Wheat germ agglutinin-conjugated fluorescent pH sensors for visualizing proton fluxes

Small-molecule fluorescent wheat germ agglutinin (WGA) conjugates are routinely used to demarcate mammalian plasma membranes, because they bind to the cell's glycocalyx. Here, we describe the derivatization of WGA with a pH-sensitive rhodamine fluorophore (pHRho; pKa = 7) to detect proton channel fluxes and extracellular proton accumulation and depletion from primary cells. We found that WGA-pHRho labeling was uniform and did not appreciably alter the voltage gating of glycosylated ion channels, and the extracellular changes in pH correlated with proton channel activity. Using single-plane illumination techniques, WGA-pHRho was used to detect spatiotemporal differences in proton accumulation and depletion over the extracellular surface of cardiomyocytes, astrocytes, and neurons. Because WGA can be derivatized with any small-molecule fluorescent ion sensor, WGA conjugates should prove useful to visualize most electrogenic and nonelectrogenic events on the extracellular side of the plasma membrane.