Digitonin does not flip across cholesterol-poor membranes.

Area of Science:

• Biochemistry
• Biophysics
• Membrane Biology

Background:

• Digitonin is widely used to permeabilize cell membranes and solubilize membrane components.
• Its interaction with cholesterol is known to form pores, but the mechanism is poorly understood.
• Understanding digitonin's membrane interaction is crucial for experimental design.

Purpose of the Study:

• To investigate the mechanism of digitonin interaction with model membranes lacking cholesterol.
• To quantitatively describe digitonin's behavior in cholesterol-depleted lipid bilayers.
• To compare digitonin's action in cholesterol-rich versus cholesterol-poor membranes.

Main Methods:

• Isothermal titration calorimetry (ITC).
• Dynamic light scattering (DLS).
• Zeta potential measurements.
• Utilized model membranes including large unilamellar vesicles (LUVs) of phosphocholine (PC) lipids.

Main Results:

• Digitonin partitions asymmetrically into the outer leaflet of PC LUVs at 20°C (partition coefficient of 0.22±0.04mM⁻¹).
• Digitonin micelles coexist with vesicles beyond a ratio of ~0.1, without causing solubilization, even at high concentrations.
• This "staying out" phenomenon was observed in various lipid compositions, including those with phosphoserine (PS) and cholesterol.

Conclusions:

• Digitonin's membrane perturbation and solubilization mechanism differs significantly between cholesterol-poor and cholesterol-rich membranes (20-30mol% cholesterol).
• The findings necessitate careful consideration of digitonin's role in experimental protocols, particularly in membrane protein studies.
• This research provides quantitative insights into digitonin-lipid interactions in the absence of cholesterol.