SUPPLEMENTARY INFORMATION

doi: 1.138/nature588 SUPPLEMENTARY INFORMATION Supplemental Information Sensory neuron sodium channel Na v 1.8 is essential for pain at cold temperatures Katharina Zimmermann*, Andreas Leffler*, Alexandru Babes, Cruz Miguel Cendan, Richard W. Carr, Jin-ichi Kobayashi, Carla Nau, John N. Wood and Peter W. Reeh Na V 1.7 Na + DRG Na + Na + cold pain Na + Na V 1.8 Na + Ca Na + TRPM8 TREK-1 K + K + Na + Na + ++ Ca++ 1ms 1ms +4 mv -5 +4 mv -4 Action potential generation in nociceptive neurons depends on Na v 1.8 at cold temperatures. Cutaneous nerve terminals with cell bodies in the dorsal root transmit nociceptive information to the central nervous system. The sensory terminals are equipped with TTX-sensitive (Na v 1.7) and TTX-resistant (Na v 1.8) voltage-gated sodium channels. At warm temperatures low-threshold TTXs channels trigger the impulse generation, and both sodium channel subtypes contribute to shaping a short and fast rising action potential. Cold temperatures activate cold transducer channels such as TRPM8, leading to membrane depolarization, and close potassium channels (e.g. TREK1 and other 2-pore potassium channels) which raises the membrane resistance. The TTXs channels undergo slow inactivation and no longer contribute to the action potential. In the cold, Na v 1.8 shapes a slow-rising action potential of long duration, sufficient to travel to warmer regions where not-blocked Na v 1.7 channels take over the (faster) impulse propagation. www.nature.com/nature 1

2 1. Supplementary information on distribution of TTX-resistance in different subclasses of cutaneous C-fibers. In the rat skin, all mechano-cold nociceptors (CMC) were blocked by TTX 1 µm at 3 C but re-gained excitability and mechanical sensitivity upon cooling in presence of TTX (blue segments in Fig. S1a). In contrast, the majority of mechano-heat sensitive C-fibers (CMH) appeared TTX-resistant at 3 C but was mostly blocked by TTX at higher temperatures (32-38 C) or, at least, strongly desensitized to mechanical and heat stimulation (for example Fig. S1b; red segments in Fig. S1a). Also, prolonged exposure to TTX (3-4 min) and/or increased concentrations (6-12.5 µm) were able to induce TTX block in C-fibers that had initially appeared TTX-resistant. TTX block at any temperature (green segments in Fig. S1a) was more frequently encountered in the fiber classes (CM and Aδ) that also comprised non-nociceptive (low-threshold) mechanoreceptors, consistent with a poor expression of Na V 1.8 in those neurons. In the WT mouse skin, most of the C-fibers were blocked by TTX at 3 C and regained excitability and mechanosensitivity upon cooling in presence of TTX. In Na V 1.8-/-, all units were rapidly blocked by TTX at all temperatures. Fig. S1a: TTX-resistance depends on sensory fiber type and ambient temperature. In the rat skin, 29 C-fibers (cv <1 m/s) and 8 A-δ fibers (cv 8 ±1.2 m/s) were randomly chosen and treated with TTX 1 µm at 3 C. The C-fibers were assigned to three different sensory fiber classes according to their temperature sensitivity: CMC (cv.45 ±.7 m/s), CMH (cv.47 ±.16 m/s) and CM (cv.77 ±.3 m/s). The bars show the percentage of TTX effects with respect to the temperature at which block or relief from block occured. Green: blocked by TTX at any temperature; blue: blocked by TTX 1 µm at 3 C, but unblocked at <27 C; red: not blocked by TTX 1 µm at 3 C but regularly blocked above 3 C or at 3 C using 6-12.5 µm TTX. In the mouse skin, 21 C-fibers were treated with TTX 1 µm and fibers were summarized with respect to genotype (WT, n=11, and Na v 1.8-/-, n=1). Importantly, all Na v 1.8 deficient C-fibers remained unexcitable in the presence of TTX, at all temperatures. Fig. S1b: Rat mechano-heat responsive nociceptor (CMH) desensitized under TTX. Upper panels show instantaneous discharge rates of a CMH fiber in response to ramp-shaped heat stimulation (lower panel, from 3 C to 46 C in 2s). After superfusion with TTX 1µM (in this case >2min), both the heat response and peak firing rate were reduced and the threshold temperature (for > 1 spike/s) increased. www.nature.com/nature 2

3 2. Supplementary information on cold- and TTX-induced slowing of the conduction velocity in WT and Na v 1.8-/- nerves. As shown in the recording in Fig. 1c, cooling of the very nerve terminals in the receptive field increased the latency of the C-fiber action potentials. The overall increase of the latency by cooling from 3 C to 1 C was ~6% in WT mouse C- fibers (from 58 ±4 ms to 93 ±5 ms, n=1). Addition of 1 µm TTX further slowed the conduction velocity by ~13%, corresponding to 7.6 ±2.5 ms (at 24 C when the excitability had recovered during cooling) and 1.3 ±1.8 ms (at 1 C). Na v 1.8-/- C-fibers showed less cold-induced slowing compared to WT (35%, from 55 ±4 ms to 74 ±5 ms, n=1; Fig. S2d). Furthermore, the onset of TTX-induced block was significantly faster in Na v 1.8-/- compared to WT C-fibers (WT: 152 ±37 s; Na v 1.8-/-: 38 ±6 s, n=9 and 1; Fig. S2c). In addition, TTX increased the latency less in Na v 1.8-/- units (before these were blocked) compared to WT fibers (Fig. S2b). These differences in the susceptibility of conduction velocity to TTX and cooling between WT and Na v 1.8-/- may not only result from the lack of Na v 1.8 but also from the reported overexpression of TTXs channels in Na v 1.8-/- 1,2. Fig. S2a: TTX slows the conduction velocity of C-fibers before block is established. Representative recording of a WT CM-fiber. The receptive field was electrically stimulated at twice threshold strength (2T, stim. width 1 ms, 2 s interstimulus interval) and superfused with TTX (1 µm) at 3 C. Action potentials generated during application of TTX show progressively prolonged latencies until the unit is blocked. Fig. S2b: Na v 1.8-/- C-fibers show less TTX-induced slowing of the conduction velocity. TTX-induced slowing of the conduction velocity depicted as the latency difference between the last action potential before and after application of TTX at 2T stimulus strength and 3 C (see Fig. S2a). Fig. S2c: Na v 1.8-/- C-fibers require less exposure time to be blocked by TTX. The onset of TTX-block depicted as the time passed between the last action potential elicited before and after application of TTX. Fig. S2d: Na v 1.8-/- C-fibers show less cold-induced slowing of the conduction velocity. Bar chart representing the average latencies of action potentials elicited with 2T at 3 C (white) and 1 C (black) in WT (left two bars) and Na v 1.8-/- (right two bars). The significance symbols (asterisks) in the Figs. refer to p<.1,.1,.1 in the Student s t-test, respectively; values are given as mean ± SEM. www.nature.com/nature 3

4 3. Supplementary information on voltage-clamp experiments. Fig. S3. Temperature-dependency of voltage-dependent activation of TTXs and TTXr sodium currents in DRG neurons. a-b. Representative families of current traces of TTXs (a) and TTXr (b) sodium currents in DRG neurons recorded at 3 C and 1 C held at -8 mv. Currents were activated by ms long test pulses from - to +3 mv in steps of 1 mv. c-d. Voltage-dependence of activation of TTXs (empty symbols) and TTXr (filled symbols) currents at 3 C and 1 C using different holding potentials; -12 mv (c) and -8 mv (d).. Table S1: Functional properties of sensory neuron sodium currents at different temperatures. Na v 1.8 was studied in wildtype mouse DRG neurons in presence of 25 nm TTX, TTXs sodium channels were studied in Na v 1.8-/- DRG neurons. Activation Steady-state Fast inactivation Steady-state Slow inactivation V 1/2, mv V 1/2, mv V 1/2, mv 3 C 1 C 3 C 1 C 3 C 1 C V h -12 mv TTXs -38.3 ±1.2-41.1 ±.6-72.7 ±.2-63.4 ±.7-6.6 ±1. -93.9 ±2.4 n= 8 n= 8 n= 1 n= 11 n= 11 n= 7 TTXr -6.2 ±1.2-7.3 ±1.3-43.1 ±.3-39.4 ±.6 65.1 ±.5-64.2 ±.4 n= 7 n= 1 n= 11 n= 9 n= 11 n= 11 Na v 1.7-26.5 ±.9-26.5 ± 1.9-56.8 ± 3.6-47.2 ± 2.7-73.9 ±.8-99.8 ± 1.4 n= 5 n= 5 n= 6 n= 6 n= 6 n= 6 Na v 1.8-1.6 ± 3.4-4.2 ± 1.4-34. ±.4-19.1 ± 1.2-55.3 ±.6-65.7 ±.9 n= 5 n= 5 n= 8 n= 7 n= 1 n= 9 V h -8 mv TTXs -34.6 ±.2-36.8 ±.2-84.4 ±.4-116.7 ±.9 n.d. n.d. n= 8 n= 7 n= 7 n= 7 TTXr -19.3 ±.8-23.6 ±1.2-39.1 ±.4-38.7 ±.6 n.d. n.d. n= 9 n= 8 n= 8 n= 7 n.d. not determined 4. Supplementary information on current-clamp experiments. Fig. S4. Resting membrane potential and action potential characteristics of WT and Na v 1.8-/- DRGs. a. The average resting membrane potential in WT- and Na v 1.8-/- DRGs did not significantly differ. At 3 C: WT: -47.1 ±1.4 mv (n=11) compared to Na v 1.8-/-: -49.1 ±2.3 mv (n=1). At 1 C: WT: -48.7 ±1.3 mv (n=11) compared to Na v 1.8-/-: -49.6 ±2.5 mv for Na v 1.8-/- (n=1). b. Action potentials in Na v 1.8-/- DRGs had a smaller overshoot than in WT DRGs at 3 C (Na v 1.8-/-: 18.2 ±2.8 mv (n=1) compared to WT: 57.3 ±2.3 mv (n=11). While the overshoot was not significantly reduced at 1 C in WT neurons (52.8 ±3.8 mv), Na v 1.8-/- neurons responded to current injections only with graded passive depolarisations. www.nature.com/nature 4

5 5. Supplementary information on the contribution of Na v 1.8 to magnitude of response, peak discharge and adaptation during constant pressure stimulation in nociceptive terminals. Fig. S5 depicts a more detailed analysis of the results described in Figs. 3e and f. Constant pressure stimuli of 3T were applied to the receptive fields of WT and Na v 1.8-/- at two different temperatures using gravity-driven von Frey-type stimulation probes (see methods). Fig. S5a: Magnitude of response in WT and Na v 1.8-/- during stimulation with 3Tconstant pressure. Box-and-whiskers plot showing median, minimum, maximum, and respective quartiles of the data points in each group. The values are calculated as total of action potentials discharged over 3s stimulus time. The actual means were in WT: 3 C: 17 ±42; 1 C: 81 ±15, n=1; Na v 1.8-/-: 3 C: 98 ±25; 1 C: 8 ±3, n=1. There was no significant difference between WT and Na v 1.8-/- of the overall response at 3 C, but at 1 C. The inset shows the respective residual response of Na v 1.8-/- fibers (calculated as percentage of the WT response) at both temperatures (3 C: 58%, 1 C: 9%). Fig. S5b: Peak firing rates of WT and Na v 1.8-/- during stimulation with 3Tconstant pressure. Box-and-whiskers plot showing median, minimum, maximum, and respective quartiles for the data points in each group. The graph depicts the maximum (peak) discharge rates reached during the first seconds of stimulation with 3T constant pressure, given in action potentials /s. The actual means were in WT: 3 C: 7 ±9; 1 C: 13 ±1, n=1; Na v 1.8-/-: 3 C: 36 ±3; 1 C: 3±1, n=1. The peak discharge was significantly smaller in Na v 1.8-/- compared to WT at both 3 C and 1 C. The inset shows the respective residual peak discharge of Na v 1.8-/- fibers (calculated as percentage of the WT response) at both temperatures (3 C: 52%, 1 C: 21%). Fig. S5c: Time to total adaptation of WT and Na v 1.8-/- during stimulation with 3T constant pressure. Bar chart showing time to total adaptation during 3T constant pressure stimulus applied for 3s to the receptive field of WT and Na v 1.8-/- at 3 C and 1 C. Na v 1.8-/- fibers stop firing at 1 C after 12 ±2.3 s. The significance symbols (asterisks) in the Figs. refer to p<.1,.1,.1 in the Student s t-test, respectively; values are given as mean ± SEM. www.nature.com/nature 5

6 6. Supplementary information on cold- and menthol-sensitivity of nociceptive terminals in Na v 1.8-/- and WT. Fig. S6a: Cold responsiveness of mechanosensitive C-fibers in WT and Na v 1.8-/-. A random sample of mechanosensitive C-fibers in WT (n=24; cv.38 ±.2 m/s, median von Frey threshold 11.4 mn) and Na v 1.8-/- (n=32; cv.53 ±.3 m/s, median von Frey threshold 8 mn), comprised 13 cold-sensitive fibers in WT and 9 in Na v 1.8-/-. Cold-induced discharge activity has previously been described as poor in C57BL/6 mice 3 and was attributed when at least 3 spikes were discharged during a 6s cold stimulus from 3 to 1 C (see methods). The cold responses of the cold-sensitive fibers (n=13 and n=9, respectively) are summarized in a histogram with a bin width of 4 s; a two-point adjacent averaging procedure was applied. The lower trace represents the cold stimulus. Although there were apparently fewer cold-sensitive C-fibers in the (larger) sample from Na v 1.8-/- (28% in comparison to 54% in WT), the magnitude of the cold-response did not significantly differ in both genotypes, and was in WT 6.9 ±1.2 (n=13) and in Na v 1.8-/- 5.3 ±.5 (n=9) spikes per 6 s cold stimulus. However, while in WT responses ranged from 3 to 18 spikes per cold stimulus, responses of C-fibers in Na v 1.8-/- were not greater than 8 spikes per stimulus. The threshold of the cold responses was not different between both genotypes (WT: 21.9 ±1.2 C, Na v 1.8-/-: 21.4 ±1.3 C). Fig. S6b: Effect of 5 µm menthol applied at 3 C on mechanosensitive C- fibers in WT and Na v 1.8-/-. The histogram summarizes the excitatory effect of menthol in both genotypes at 3 C for the first 2 min of the 5 min long application of menthol and gives a timepoint-analysis of Fig. 3h. Values are summarized in bins of 4 s and a two-point adjacent averaging procedure was applied. 24 (WT) and 23 (Na v 1.8-/-) mechanosensitive fibers were randomly chosen, and the receptive fields were superfused with menthol (5µM) at 3 C for 5 minutes before a cold stimulus was applied. 7 WT and 12 Na v 1.8-/- fibers were sensitized to cold in the presence of menthol (see also Fig. 3g, h), only these fibers were included in this histogram. Of these cold-sensitized fibers, 4 WT and 7 Na v 1.8-/- were also excited by menthol at 3 C. The actual values were; in WT 27 ±11 spikes/ 2min (n=7) and 22 ±8 spikes/ 2min (n=12). www.nature.com/nature 6

7 7. Electrical stimulus strength duration measurements (according to Hugh Bostock, 1983 4 ). Membrane parameters can not be measured directly in cutaneous C-fiber terminals, but the effect of changes in variables such as temperature on electrical excitability can provide indirect evidence of changes in membrane parameters. Therefore we have analyzed the effect of temperature and TTX on excitability changes in the C- fiber terminals. The SD-relationship (see methods) was used to determine two key parameters of C-fiber excitability; rheobase current and chronaxy Changes in the excitability of the terminals in response to cooling and TTX were expressed as changes in rheobase current and chronaxy. Rheobase current and chronaxy are both dependent upon the passive (i.e. resting or sub-threshold) electrical properties of the nerve endings as well as on sodium channel properties 4,5. Cooling affects both of these and it has previously been shown that a decrease in temperature increases chronaxy (Q 1 1.4) and decreases rheobase current (Q 1.86) 4. In accord with this, cooling the nerve terminal region by 2 C decreased rheobase current (I rh 17 ±2.2 µa at 3 C and 1 ±.9 µa at 1 C; p<.1; n=9, paired t-test) and increased chronaxy. The increase in chronaxy was most pronounced in cold-sensitive fibres (τ SD 3.8-fold ±.4 CMC, n=5; other units (CMH, CM): 1.2-fold ±.3, n=11; p<.1, paired t-test). In addition to its dependence on sodium channel properties chronaxy also reflects the passive membrane time constant (i.e. τ M = R M C M ). Since membrane capacitance should not change for a fixed electrode geometry, this large increase in chronaxy is i. a. consistent with a relative increase in sodium conductance 5 in the cold, as suggested by the leftward shift of the (I-V) activation curves (at -8 mv holding potential) in Figs. S3c,d. However, it may as well reflect a cooling-induced increase in membrane resistance, i.a. due to closure of leak potassium channels (e.g. two-pore domain potassium channels) expressed in cold-sensitive units 6-8. In the presence of TTX (1 µm) cooling did not further prolong chronaxy (control 1 C: 3.6 ±.3, and TTX 1 C: 2.7 ±.9 n=5; p=.2, paired t-test), but produced a large decrease in rheobase and all other threshold currents consistent with increased depolarizations and the availability of TTXr current during cooling (Fig. S7b, lower panel). Fig. S7: Cooling increases TTXr excitability in native nociceptors. Strengthduration measurements (upper panels) for a single CMH fiber at different temperatures (3 C and 1 C). This unit was extraordinary, as it retained excitability at 3 C in presence of 6 µm TTX, although 65-fold greater current (1 ms) than in absence of TTX was required to reach the threshold. a. before and b. in the presence of TTX 6µM. Rheobase current and chronaxy were determined by the gradient and zero charge intercept of the regression of charge (Q) on stimulus duration (t) in the lower panels. In the absence of TTX (a) cooling increased threshold currents, but decreased rheobase current and prolonged chronaxy (lower left panel), while in the presence of TTX (b) cooling strongly decreased rheobase, and threshold currents at all stimulus durations. Like cooling, TTX also increased chronaxy (lower panel, a versus b), consistent with the slower kinetics of Na v 1.8. www.nature.com/nature 7

8 References used in Supplement 1. Matsutomi,T., Nakamoto,C., Zheng,T., Kakimura,J. & Ogata,N. Multiple types of Na(+) currents mediate action potential electrogenesis in small neurons of mouse dorsal root ganglia. Pflügers Arch. 453, 83-96 (26). 2. Akopian,A.N. et al. The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat. Neurosci. 2, 541-548 (1999). 3. Alloui,A. et al. TREK-1, a K+ channel involved in polymodal pain perception. EMBO J. 25, 2368-2376 (26). 4. Bostock,H. The strength-duration relationship for excitation of myelinated nerve: computed dependence on membrane parameters. J. Physiol 341, 59-74 (1983). 5. Bostock,H. & Rothwell,J.C. Latent addition in motor and sensory fibres of human peripheral nerve. J. Physiol 498, 277-294 (1997). 6. Viana,F., de la,p.e. & Belmonte,C. Specificity of cold thermotransduction is determined by differential ionic channel expression. Nat. Neurosci. 5, 254-26 (22). 7. Kang,D., Choe,C. & Kim,D. Thermosensitivity of the two-pore domain K+ channels TREK-2 and TRAAK. J. Physiol 564, 13-116 (25). 8. Reid,G. & Flonta,M. Cold transduction by inhibition of a background potassium conductance in rat primary sensory neurones. Neurosci. Lett. 297, 171-174 (21). www.nature.com/nature 8

9 Fig. S1 a b TTX resistance depends on sensory fiber-type and ambient temperature Percentage of fibres 8 6 4 2 1 Rat (n=37, C and A ) 1 2 7 4 3 6 Mouse (n=11/1) 2 2 1 CMC CMH CM A-delta WT NaV1.8-/- Rat CMH-fiber,.81 m/s, 45 mn 1 9 1 Temp., C Inst. discharge, 1/s 2 1 5 4 3 2ms μv 1 2 3 TTX 1μM 136 137 138 139 14 s www.nature.com/nature 9

1 Fig. S2 a C57BL/6, CM-fiber,.4 m/s, 11.4 mn Time TTX1μM, 3 C, 2T 4s 8s 12s 16s 2s 6s 12s 13s 53.4ms b TTX-induced slowing of CV Shift in Latency, ms d 6 5 4 3 2 1 Cold-induced slowing of the conduction velocitiy (CV) 12 * WT Nav1.8-/- WT ** n.s. c Time to onset of TTX-block Time, s 18 12 6 Nav1.8-/- * WT Nav1.8-/- *** ** 14s Block after 152s at 3 C 59.2ms Latency, ms 8 6 4 2 1 2 3 4 5 6 7 8 Latency ms 3 C 1 C 3 C 1 C www.nature.com/nature 1

Fig. S3 a TTXs - DRG TTXr - DRG b 3 C 1nA 1nA 1 ms 1 ms 1 C 1nA 1nA c V -12 mv h 1 ms d V -8 mv h 1 ms normalized current 1..8.6.4.2. TTXs TTXr 3 C 1 C normalized current 1..8.6.4.2. TTXs TTXr 3 C 1 C - -8-6 -4-2 2 Voltage, mv - -8-6 -4-2 2 Voltage, mv www.nature.com/nature 11

12 Fig. S4 a Voltage, mv Resting Membrane Potential -6-5 -4-3 -2-1 n.s. n.s. Voltage, mv Action Potential Overshoot 7 n.s. 6 5 4 3 2 1 3 C 1 C 3 C 1 C 3 C 1 C 3 C 1 C WT Nav1.8-/- WT Nav1.8-/- b * not excitable www.nature.com/nature 12

aspikes per 3s stimulus Fig. S5 Magnitude of Response 4 3 2 WT Nav1.8-/- Nav1.8-/- n.s. * ** 3 C 1 C 3 C 1 C Median 25%-75% Min-Max * percentage of WT value 25 5 75 3 C 1 C b Peak frequency, spikes /s Peak Firing Rate 12 8 6 4 2 WT Nav1.8-/- Median 25%-75% Min-Max ** * ** *** 3 C 1 C 3 C 1 C percentage of WT value 25 5 75 3 C 1 C Time, s c Time to total adaptation 3 C 1 C n.s. n.s. 3 25 2 15 1 5 WT ** ** www.nature.com/nature 13

14 Fig. S6 a Spikes /4s Temp, C Cold Responsiveness of nociceptive terminals in WT and Nav1.8-/- 1.4 1.2 1..8.6.4.2 3 1 WT Nav1.8-/- 2 4 6 8 s Spikes per 6s cold stimulus 9 n.s. 8 7 6 5 4 3 2 1 WT NaV1.8-/- b Excitatory effects of menthol on nociceptive terminals at 3 C in WT and Nav1.8-/- 5 Menthol 5μM Spikes /4s 4 3 2 1 WT Nav1.8-/- 2 4 6 8 12s www.nature.com/nature 14

15 Fig. S7 a Rat CMH-fiber, 11.4 mn,.41 m/s b Same fibre as (a) under TTX Threshold current, μa 4 3 2 SD - Curves 1 C 3 C Threshold current, μa 7 6 5 4 3 2 SD - Curves TTX 6μM 3 C 1 C.1 1 1 ms.1 1 1 ms Charge - Threshold Plot Charge - Threshold Plot Threshold charge, μc 3 25 2 15 5-3.46 3 C 1 C 1 2 3 4 5 6 7 8 9 1 -.96 Stimulus width, ms Threshold charge, μc 8 6 4 2-4.98-2.32 3 C 1 C 1 2 3 4 5 6 7 8 9 1 Stimulus width, ms www.nature.com/nature 15