conductive chloride transport. Reaction of total brush-border membrane protein with phenyl-isothiocyanate inhibited conductive chloride transport into brush-border membrane vesicles. The conductive transport process was protected from this inhibition by including the ligands Cl- and alpha-phenylcinnamate in the reaction mixture. Brush-border membrane protein protected by this procedure and labeled with fluorescein had an apparent molecular mass in the region of 130 and 23 kDa on separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation of brush-border membrane protein with [gamma-32P] ATP and endogenous protein kinase under conditions causing activation of chloride conductance in membrane vesicles caused the transfer of 32P to several proteins, including ones in the same molecular size range (130 and 23 kDa) as those identified by the fluorescein labeling procedure. Conductive chloride transport in porcine intestinal brush-border vesicles may occur via proteins identified by this differential labeling procedure.