A photoactivatable probe for the Na⁺/H⁺ exchanger cross-links a 66-kDa renal brush border membrane protein

Earlier studies on LLC-PK₁ cells have demonstrated two pharmacologically distinct Na⁺/H⁺ exchangers in renal epithelia. In addition, the cDNA clone for the human Na⁺/H⁺ antiporter which is growth factor activatable has been isolated and expressed (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280). We report here the synthesis of an amiloride analogue that can be photoactivated and labeled with ¹²⁵I. This analogue covalently cross-links a 66-kDa protein of bovine renal brush border membranes. A rabbit polyclonal antibody that was directed against a 20-amino acid peptide of the cytoplasmic domain of its human Na⁺/H⁺ antiporter also gives a positive Western against 66-kDa protein of bovine brush border membranes. Thus, the photoactive probe may be helpful in the isolation and purification of the brush border Na⁺/H⁺ exchanger.