Figure 6.
GxTX-594 labeling responds to transmembrane voltage.(A) Fluorescence from an optical section of a voltage-clamped Kv2.1-CHO cell in 9 nM GxTX-594. Color progression for pseudocoloring of fluorescence intensity is shown in vertical bar on right. Middle column in each row indicates voltage step taken from a holding potential of −80 mV. Times listed at top of each column correspond to time axis in panel B. Scale bar, 10 μm. (B) GxTX-594 fluorescence during steps to indicated voltages. Smooth lines are monoexponential fits (Eq. 1): −40 mV kΔF = 2.15 × 10−2 ± 0.22 × 10−2 s−1; 0 mV kΔF = 1.279 × 10−1 ± 0.023 × 10−1 s−1; 40 mV kΔF = 2.492 × 10−1 ± 0.062 × 10−1 s−1; and 80 mV kΔF = 4.20 × 10−1 ± 0.11 × 10−1 s−1. ROIs were hand-drawn around the apparent cell surface membrane based on GxTX-594 fluorescence. 0% was set by subtraction of background, which was the average intensity of a region that did not contain cells over the time course of the voltage protocol. For each trace, 100% was set from the initial fluorescence intensity at −80 mV before the subsequent voltage step. Raw initial fluorescence values before normalization were within 10% of one another. (C) Fluorescence intensity remaining at the end of 50-s steps to +80 mV. Each circle represents one cell. Background subtraction as in B. (D) Voltage dependence of fluorescence intensity at the end of 50-s steps. For each cell, 100% was set from the initial fluorescence intensity at −80 mV before the first step to another voltage. Cells did not always recover to initial fluorescence intensity during the −80-mV holding period between voltage steps. Top: Circle coloring indicates data from the same cell, and lines connect points from the same cell. Gray circles represent data shown in B. Bottom: Black bars represent the mean F/Finit at each voltage, and error bars represent the SEM. Black line is the fit of a first-order Boltzmann equation (Eq. 2): V1/2 = −27.4 ± 2.5 mV, z = 1.38 ± 0.13 e0. Green line is the prediction from the EVAP model at 9 nM GxTX. (E) Voltage dependence of fluorescence intensity kinetics (kΔF). Top: Circle coloring is the same as D. Bottom: Black bars represent the average kΔF at each voltage, and error bars represent the SEM. Black line is a first-order Boltzmann equation fit to the kΔF–voltage relation: V1/2 = +38 ± 15 mV, z = 1.43 ± 0.35 e0. Green line is the prediction from the EVAP model at 9 nM GxTX.

GxTX-594 labeling responds to transmembrane voltage. (A) Fluorescence from an optical section of a voltage-clamped Kv2.1-CHO cell in 9 nM GxTX-594. Color progression for pseudocoloring of fluorescence intensity is shown in vertical bar on right. Middle column in each row indicates voltage step taken from a holding potential of −80 mV. Times listed at top of each column correspond to time axis in panel B. Scale bar, 10 μm. (B) GxTX-594 fluorescence during steps to indicated voltages. Smooth lines are monoexponential fits (Eq. 1): −40 mV kΔF = 2.15 × 10−2 ± 0.22 × 10−2 s−1; 0 mV kΔF = 1.279 × 10−1 ± 0.023 × 10−1 s−1; 40 mV kΔF = 2.492 × 10−1 ± 0.062 × 10−1 s−1; and 80 mV kΔF = 4.20 × 10−1 ± 0.11 × 10−1 s−1. ROIs were hand-drawn around the apparent cell surface membrane based on GxTX-594 fluorescence. 0% was set by subtraction of background, which was the average intensity of a region that did not contain cells over the time course of the voltage protocol. For each trace, 100% was set from the initial fluorescence intensity at −80 mV before the subsequent voltage step. Raw initial fluorescence values before normalization were within 10% of one another. (C) Fluorescence intensity remaining at the end of 50-s steps to +80 mV. Each circle represents one cell. Background subtraction as in B. (D) Voltage dependence of fluorescence intensity at the end of 50-s steps. For each cell, 100% was set from the initial fluorescence intensity at −80 mV before the first step to another voltage. Cells did not always recover to initial fluorescence intensity during the −80-mV holding period between voltage steps. Top: Circle coloring indicates data from the same cell, and lines connect points from the same cell. Gray circles represent data shown in B. Bottom: Black bars represent the mean F/Finit at each voltage, and error bars represent the SEM. Black line is the fit of a first-order Boltzmann equation (Eq. 2): V1/2 = −27.4 ± 2.5 mV, z = 1.38 ± 0.13 e0. Green line is the prediction from the EVAP model at 9 nM GxTX. (E) Voltage dependence of fluorescence intensity kinetics (kΔF). Top: Circle coloring is the same as D. Bottom: Black bars represent the average kΔF at each voltage, and error bars represent the SEM. Black line is a first-order Boltzmann equation fit to the kΔF–voltage relation: V1/2 = +38 ± 15 mV, z = 1.43 ± 0.35 e0. Green line is the prediction from the EVAP model at 9 nM GxTX.

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