Concentration – effect characteristics of GxTX-594 labeling. (A) Fluorescence from confluent Kv2.1-CHO cells incubated in indicated concentrations of GxTX-594 for 15 min then washed out before imaging. Imaging plane was near the glass-adhered cell surface. Fluorescence shown corresponds to GxTX-594 (magenta). Scale bar, 20 μm. (B) Time-lapse fluorescence intensity from the concentration–effect experiment shown in A. Fluorescence was not background subtracted. Fmax is intensity while cells were incubated in 1,000 nM GxTX-594. (C) Relative fluorescence intensity of GxTX-594 that remains on cells immediately after washout of indicated concentrations of GxTX-594 from the extracellular solution. Symbols correspond to each of three experiments. Black line is the fit of a Langmuir isotherm for Kd = 26.9 nM ± 8.3. (D) Labeling or unlabeling kinetics for GxTX-594 at indicated concentrations. Symbols correspond to the same experiments as panel C. kΔF values obtained from monoexponential fits (Eq. 1). Error bars represent the SD of kΔF fitting. Black line is fit of the kΔF–concentration relation with Eq. 4, kon = 6.372 × 10−5 ± 0.049 × 10−5 nM−1 s−1; koff = 5.929 × 10−4 ± 0.043 × 10−4 s−1.
