Fluorescent Visualization of Cellular Proton Fluxes

Abstract

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.

Keywords: glycocalyx; membrane transport; pH; proton-coupled transport; voltage-gatedion channels.

Figure 1

Glycocalyx engineering strategy to visualize plasma membrane proton fluxes: (A) Cells expressing ion channels or membrane transporters are incubated with an azidosugar (azido group in blue) and the cell surface is subsequently labeled with a fluorescent pH sensor (pH-DIBO) to detect proton fluxes. The azide-reactive and fluorescent pH-sensitive groups in the chemical structure and label are colored blue and red, respectively. (B) Scheme for the synthesis of pH-DIBO.

Figure 2

Visualization of proton efflux from CHO cells expressing Hv-1: (A) Voltage-clamp fluorometry (VCF) traces: the cell was held at − 80 mV, and currents and fluorescence were elicited from 4-s command voltages from 0 to 100 mV in 20-mV increments. (B) Average normalized fluorescence at − 80 mV and after a 4-s depolarization (100 mV) in the presence (+) or absence (−) of azidosugar. Data were averaged from 5 cells ± SEM; asterisk indicates significance of at least P < 0.05. Inset: Images of cells labeled with (top) or without (bottom) azidosugar. (C) Fluorescence traces of cells expressing similar total current (150 – 300 pA) in bath solutions with various buffer concentrations (HEPES); n = 5; error bars are ± SEM. (D) Change in pH-DIBO fluorescence attached to the cell surface (filled circles) and in a solution (open squares) versus pH. Buffer concentration 10 mM; ΔF/F₀ at pH 7.5 was defined as 0; data were averaged 5 – 10 experiments ± SEM. (E) GFP and pH-DIBO fluorescence images (10 ms) of CHO cells expressing Hv-1. (F) ΔF/F₀ snapshots of pH-DIBO fluorescence at 100 mV at time points indicated in (A). VCF scale bars represent 50 pA, 2% ΔF/F₀, and 1 s; fluorescent image scale bars are 5 μm; pH_o/pH_i = 7.5/6.0; bath solution 0.1 mM HEPES except in (C and D).

Figure 3

Visualization of proton efflux from co-transport proteins expressed in CHO cells: (A) Cartoons of Hv-1, ClC-5 antiporter, and Shaker omega pore mutant (R371H). (B) Voltage-clamp fluorometry (VCF) current and fluorescence traces of cells expressing Shaker R371H (scale bars 100 pA, 1%, 1 s) from 0 to 120 mV in 40-mV increments and WT Shaker-IR at 120 mV (scale bars 1 nA, 1%, 1 s); holding potential − 80 mV; pH_o/pH_i = 7.5/6.0 (bath: 0.1 mM HEPES). (C) VCF current and fluorescence traces of cells expressing ClC-5 from 60 to 140 mV in 20-mV increments or ClC-5 E211A at 140 mV. Scale bars 50 pA, 2%, 1 s; holding potential − 80 mV; pH_o/pH_i = 7.5/7.5 (bath: 0.1 mM HEPES). (D) Plot of ΔF/F₀ as a function of current for Hv-1 (filled circles) and ClC-5 (open squares).

Figure 4

Visualization of extracellular proton depletion and wavefronts in CHO cells: (A) Voltage-clamp fluorometry traces of cells expressing Shaker R362H: the cell was held at 30 mV, and currents and fluorescence were elicited from 4-s command voltages from − 40 to − 120 mV in 20-mV increments; scale bars 50 pA, 2%, 1 s, pH_o/pH_i = 6.0/7.5 (bath: 0.1 mM MES); (B) Left: Cartoon of proton diffusion to a neighboring cell. Right: Fluorescence signals of pH-DIBO-labeled cells. A voltage-clamped Hv-1 expressing cell (− 80 mV) was depolarized to 100 mV and the fluorescent signals from the clamped (d = 23 μm) and a neighboring cell (near and far side, d = 18 μm) were plotted versus time. The distance of the two cells is ~ 4 μm; scale bars 2%, 1 s, pH_o/pH_i = 7.5/6.0 (bath: 0.1 mM HEPES). (C) Buffer concentration dependence of the fluorescent signals of a neighboring cell ~ 8 μm away from an Hv-1 expressing cell. The clamped cells were held at − 80 mV, depolarized to 100 mV, and the fluorescence of the clamped and neighboring cells were plotted versus time. Scale bars 2%, 1 s, pH_o/pH_i = 7.5/6.0; (bath: 0.1 mM HEPES). (D) Cartoon diagram and proton diffusion equation of the concentric volume model with an unstirred layer (USL). (E) Fits of the fluorescent data in (A). Bulk = 1 mL; USL = 1.5 fL; shell = 0.08 fL; K_USL = K_B = 1; k_f = k_r = 1.4 s⁻¹; k_in (black) = 0.090 s⁻¹; k_in (red) = 0.070 s⁻¹; k_in (blue) = 0.050 s⁻¹; k_in (magenta) = 0.030 s⁻¹; k_in (green) = 0.013 s⁻¹..