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J Gen Physiol. 2020 Jun 1;152(6). pii: e201912498. doi: 10.1085/jgp.201912498.

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

Author information

1
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA.
2
Biomedical Imaging Group, Molecular Medicine, University of Massachusetts Medical School, Worcester, MA.
3
Department of Biochemistry and Microbiology, Universidad Austral de Chile, Campus Isla Teja, Los Rios, Chile.
4
Center for the Interdisciplinary Studies on Nervous System, Universidad Austral de Chile, Campus Isla Teja, Los Rios, Chile.
5
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA.
6
Department of Physiology, Universidad Austral de Chile, Campus Isla Teja, Los Rios, Chile.
7
Universidad Austral de Chile, Campus Isla Teja, Los Rios, Chile.
8
Programs in Neuroscience and Chemical Biology, University of Massachusetts Medical School, Worcester, MA.

Abstract

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.

PMID:
31978216
DOI:
10.1085/jgp.201912498

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