R.M. Kühnel, M.G. Junyent, I.L. Jørgensen, G.C. Kemmer, J. Schiller, M.B. Palmgren, B.H. Justesen and T. Günther Pomorski (2019).
Short-chain lipid-conjugated pH-sensors for imaging of transporter activities in reconstituted systems and living cells.
Analyst 144: 3030
doi: 10.1039/C8AN02161A
The design of ion sensors has gained importance for the study of ion dynamics in cells, with fluorescent proton nanosensors attracting particular interest because of their applicability in monitoring pH gradients in biological microcompartments and reconstituted membrane systems. In this work we describe the improved synthesis, photophysical properties and application of pH sensors based on amine-reactive pHrodo esters and short-chain lipid derivatives of phosphoethanolamine. The major features of these novel probes include strong fluorescence under acidic conditions, efficient partitioning into membranes, and extractibility by back exchange to albumin. These features allow for selective labeling of the inner liposomal leaflet in reconstituted membrane systems for studying of proton pumping activities in a quantitative fashion, as demonstrated by assaying the activity of a plant plasma membrane H+-ATPase. Furthermore, the short-chain lipid-conjugated pH sensors enable monitoring pH changes from neutral to acidic conditions in the endocytic pathway of living cells. Collectively, our results demonstrate the applicability of short-chain lipid-conjugated sensors for in vivo and in vitro studies, and thus pave the way for the design of lipid-conjugated sensors selective to other biological relevant ions, e.g. calcium and sodium.