CCCP activation of the reconstituted NaK-pump

Area of Science:

• Biochemistry
• Membrane Transport
• Enzymology

Background:

• The NaK-ATPase (sodium-potassium adenosine triphosphatase) is a crucial ion pump in cell membranes.
• Membrane potential (Vm) is known to influence protein function, but its specific role in NaK-ATPase activity is complex.
• Ionophores like CCCP can alter membrane properties, offering a tool to probe Vm effects.

Purpose of the Study:

• To investigate the effect of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on NaK-ATPase activity in proteoliposomes (PLs).
• To determine if membrane potential (Vm) influences NaK-pump function and how CCCP modulates this interaction.
• To elucidate the specific step in the NaK-ATPase catalytic cycle affected by Vm and CCCP.

Main Methods:

• Utilized NaK-ATPase proteoliposomes (PLs) to study pump activity, Na+ uptake, and ATP hydrolysis.
• Applied ionophores, including CCCP, in the absence of ion gradients.
• Analyzed changes in phosphorylated intermediates of NaK-ATPase (EP forms) under varying conditions.

Main Results:

• CCCP and other ionophores enhanced NaK-pump activity, Na+ uptake, and ATP hydrolysis without ion gradients.
• CCCP abolished the rapid decrease in ATPase activity observed without ionophores, suggesting Vm inhibition.
• CCCP's effects were dependent on extracellular K+ and ATP concentration, impacting the conversion between phosphorylated NaK-ATPase states (E1P, E*P, E2P).

Conclusions:

• A build-up of membrane potential (Vm) partially inhibits NaK-ATPase activity by altering pump conformation.
• CCCP eliminates this Vm-induced inhibition by enhancing proton mobility and dissipating Vm.
• The E1P to E*P conversion in the NaK-ATPase cycle is identified as a voltage-sensitive step inhibited by Vm.