Related Experiment Videos
Coupling of CFTR Cl- channel gating to an ATP hydrolysis cycle
T Baukrowitz1, T C Hwang, A C Nairn
1Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, New York 10021.
Abstract:
For cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels to open, they must be phosphorylated by protein kinase A and then exposed to a hydrolyzable nucleoside triphosphate, such as ATP. To test whether channel opening is linked to ATP hydrolysis, we applied VO4 and BeF3 to CFTR channels in inside-out patches excised from cardiac myocytes. These inorganic phosphate analogs interrupt ATP hydrolysis cycles by binding tightly in place of the released hydrolysis product, inorganic phosphate. The analogs acted only on CFTR channels opened by ATP and locked them open, increasing their mean open time by 2-3 orders of magnitude. These findings establish that opening and closing of CFTR channels are coupled to an ATP hydrolysis cycle.
Insights
Cystic fibrosis transmembrane conductance regulator (CFTR) channel opening requires ATP. Inorganic phosphate analogs locked opened CFTR channels, demonstrating that CFTR channel gating is coupled to ATP hydrolysis.
Area of Science:
- Biochemistry
- Molecular Biology
- Ion Channel Physiology
Background:
- Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels require protein kinase A phosphorylation and nucleoside triphosphate binding for opening.
- The precise role of ATP hydrolysis in CFTR channel gating remains incompletely understood.
Purpose of the Study:
- To investigate the direct link between ATP hydrolysis and the opening/closing cycle of CFTR channels.
- To determine if ATP hydrolysis is a necessary event for CFTR channel gating.
Main Methods:
- Utilized inside-out patch-clamp electrophysiology on CFTR channels from cardiac myocytes.
- Applied vanadate (VO4) and beryllium fluoride (BeF3) inorganic phosphate analogs to CFTR channels.
- These analogs mimic inorganic phosphate, inhibiting ATP hydrolysis by binding to the active site.
Main Results:
- VO4 and BeF3 analogs were applied to ATP-activated CFTR channels.
- The analogs locked the CFTR channels in an open state.
- This resulted in a 2-3 order of magnitude increase in the mean open time of the channels.
Conclusions:
- CFTR channel opening and closing are intrinsically coupled to the ATP hydrolysis cycle.
- ATP hydrolysis is essential for the dynamic gating of CFTR channels, not just for initial activation.