• Figure 1.
    Download figureOpen in new tabFigure 1. NaV1.7 Optopatch Spiking (NaV1.7-OS) HEK cells.

    (A) Genes expressed heterologously in NaV1.7-OS HEK cells. Kir2.1 maintains a hyperpolarized resting potential close to the K+ reversal potential. NaV1.7 imparts electrical excitability. CheRiff depolarizes the cells upon optical excitation and can trigger a NaV1.7-mediated action potential. QuasAr2 is excited by red light and emits near infrared fluorescence in a voltage-dependent manner. (B) Epifluorescence images of QuasAr2 and CheRiff-eGFP expressed in NaV1.7-OS HEK cells. Scale bar 10 μm.

    DOI: http://dx.doi.org/10.7554/eLife.15202.003

    Figure 4.
    Download figureOpen in new tabFigure 4. Effect of PF-04856264, a subtype-specific blocker, on NaV1.7-OS HEK cells.

    (A) Dose-response curves for PF-04856264 when stimulated with prepulses of different durations and with different bath K+ concentrations (n = 4 wells for each concentration). The optical protocol was as in Figure 3B, with prepulse duration specified in figure legends. (B) Comparison between membrane voltage predicted by the Nernst Equation (assuming pure K+ conductance) and recorded by manual patch clamp, as a function of bath [K+] (n = 4–7 cell clusters per data point). (C) Use-dependent inhibition of spiking in NaV1.7-OS HEK cells by PF-04856264, at 8 mM external K+. Cells were stimulated with eight pulses of blue light (20 ms, 50 mW/cm2) at 5 Hz and 10 Hz and QuasAr2 fluorescence was monitored with 635 nm excitation, 400 W/cm2. After photobleaching correction, the QuasAr2 fluorescence in the absence or in the presence of 100 nM PF-04856264, was normalized to peak amplitude of the first spike at 5 Hz in the absence of the drug. Each trace was averaged from 4 wells. Inset: structure of PF-04856264.

    DOI: http://dx.doi.org/10.7554/eLife.15202.011

    Figure 6.
    Download figureOpen in new tabFigure 6. Optopatch assay of KV4.3 function.

    (A) Voltage clamp recording of KV4.3 current in NaV1.5-KV4.3 Optopatch HEK cells. The bath contained 30 μM TTX to block the NaV1.5 current. Cells were held at −70 mV and then subjected to 1 s steps to −60 mV to +40 mV in 10 mV increments. Peak KV4.3 current densities were 218 pA/pF. (B) NaV1.5-KV4.3-OS HEK cells were probed with simultaneous current clamp and QuasAr2 fluorescence. The cells were stimulated with a pulse of blue light (100 ms, 50 mW/cm2), and QuasAr2 fluorescence was monitored with 640 nm excitation, 400 W/cm2. KV activation led to a narrow action potential width, followed by KV inactivation and a return to steady-state depolarization. (C) Average QuasAr2 fluorescence traces from NaV1.5-KV4.3-OS HEK cells treated with HpTx-2 (n = 3–4 wells for each concentration). (D) Dose-response curve of HpTx-2 on NaV1.5-KV4.3-OS HEK cells. Drug effect was quantified by the fluorescence at the peak repolarization (~40 ms after onset of stimulus) relative to peak fluorescence intensity under 1200 nM HPTX2 treatment.

    DOI: http://dx.doi.org/10.7554/eLife.15202.016