Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis, Axonal Conduction and Presynaptic Release.

Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7's role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.The funder provided support in the form of salaries for authors [AA, AB, MC, JT, MM, AW, EP, AG, PJC, RD, DP, ZL, BM, CW, NS, RS, PS, NC, DK, RB, ES], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section

Subtype-selective small molecule inhibitors reveal a fundamental role for Nav1.7 in nociceptor electrogenesis, axonal conduction and presynaptic release

Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7's role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission

Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis, Axonal Conduction and Presynaptic Release

<div><p>Human genetic studies show that the voltage gated sodium channel 1.7 (Na_v1.7) is a key molecular determinant of pain sensation. However, defining the Na_v1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Na_v1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Na_v1.7’s role in nociceptor physiology. We report that Na_v1.7 is the predominant functional TTX-sensitive Na_v in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Na_v1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Na_v1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.</p></div

Potency of PF-05089771 across hNa_v1.7 splice variants.

<p>Potency of PF-05089771 across hNa_v1.7 splice variants.</p

PF-05198007 reduces the capsaicin flare response in WT, but not Na_v1.7^Nav1.8Cre mice.

<p>A, B.Time-course plots showing the effects of PF-05198007 on skin blood flow measured before and after topical capsaicin application for WT (A) and Nav1.7^Nav1.8Cre (B) mice (for each genotype, n = 8 per group). C, D. Corresponding summary bar graphs showing flare response measured as area under the curve for WT (C) and Nav1.7^Nav1.8Cre (D) mice before and after PF-05198007 treatment. 1 mg/kg and 10 mg/kg PF-05198007 significantly reduced capsaicin-induced flare in WT mice (C, both 1 mg/kg and 10 mg/kg, p < 0.01, ANOVA) but had no effect in Na_v1.7^Nav1.8Cre mice (D, both 1 mg/kg and 10 mg/kg, p > 0.05, ANOVA).</p

Na_v1.7 is the major TTX-sensitive Na_v channel in small diameter mDRG neurons.

<p>A. RNASeq analysis of Na_v channel mRNA from pooled small diameter mouse DRG neurons. B. Structure of PF-05198007 (4-(2-(3-amino-1H-pyrazol-4-yl)-4-(trifluoromethyl)phenoxy)-5-chloro-2-fluoro-N-(thiazol-4-yl)benzenesulfonamide C. Patch clamp data showing concentration-response relationship for PF-05198007 assessed against recombinantly expressed mouse Na_v1.7, Na_v1.6 and Na_v1.1 (IC₅₀, Slope: 5.2 nM, 1.1; 149 nM, 1.5; 174 nM, 0.7 respectively; n = 3–4 per concentration). D. Representative patch clamp current traces of peak sodium current from small diameter mouse DRG neurons in the presence of A-803467 and following concurrent application of PF-05198007 and TTX. E. Representative peak TTX-S current <i>vs</i> time plot before and after 30 nM PF-05198007 and 500 nM TTX. G. Scatter plot of cell capacitance <i>vs</i> Na_v1.7/TTX-S ratio (n = 35). Note that in every cell tested, Na_v1.7 provided the predominant TTX-S sodium conductance.</p

Evidence for functional Na_v1.7 in human DRG neurons.

<p>A. Representative TTX-S current traces (recorded in the presence of 1 μM A-803467 and following graded voltage steps from -110 mV to 10 mV. B. Voltage dependence of activation (red, n = 4 for each voltage) generated from the protocol described in A and steady state fast inactivation (blue) generated by conditioning 500 msec prepulses to voltages between -110 mV and +10 mV followed by a test pulse to 0 mV from a holding potential of -110 mV (n = 4 for each voltage). Both datasets are fitted with Boltzmann functions. C. Representative timecourse relationship for peak TTX-S current following the application of 100 nM PF-05089771 and 500 nM TTX. D. Concentration-response relationship for PF-05089771 block of TTX-S current (IC₅₀, slope: 8.4 nM, 1.1; n = 3–6 per concentration) E. Example voltage traces from a current clamp recording. Single action potentials were evoked by a 20 ms suprathreshold current step at 0.1 Hz. The scale bar refers to the voltage traces whereas the start-to-start interval is 10 s. F. Summary pie charts showing that the application of 30 and 100 nM PF-05089771 resulted in action potential block in 3/7 and 5/8 DRG neurons respectively.</p

PF-05198007 increases action potential rheobase in small diameter mDRG neurons.

<p>A. Overlayed representative voltage traces in response to graded current step injections before (blue) and after PF-05198007 application (red). Current step stimulations are shown below. B. Example timecourse of change in rheobase following PF-05198007 application and washout. C. Summary bar graph, n = 8 neurons, ** p < 0.01, ANOVA. Data are shown ±SEM.</p

Potency of PF-05089771 assessed at orthologous channels from selected species.

<p>Potency of PF-05089771 assessed at orthologous channels from selected species.</p