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Differential regulation of single CFTR channels by PP2C, PP2A, and other phosphatases
1Department of Physiology, McGill University, Montreal, Quebec, Canada.
Abstract:
Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity declines rapidly when excised from transfected Chinese hamster ovary (CHO) or human airway cells because of membrane-associated phosphatase activity. In the present study, we found that CFTR channels usually remained active in patches excised from baby hamster kidney (BHK) cells overexpressing CFTR. Those patches with stable channel activity were used to investigate the regulation of CFTR by exogenous protein phosphatases (PP). Adding PP2A, PP2C, or alkaline phosphatase to excised patches reduced CFTR channel activity by > 90% but did not abolish it completely. PP2B caused weak deactivation, whereas PP1 had no detectable effect on open probability (Po). Interestingly, the time course of deactivation by PP2C was identical to that of the spontaneous rundown observed in some patches after excision. PP2C and PP2A had distinct effects on channel gating Po declined during exposure to exogenous PP2C (and during spontaneous rundown, when it was observed) without any change in mean burst duration. By contrast, deactivation by exogenous PP2A was associated with a dramatic shortening of burst duration similar to that reported previously in patches from cardiac cells during deactivation of CFTR by endogenous phosphatases. Rundown of CFTR-mediated current across intact T84 epithelial cell monolayers was insensitive to toxic levels of the PP2A inhibitor calyculin A. These results demonstrate that exogenous PP2C is a potent regulator of CFTR activity, that its effects on single-channel gating are distinct from those of PP2A but similar to those of endogenous phosphatases in CHO, BHK, and T84 epithelial cells, and that multiple protein phosphatases may be required for complete deactivation of CFTR channels.
Insights
Protein phosphatases, particularly PP2C, regulate cystic fibrosis transmembrane conductance regulator (CFTR) channel activity. Different phosphatases impact CFTR gating uniquely, with PP2C mimicking spontaneous rundown and PP2A shortening burst duration.
Area of Science:
- Molecular biology
- Ion channel physiology
- Cellular signaling
Background:
- Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity is known to decline rapidly upon cell membrane excision due to endogenous phosphatases.
- Previous studies indicate membrane-associated phosphatase activity is responsible for the rapid rundown of CFTR channel function in excised patches.
Purpose of the Study:
- To investigate the regulation of CFTR channel activity by exogenous protein phosphatases (PP) in excised membrane patches.
- To compare the effects of different exogenous phosphatases (PP1, PP2A, PP2B, PP2C, alkaline phosphatase) on CFTR gating kinetics.
- To elucidate the role of specific phosphatases in CFTR rundown and deactivation mechanisms.
Main Methods:
- Utilized excised membrane patches from baby hamster kidney (BHK) cells overexpressing CFTR to maintain stable channel activity.
- Applied exogenous protein phosphatases (PP1, PP2A, PP2B, PP2C) and alkaline phosphatase to excised patches containing CFTR channels.
- Analyzed single-channel gating parameters, including open probability (Po) and mean burst duration, before and after phosphatase treatment.
- Investigated CFTR-mediated current rundown in intact T84 epithelial cell monolayers using a PP2A inhibitor (calyculin A).
Main Results:
- Exogenous PP2A, PP2C, and alkaline phosphatase significantly reduced CFTR channel activity (>90%), while PP2B caused weak deactivation and PP1 had no effect.
- PP2C exposure led to a decline in Po without altering burst duration, mirroring spontaneous rundown kinetics observed in some patches.
- PP2A deactivation was characterized by a significant shortening of burst duration, distinct from PP2C effects.
- Rundown of CFTR current in intact T84 cells was unaffected by high concentrations of the PP2A inhibitor calyculin A.
Conclusions:
- Exogenous PP2C is a potent regulator of CFTR activity, exhibiting distinct single-channel gating effects compared to PP2A.
- The effects of PP2C on CFTR gating resemble those of endogenous phosphatases in various cell types (CHO, BHK, T84).
- Complete deactivation of CFTR channels likely involves the action of multiple protein phosphatases.