Specific stabilization of CFTR by phosphatidylserine

Area of Science:

• Biochemistry
• Structural Biology
• Membrane Protein Research

Background:

• The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a critical chloride channel and a primary target for cystic fibrosis therapies.
• Structural elucidation of CFTR is hindered by its limited in vitro and in vivo stability, particularly of its nucleotide-binding domains.

Purpose of the Study:

• To investigate the effect of lipids on the stability and function of purified human CFTR.
• To identify specific lipids that can enhance CFTR stability for structural and biophysical studies.

Main Methods:

• Purified human CFTR was incubated with various lipids, including phosphatidylserine.
• Thermal stabilization was assessed by measuring the shift in the melting temperature (Tm) of ATPase activity.
• ATP hydrolysis rates (Vmax) were measured at 37°C in the presence of phosphatidylserine.

Main Results:

• Phosphatidylserine, particularly brain phosphatidylserine, significantly enhanced the thermal stability of CFTR's ATP hydrolysis function.
• The Tm for ATPase activity increased from 22.7°C to 35.0°C for wild-type CFTR and from 26.6°C to 42.1°C for a stabilized mutant.
• Vmax for wild-type CFTR at 37°C was 240±60 nmol/min/mg, a rate comparable to other purified ABC transporters.

Conclusions:

• Phosphatidylserine uniquely stimulates and thermally stabilizes purified human CFTR.
• This lipid-mediated stabilization offers a promising approach for future structural and biophysical investigations of CFTR.