Phosphatidylserine affinity for and flip-flop dependence on Ca2+ and Mg2+ ions

Abstract

Ca²⁺ ions are believed to play a crucial role in regulating lipid membrane asymmetry by modulating the activity of flippases, floppases, and scramblases. Dysregulation of Ca²⁺ homeostasis, and subsequent loss of phosphatidylserine (PS) lipid asymmetry, is associated with physiological conditions such as blood clotting, neurodegeneration, and apoptosis. Yet, despite the prominence of Ca²⁺ with regards to PS flip-flop, the specific actions of Ca²⁺ are not fully understood and detailed mechanisms remain elusive. Much focus has been placed on enzymatic interactions, while the endogenous interactions of Ca²⁺ ions with PS and the direct role Ca²⁺ ions play on maintaining PS asymmetry have not been characterized in detail, a potentially crucial gap in understanding. In the current study the binding affinities of Ca²⁺ ions to planar supported lipid membranes containing PS were measured via sum-frequency vibrational spectroscopy (SFVS). Evaluation of binding affinity obtained from SFVS peak area analysis yielded an affinity of 1.3 × 10⁵ M⁻¹. The rate of PS flip-flop was also measured in the presence and absence of Ca²⁺ via SFVS, with a nearly five-fold decrease in the rate of translocation when Ca²⁺ ions are present. Controls which tested Mg²⁺ with PS or phosphatidylcholine (PC) with Ca²⁺ did not show similar slowing effects, highlighting the specificity of the PS–Ca²⁺ interaction. For the binary lipid mixture tested, the disparity in the PS flip-flop rate would be sufficient to produce an 82% PS asymmetry if Ca²⁺ ions are localized to one side of the membrane. These studies have important implications for the non-enzymatic role Ca²⁺ ions may play in the maintenance of PS asymmetry.