The effect of rapamycin on single ENaC channel activity and phosphorylation in A6 cells

Rapamycin and FK-506 are immunosuppressive drugs that bind a ubiquitous imm unophilin, FKBP12, but immunosuppressive mechanisms and side effects appear to be different. Rapamycin binds renal FKBP12 to change renal transport. W e used cell-attached patch clamp to examine rapamycin's effect on Na+ chann els in A6 cells. Channel NPo was 0.5 +/- 0.08 (n = 6) during the first 5 mi n but fell close to zero after 20 min. Application of 1 mu M rapamycin reac tivated Na+ channels (NPo = 0.47 +/- 0.1; n = 6), but 1 mu M FK-506 did not . Also, GF-109203X, a protein kinase C (PKC) inhibitor, mimicked the rapamy cin-induced reactivation in a nonadditive manner. However, rapamycin did no t reactivate Na+ channels if cells were exposed to 1 mu M FK-506 before rap amycin. In PKC assays, rapamycin was as effective as the PKC inhibitor; how ever, epithelial Na+ channel (ENaC) phosphorylation was low under baseline conditions and was not altered by PKC inhibitors or activators. These resul ts suggest that rapamycin activates Na+ channels by binding FKBP12 and inhi biting PKC, and, in renal cells, despite binding the same immunophilin, rap amycin and FK-506 activate different intracellular signaling pathways.