Protein carboxyl methylation controls intracellular pH in human platelets

Area of Science:

• Cellular Biology
• Biochemistry
• Physiology

Background:

• Carboxyl methylation is a key post-translational modification regulating protein function.
• The human Na+-H+ antiporter (NHE-1) is crucial for maintaining intracellular pH homeostasis.
• NHE-1 contains a consensus sequence for carboxyl methylation, suggesting its regulation by this process.

Purpose of the Study:

• To investigate the role of protein carboxyl methylation in regulating intracellular pH (pHi) in human platelets.
• To determine how inhibiting carboxyl methylation affects the kinetic properties of the Na+-H+ antiporter (NHE-1).

Main Methods:

• Human platelets were treated with N-acetyl-S-trans,trans-farnesyl-L cysteine (AFC), a specific inhibitor of prenylcysteine methyltransferase.
• Intracellular pH (pHi) was measured using a fluorescent pH indicator.
• Na+-H+ antiporter kinetics were analyzed by acidifying platelets and challenging them with varying extracellular sodium concentrations.

Main Results:

• AFC treatment significantly decreased basal pHi in human platelets.
• Inhibition of carboxyl methylation altered the kinetic properties of the Na+-H+ antiporter, including maximal recovery rate and sodium affinity.
• The effects were dose-dependent and specific to Na+-dependent recovery.

Conclusions:

• Carboxyl methylation plays a significant role in regulating intracellular pH homeostasis.
• Inhibition of carboxyl methylation disrupts normal pHi regulation by altering NHE-1 activity.
• These findings highlight carboxyl methylation as a potential target for modulating cellular pH balance.