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, did not appreciably alter the voltage-gating of glycosylated ion channels, and the extracellular changes in pH directly 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 non-electrogenic events on the extracellular side of the plasma membrane.