Extracellular protons regulate the extracellular cation selectivity of the sodium pump

Area of Science:

• Biochemistry
• Cell Physiology
• Membrane Transport

Background:

• The Na+/K+-ATPase (sodium-potassium pump) is crucial for maintaining cellular ion gradients.
• Extracellular proton concentration (pH) can influence ion pump activity.
• Understanding ion binding to the pump is key to elucidating its transport mechanisms.

Purpose of the Study:

• To investigate the effect of extracellular protons on the binding of various cations to the Na+/K+-ATPase.
• To determine how protonation state of the pump affects the affinity for different ions, including rubidium and sodium.

Main Methods:

• Utilized 4,4'-diisocyanostilbene-2,2'-disulfonate (DIDS)-treated human and rat erythrocytes to clamp intracellular pH.
• Measured ouabain-sensitive rubidium influx under varying extracellular proton concentrations (pH 9.5-8.0).
• Assessed the binding of other cations (bretylium, guanidinium, sodium, tetrapropylammonium) to protonated and unprotonated pump states.

Main Results:

• Rubidium binding affinity increased significantly (13-34 fold) with pump protonation.
• Protonation enhanced binding of potassium-like ions (bretylium, tetrapropylammonium) but not sodium or guanidinium in human cells.
• Protonation decreased sodium binding in rat cells, suggesting differential ion interactions.
• Dissociation constants (K(d)) for proton binding varied significantly depending on whether the pump's extracellular site was empty, sodium-loaded, or rubidium-loaded.

Conclusions:

• Extracellular protons modulate cation binding to the Na+/K+-ATPase, with distinct effects on different ions.
• Protonation favors rubidium binding, indicating a non-competitive interaction mechanism.
• Conformational changes in the pump's extracellular surface occur upon ion binding and protonation, influencing ion selectivity.