Nav1.7 gating in human iPSC derived sensory neurons: an experimental and computational study

Chronic pain is a global healthcare problem with a huge societal impact. Its management remains unsatisfactory, with no single treatment clinically approved in most cases. In this study we use an in vitro experimental model of erythromelalgia consisting of sensory neurons derived from human induced pluripotent stem cells obtained from a patient (carrying the mutation F1449V) and a control subject. We combine neurophysiology and computational modelling to focus on the Nav1.7 voltage gated sodium channel, which acts as an amplifier of the receptor potential in nociceptive neurons and plays a critical role in erythromelalgia due to gain of function mutations causing the channel to open with smaller depolarisations. Using multi-electrode array (extracellular) recordings, we found that the scorpion toxin OD1 increases the excitability of sensory neurons in cultures obtained from the control donor, evidenced by increased spontaneous spike rate and amplitude. In erythromelalgia cultures, the application of the Nav1.7 blocker PF-05089771 effectively stopped spontaneous firing. These results, which are in accordance with current clamp and voltage clamp recordings reported in the literature, are explained with a conductance-based computational model of a single human nociceptive neuron. The disease was simulated through a decrease of the Nav1.7 half activation voltage, which decreased the rheobase and increased the response to supra threshold depolarizing currents. This enhanced response could be successfully supressed by blocking the Nav1.7 channels. The painful effects of OD1 were simulated through a slower establishment and a quicker removal of Nav1.7 inactivation, reproducing the effects of the toxin on the spike frequency and amplitude. Our model simulations suggest that the increase in extracellular spike amplitude observed in the MEA after OD1 treatment can be due mainly to a slope increase in the ascending phase of the intracellular spike caused by impaired inactivation gating.


Introduction
Chronic pain is a global healthcare problem, particularly affecting elderly people, women and persons 27 with lower socio-economic status (Van Hecke et al., 2013). It is one of the most common reasons for 28 physician consultation in developed countries, interfering with quality of life and causing large socio-29 economic impacts that include significant loss of working hours and the need of clinical care. Current 30 therapies have limitations in their effectiveness and side effects, creating an urgent need to develop 31 more precise and effective treatments for pain management (Khouzam, 2000). In this study we focus 32 on the voltage dependent properties of the sodium channel Nav1.7 that is implicated in inherited    ) 10

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Pairing all the units that were active before and after OD1 application in the control cells (n = 7), we 224 found that both the spike frequency and amplitude increased significantly (Fig 1-B). Although the 225 increase in amplitude was not dramatic (20 % in average), it was consistently found in every pair of 226 units. The extent of the increase in firing frequency was more pronounced (70 %). The unpaired units 11 227 that were initially silent and started to fire after the OD1 were not significantly different in rate and 228 amplitude from those already active before the treatment.

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The cells from the patient were treated with PF05089771 (100 nM). The percentage of channels with 230 spiking activity fell from 5.9 to 2.08 %. Twenty four out of thirty of the units sorted stopped firing in 231 the presence of the drug. A typical example of the raw voltage trace before and after treatment is 232 presented in Fig 2-A, note the suppression of the spiking activity. Pairing the few units that remained 233 active after the treatment (n=6), we found that the spike frequency significantly decreased but the 234 amplitude did not change (Fig 2-B). The unpaired units that stopped firing as a result of the 235 PF05089771 application were not significantly different in rate and amplitude to the ones that 236 remained active after treatment. Methods. The parameter set is provided in Table 1 and the model equations in S1 Appendix.

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The possible mechanism behind the enhanced extracellular spike amplitude observed after OD1 274 treatment (Fig. 1) is addressed in the simulation of Fig. 4. We focused on the third spike of the 13 275 simulated train because it is representative of the continuous firing regime. The order of the spike is 276 not important providing that we avoid the first one after the onset of the current step, which is 277 slightly taller. This is due to less Na + inactivation, as the first spike is the only one that starts from the 278 resting potential before the stimulus. In the spikes that follow this initial transient, the peak value of 279 the membrane voltage and Na + currents reaches a stable level. By choosing the third spike of the 280 train we make sure that our assessment applies to a typical spike within a train of repetitive firing, 281 without being influenced by the initial conditions of the stimulus.  (Fig. 4, left panel). Note that the red 295 spike (OD1) occurs before the blue spike (control), due to its higher firing rate (or shorter inter spike 296 interval) with little amplitude difference. This picture changes when we look at the minus first 297 derivative of the intracellular spike, which has been shown to constitute a reasonable estimation of 298 the extracellular spike (e.g., Figure 1 in Henze et al., 2000). The thick traces in Fig. 4

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To summarize, we considered the hypothesis that the extracellular spike was taller after OD1 321 treatment mainly because the intracellular spike reached higher slope in the ascending phase. The 322 model simulation of Fig. 4 confirmed the plausibility of the proposed mechanism and showed that 323 the amplitude effect is expected to be larger when the rate increase is created through the inactivation parameters. However, our hypothesis cannot be truly confirmed without a simultaneous 325 intracellular recording, which we aim to perform in the future.

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In this study, we performed to our knowledge the first test of the scorpion toxin OD1 in hiPSC-derived

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According to these reports, we adopted a plausible value of -6 mV in our schematic model simulations 346 of the disease. After this manipulation, the rheobase and the threshold for sustained spiking