Fluorescent Visualization of Cellular Proton Fluxes

Cells use plasma membrane proton fluxes to maintain cytoplasmic and extracellular pH and to mediate the co-transport of metabolites and ions. Because proton-coupled transport often involves movement of multiple substrates, traditional electrical measurements provide limited information about proton transport at the cell surface. Here we visualize voltage-dependent proton fluxes over the entire landscape of a cell by covalently attaching small-molecule fluorescent pH sensors to the cell's glycocalyx. We found that the extracellularly facing sensors enable real-time detection of proton accumulation and depletion at the plasma membrane, providing an indirect readout of channel and transporter activity that correlated with whole-cell proton current. Moreover, the proton wavefront emanating from one cell was readily visible as it crossed over nearby cells. Given that any small-molecule fluorescent sensor can be covalently attached to a cell's glycocalyx, our approach is readily adaptable to visualize most electrogenic and non-electrogenic transport events at the plasma membrane.