Saponin Selectively Permeabilizes the Sweat Duct Basolateral Membrane (BLM)

Understanding the role of endogenous phosphatases and kinases in regulating CFTR in the native epithelial cells is hindered by our inability to add or delete large, putative regulatory proteins to and from native cells at will. Therefore, we investigated the ability of Saponin to selectively permeabilize the BLM to macromolecules. We measured the CFTR Cl‐ conductance (CFTR‐gCl) in the sweat ducts as previously described (Quinton, 1986) and permeabilized the BLM either with α‐toxin (1000 U/ml) or with Saponin (0.01%) in the basolateral bath. ATP (5mM), cAMP (0.1 mM), PP2A inhibitor Okadaic acid (10 −8 M) was added to the cytoplasmic bath as needed. α‐toxin permeabilization of the BLM spontaneously inhibited CFTR‐gCl (3.5±2 mS/cm 2 , n=12) Such deactivation was prevented either by the application Okadaic acid (CFTR‐gCl= 38± 4 mS/cm2, n=5) or cAMP CFTR‐gCl=45±6 mS/cm 2 , n=12). In contrast, after permeabilizing the BLM with saponin, CFTR‐gCl failed to deactivate if ATP was present. These results seem to indicate that: a.) α‐toxin pores in the BLM are small enough to retain endogenous phosphatases in the cytosol that dephosphorylate and deactivate CFTR even while depleting cytosolic cAMP through α‐toxin pores, b.) Saponin appears to make larger pores in the BLM so that endogenous phosphatases are lost from the cytosol that prevents deactivation by dephosphorylation of CFTR‐gCl. We thank Mr. K. Taylor for technical assistance. Supported by the NIH