DIFFERENTIAL REGULATION OF SINGLE CFTR CHANNELS BY PP2C, PP2A, AND OTHER PHOSPHATASES

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity declines rapidly when excised from transfected Chinese hamst er ovary (CHO) or human airway cells because of membrane-associated ph osphatase activity. In the present study, we found that CFTR channels usually remained active in patches excised from baby hamster kidney (B HK) cells overexpressing CFTR. Those patches with stable channel activ ity were used to investigate the regulation of CFTR by exogenous prote in phosphatases (PP). Adding PP2A, PP2C, or alkaline phosphatase to ex cised patches reduced CFTR channel activity by >90% but did hot abolis h it completely. PP2B caused weak deactivation, whereas PPI had no det ectable effect on open probability (P-o). Interestingly, the time cour se of deactivation by PP2C was identical to that of the spontaneous ru ndown observed in some patches after excision. PP2C and PP2A had disti nct effects on channel gating; P-o declined during exposure to exogeno us PP2C land during spontaneous rundown, when it was observed) without any change in mean burst duration. By contrast, deactivation by exoge nous PP2A was associated with a dramatic shortening of burst duration similar to that reported previously in patches from cardiac cells duri ng deactivation of CFTR by endogenous phosphatases. Rundown of CFTR-me diated current across intact T84 epithelial cell monolayers was insens itive to toxic levels of the PP2A inhibitor calyculin A. These results demonstrate that exogenous PP2C is a potent regulator of CFTR activit y, that its effects an single-channel gating are distinct from those o f PP2A but similar to those of endogenous phosphatases in CHO, BHK, an d T84 epithelial cells, and that multiple protein phosphatases may be required for complete deactivation of CFTR channels.