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. 2016 May 24;5:e15202. doi: 10.7554/eLife.15202

Figure 2. Biophysical characterization of NaV1.7-OS HEK cells.

(A) CheRiff current in a NaV1.7-Optopatch HEK cell. Membrane potential was held at −80 mV and then stepped for 2 s to −80 to +40 mV in 20 mV increments. During each depolarization, the cell was exposed to 5 pulses of blue light, 100 ms duration, with increasing intensity (1.7, 18, 50, 79, 93 mW/cm2). The horizontal dashed line indicates zero current. (B) I-V relation of CheRiff, under different light intensities. Currents were measured relative to baseline without blue light. Inset: Steady state photocurrent density as a function of blue light intensity, with a holding potential of −60 mV. (C) Peak NaV1.7 current densities as a function of depolarization potential. Membrane potential was held at -100 mV and then stepped for 100 ms to −90 mV to + 30 mV in 10 mV increments. These measurements were performed prior to transient expression of Kir2.1. Inset: currents in the 10 ms interval following each voltage step. (D) I-V relationship of Kir2.1 expressed in NaV1.7-OS HEK cells. Membrane potential was held at -100 mV and stepped for 500 ms to −130 mV to +30 mV in 10 mV increments. Inset: representative Kir2.1 current recording. Red line indicates the time point (4 ms after voltage step) at which the current was quantified. (E) Simultaneous voltage and QuasAr2 fluorescence recording from NaV1.7-OS HEK cells. The cell was exposed to a series of blue laser pulses, 500 ms duration, with increasing intensities (1.1, 2.3, 4.3, 7.0, 11, 15, 20, 26 mW/cm2) and QuasAr2 fluorescence was monitored with 640 nm excitation, 400 W/cm2. Inset: overlay of the voltage and fluorescence recordings from the most intense blue pulse (26 mW/cm2).

DOI: http://dx.doi.org/10.7554/eLife.15202.004

Figure 2.

Figure 2—figure supplement 1. Current clamp recording of light triggered action potentials in Nav1.7-OS HEK cells.

Figure 2—figure supplement 1.

(A) An action potential recorded via manual patch clamp from a Nav1.7-OS HEK cell cluster stimulated by 20 ms blue light pulse at 50 mW/ cm2. The dashed line indicates the firing threshold. (B) Plateau potential induced by different intensities of blue light stimulation. Current clamp recordings were performed on Nav1.7-OS HEK cell clusters stimulated with 500 ms blue light ranging from 1.1 to 84 mW/ cm2. (C) Membrane potential at 400 ms after onset of blue light stimulus as a function of the blue light intensity.
DOI: http://dx.doi.org/10.7554/eLife.15202.005
Figure 2—figure supplement 2. Relationship between Nav1.7 current density and spike height.

Figure 2—figure supplement 2.

(A) Combined current clamp and voltage clamp protocol in the presence of 3 μM amitriptyline to prepare cells with varying NaV1.7 capacities. Initially, a current clamp protocol was applied in which a depolarizing pulse led to amitriptyline binding and complete channel block. After a variable recovery period, Δt, a test pulse of current induced a voltage spike whose amplitude was recorded. The voltage during the prepulse and recovery periods was then replayed in voltage clamp mode to induce an identical level of channel block. A step depolarization to -20 mV induced a spike in NaV1.7 current whose amplitude was recorded. For details see Materials and methods. (B) Upper trace: current clamp recording showing voltage during 500 ms prepulse intervals, variable recovery period, and test pulses (asterisks). Bottom left: magnified view of voltage during test pulses. Bottom right: magnified view of NaV1.7 current during test pulse under voltage clamp. (C) Voltage spike amplitude as a function of NaV1.7 current amplitude for paired current-clamp and voltage-clamp recordings. Error bars represent s.e.m. of n = 8 cells.
DOI: http://dx.doi.org/10.7554/eLife.15202.006
Figure 2—figure supplement 3. Cell-to-cell variability in Optopatch measurements.

Figure 2—figure supplement 3.

(A) Nav1.7-OS HEK cells in a confluent monolayer were imaged in the presence of 3 μM amitriptyline with a 60× oil immersion objective (numerical aperture 1.45). Cells were illuminated with a red laser at 400 W/cm2. A blue prepulse (500 ms, 50 mW/cm2) depolarized membrane potential and allowed drug binding. A variable recovery period (40 ms to 5120 ms) led to partial unbinding. A blue test pulse (20 ms, 50 mW/cm2) probed the voltage spike induced by residual NaV1.7 capacity. Traces show fluorescence of QuasAr2 from five different single cells recorded in parallel. (B) Ratio of spike amplitude in test pulse to spike amplitude at the longest recovery time (5120 ms), as a function of recovery time for the five single cells shown in (A).
DOI: http://dx.doi.org/10.7554/eLife.15202.007
Figure 2—figure supplement 4. Effects of intense red laser illumination.

Figure 2—figure supplement 4.

(A) Heating by red laser illumination (635 nm, 400 W/cm2). Temperatures were recorded with a small thermocouple not directly illuminated by the laser. Measurements were performed in a single well of a 384-well plate with 36 μL buffer, and in a 35 mm dish with 2 mL buffer (error bars represent s.e.m., n = 4 replicates). (B) Test for photobleaching and phototoxicity. Nav1.7-OS HEK cell were illuminated with red laser (635 nm, 400 W/cm2) continuously for 10 min. Every one minute, the cells were stimulated with blue light (6 pulses of 20 ms, 5 Hz, 50 mW/cm2). The first, sixth and the tenth spike trains are shown above. (C) SNR and spike height (ΔF/F0) for each spike train in (B) as a function of red laser illumination time. Error bars represent s.d. of n = 6 spikes. (D) Effect of red laser intensity on SNR of QuasAr2 fluorescence spikes. Cells were illuminated with red laser at varying intensities and stimulated with pulses of blue light (8 pulses of 20 ms, 10 Hz, 50 mW/cm2). (E) SNR of spikes recorded in (D) as a function of red laser intensity. Error bars represent s.d. of n = 8 spikes.
DOI: http://dx.doi.org/10.7554/eLife.15202.008