Fluorescent Visualization of Cellular Proton Fluxes

Abstract

SummaryCells 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.

Wheat Germ Agglutinin Conjugated Fluorescent pH Sensors for Visualizing Proton Fluxes

“…This distinction between channel gating and proton accumulation (or depletion) was evident upon Hv-1 deactivation at -80 mV: no current remained after 300 ms whereas it required seconds for the fluorescent signal to fully decay. As we have had previously observed (Zhang et al, 2016), the fluorescent signal decay has two time constants: the fast component corresponds to protons rushing into the cell before Hv-1 channel closing; the slow component corresponds to diffusion into bulk solution. A movie of F/Fo snapshots before, during, and after a 100-mV test pulse highlights the kinetic differences in proton accumulation and depletion at the cell surface (Movie S1).…”

“…WGA-pHRho synthesis: pH-Rho was synthesized as described previously (Zhang et al, 2016). pH-Rho (1.0 mg, 1.4 µmol) was reacted with 14 µmol of N,N,N',N-Tetramethyl-O-(Nsuccinimidyl) uronium tetrafluoroborate (TSTU) in 200 L of anhydrous N,Ndimethylformamide (DMF) at RT for 1 hr.…”

“…CHO cell labeling with pH sensitive WGA conjugates, cell surface calibration, and wholecell patch-clamp fluorometry. CHO cell culture and transfection procedures were conducted as previously described (Zhang et al, 2016). Transfected CHO cells were trypsinized and seeded on a glass-bottomed culture dish for 2 h, labeled with WGA conjugates (50 µg/mL) in HBSS at RT for 10 min, and then rinsed with bath solution (in mM): 145 NaCl, 5.4 KCl, 2 CaCl2, and 0.1 buffer (HEPES for pH 7.5, MES for pH 6.0) with NaOH.…”

“…Transfected CHO cells were trypsinized and seeded on a glass-bottomed culture dish for 2 h, labeled with WGA conjugates (50 µg/mL) in HBSS at RT for 10 min, and then rinsed with bath solution (in mM): 145 NaCl, 5.4 KCl, 2 CaCl2, and 0.1 buffer (HEPES for pH 7.5, MES for pH 6.0) with NaOH. Whole-cell VCF was performed (Zhang et al, 2016) CaCl2, 1 MgCl2 and 0.1 HEPES, pH 7.5 and then with 10 mM lactate in Tyrode's. The MCT-1 inhibitor, AR-C155858 (50 nM) was incubated 30 min at RT before lactate perfusion.…”

“…A substantial stumbling block is optimizing the protein biosensor to properly fold in the ER and traffic uniformly to the plasma membrane. To avoid the secretory pathway, we previously described a bioorthogonal chemistry approach that targets small molecule fluorescent pH sensors to the cell's glycocalyx for the real-time visualization of extracellular proton accumulation and depletion (Zhang et al, 2016). Although our first approach detected proton fluxes at the plasma membrane from ion channels and membrane transport proteins, it required cells to utilize and efficiently incorporate an unnatural sialic acid precursor into the glycocalyx.…”