Transmembrane Cu(I) P-type ATPase pumps are electrogenic uniporters

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Copper(I) P-type ATPases (Cu-pumps) are essential for cellular copper homeostasis across all life.
• Previous work elucidated the structural basis of Cu-pump function, but transport mechanism and electrogenicity remained unclear.
• Understanding these pumps is crucial for comprehending cellular ion transport and developing targeted therapies.

Purpose of the Study:

• To quantitatively characterize the transport mechanism, including substrate translocation, ion movement, and charge transfer, of the E. coli Cu(i)-pump (EcCopA).
• To determine kinetic parameters such as transport rate and affinity for Cu(i).
• To elucidate whether Cu(i) pumps function as uniporters or cotransporters and their electrogenic properties.

Main Methods:

• Reconstitution of the EcCopA model Cu(i)-pump into artificial lipid bilayer small unilamellar vesicles (SUVs).
• Utilization of a multi-fluorescence reporter system (CTAP-3, pyranine, oxonol VI) to monitor Cu(i), pH, and membrane potential changes in real-time.
• Real-time kinetic analysis of wild-type and mutant EcCopA proteoliposomes to correlate substrate, ion, and charge translocation events.

Main Results:

• Established EcCopA as a primary-active uniporter, demonstrating Cu(i) translocation without the requirement for proton cotransport.
• Quantified maximal initial Cu(i) transport rate at 165 nmol Cu(i) mg-1 min-1 and determined the Michaelis constant (KM) for Cu(i) to be 0.15 ± 0.07 μM.
• Revealed that EcCopA is electrogenic, generating a transmembrane potential upon translocation of one Cu(i) per ATP hydrolysis cycle.

Conclusions:

• Cu(i) pumps are electrogenic uniporters, differing mechanistically from other characterized P-type ATPases.
• The developed fluorescence-based platform enables real-time mechanistic studies of transition metal P-type ATPase pumps.
• Findings provide critical insights into the fundamental mechanisms of copper transport and cellular ion homeostasis.