Endogenous Aquaporin-0 Lipid Binding in Ocular Lens Tissue via Native Mass Spectrometry

The ocular lens microcirculation system (MCS) is required to maintain transparency; however, how this system is established and maintained as a function of age is not well understood. Through its role in cell adhesion and water permeability, Aquaporin-0 (AQP0) is an important protein in the generation and regulation of the MCS. AQP0 permeability studies have shown that the lipid composition surrounding AQP0 has a direct effect on its function; nevertheless, interactions of native lens lipids with AQP0 have yet to be elucidated. In this study, we used native mass spectrometry (nMS) analysis of ocular lens membrane preparations to identify endogenous lipids bound to AQP0 to inform our understanding of how AQP0-lipid interactions regulate AQP0 function in the lens. We found that a variety of endogenous lens lipids (phosphatidylcholines (PCs) and sphingomyelins (SMs)) differentially bind AQP0 in a regionally dependent manner (cortex vs nucleus). Furthermore, spike-in experiments using native lens lipid extracts allowed us to uncover new AQP0-lipid assemblies not detected in the crude AQP0 experiments, including AQP0-ether-linked PC and AQP0-SM interactions.