0.002% DMSO 10µM (S)-LCM

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0.002% DMSO 10µM (S)-LCM

A

B

C

100

+ AT7519 100 nM 50

0

NS

100

50

0 + ω-conotoxin GVIA (500 nM) NS

50 0 100 150

+ myr-tat-CBD3 (10 µM) NS

Calcium influx (% of control) Calcium influx (% of control) Calcium influx (% of control)

Supplementary Figure 1: (S)-LCM inhibition of N-type channels Ca

²⁺

channels in dorsal root ganglion (DRG) neurons through block of CRMP2 phosphorylation and uncoupling of CRMP2–CaV2.2 interaction. Following a 1-min baseline measurement, neurons were stimulated with 90mM KCl for 15 s and the response measured for 3 additional minutes. Bar graphs show the normalized peak fluorescence response (normalized to vehicle) of DRGs incubated for 30 min with vehicle (0.002%

DMSO) or 10 M (S)-LCM together with the Cdk5 inhibitor AT7519 (A); myr-tat-CBD3, a peptide uncoupling the CRMP2–

CaV2.2 interaction (B); or the N-type calcium channel-specific

blocker -conotoxin GVIA (C). Values represent the average ±

S.E.M., n=71–151cells per condition. No difference was detected

between the groups (p>0.05, Student’s t-test).

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500pA 50ms

-60 -40 -20 0

Peak current density (pA/pF)

n=8 n=9

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Post/Pre pulse ratio

n=7 n=8

1nA 20ms

A

B

D C

0.002% DMSO 10 µM (S)-LCM

Supplementary Figure 2: (S)-LCM does not affect low voltage activated Ca²⁺channels currents or involve GPCR signaling. (A) Representative family of current traces for a low voltage activated Ca²⁺

channels are illustrated for control- (0.002% DMSO) or 10 M (S)-LCM-treated. (B) Peak current density, at –10 mV from a holding potential of –110 mV, for the indicated conditions (n=8 cells for control and n=9 cells for 10 M (S)-LCM treated cells; asterisk indicates statistical significance compared with control cells (p<0.05, Student’s t-test). (C) Representative family of current traces for a double pulse protocol are illustrated for control- (0.002% DMSO) or 10 M (S)-LCM-treated. (D) Bar graph shows the ratio of the peak current density before and after the depolarizing pulse. No difference was detected between control- (0.002% DMSO) and 10 M (S)-LCM indicating a GPCR–independent inhibition of voltage gated Ca²⁺ channel by (S)-LCM.!!

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0 200 400 600 800

17°C KCl

A B

0.9

0.3

1.3

0.3

0.002% DMSO 10 µM (S)-LCM

1.2

0.3 1.0

0.3

1.3

0.3 1.3

0.5

4°C

0 200 400 600 800

17°C 4°C KCl

^{Time (s)}

Cell count

17°C responders 12

10 8 6 4 2 0

400-499 500-599 600-649 650-699 700-849 850-999 >1000 0-399

Cell count

30 25 20 15 10 5 0

400-499 500-599 600-649 650-699 700-849 850-999 >1000 0-399

4°C responders

F340/F380 ratio F340/F380 ratio

Time (s)

C D

*

* *

n.s.

Supplementary Figure 3: Profiling Ca responses to innocuous and noxious cold responders. Each trace represents the response of a different neuron. In a typical ²⁺

experimental trial, the responses of >100 individual neurons were monitored simultaneously. Selected traces are shown. Arrows indicate 15-s challenges of Tyrode’s at 17°C and then 4°C followed by high [K+]

o

; after each application the cells were continuously washed out of the well with room-temperature bath solution (i.e., Tyrode’s).

The y-axis shows the Fura-2AM fluorescence ratio (F

₃₄₀

/F

380

) for each trace. Raw, unfiltered traces are presented. DRG neurons were treated with either vehicle (0.002%

DMSO) (A) or 10 M (S)-LCM (B). All cells were selected based on their response to the depolarizing pulse of KCl. Size class data for select neuronal classes is shown.

For both panels, the x-axis represents the cell area (classified according to the indicated intervals) and the y-axis denotes the number of cells corresponding to the indicated

temperature at 17°C (C) and then 4°C (D) for each condition (0.002% DMSO or 10 M (S)-LCM). !

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Supplementary Table 1:

Average peak KCl responses of DRG neurons responding to constellation pharmacology triggers

0.002% DMSO 10 µM (S)-LCM

Trigger(s) average SEM count average SEM count

responder Ach 0.277820786 0.022073543 246 0.140108593 0.020787319 90 responder AITC 0.333781853 0.031876486 120 0.127116706 0.02591476 45 responder ATP 0.450693521 0.048544016 87 0.115170833 0.02734834 51 responder Histamine 0.346054601 0.115682922 6 0.116960788 0.042078763 27 responder Menthol 0.344177003 0.047281762 45 0.090915692 0.009727203 224 responder Capsaicin 0.588745407 0.021510959 316 0.131589385 0.010372696 305 responder kcl 0.347848278 0.012593659 796 0.115217302 0.005839222 858 KCL only 0.217520115 0.014341635 209 0.114773236 0.010269473 289

all triggers 0 0

ACh 0.140835741 0.018436599 159 0.205606431 0.029366836 46 AITC 0.194015096 0.037935399 46 0.268826028 0.061466143 10 ATP 0.115898828 0.042029271 15 0.039284924 0.012450753 32 Hist 0.191268998 0.13360721 2 0.007998625 0.003276891 6 Ment 0.273122512 0.045032206 13 0.057643062 0.007841677 138

Cap 0.573506368 0.027594287 195 0.137588891 0.012726344 197

ACh/ AITC 0.193590561 0.077045438 6 0.163290362 0 1

ACh/ ATP 0.090268253 0.035300572 9 0.200047056 0.128231908 3

ACh/ Hist 0.627172196 0 1 0.920441001 0 1

ACh/ Ment 0.097053792 0.016008256 4 0.028724826 0.004547346 13 ACh/ Cap 0.672288795 0.044371714 48 0.025813314 0.008180829 20 AITC/ ATP 0.493994261 0.166726713 8 0.460999759 0.024244176 2

AITC/ Hist 0 0.144782402 0.013187269 2

AITC/ Ment 0.438702809 0.053253657 7 0.158027802 0.079963805 2 AITC/ Cap 0.499576538 0.061638599 15 0.044110199 0.02569513 20

ATP/ Hist 0 0.008931826 0 1

ATP/ Ment 1.687699681 0 1 0.293413927 0.124882595 4

ATP/ Cap 0.608198463 0.094761976 20 0.178968943 0.069313281 3

Hist/ Ment 0 0.030362148 0.007383116 9

Hist/ Cap 0 0.030789826 0 1

Ment/ Cap 0.149760227 0.084187233 4 0.174473228 0.02539487 43

ACh/ AITC/ ATP 0.004126547 0 1 0

ACh/ AITC/ Hist 0.116339608 0 1 0

ACh/ AITC/ Ment 0.243275553 0 1 0

ACh/ AITC/ Cap 0.081830791 0 1 0.008346492 0 1

ACh/ ATP/ Hist #DIV/0! #DIV/0! 0 0

ACh/ ATP/ Ment #DIV/0! #DIV/0! 0 0

ACh/ ATP/ Cap 0.901513068 0.114029741 7 0

ACh/ Hist/ Ment #DIV/0! #DIV/0! 0 0.041320351 0 1

ACh/ Hist/ Cap #DIV/0! #DIV/0! 0 0

ACh/ Ment/ Cap 0.666179724 0.009886922 2 0

AITC/ ATP/ Hist 0 0

AITC/ ATP/ Ment 0.330533482 0.090234815 9 0

AITC/ ATP/ Cap 0.724680755 0.156111856 6 0.07972672 0.039078956 2

AITC/ Hist/ Ment 0 0

AITC/ Hist/ Cap 0 0

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AITC/ Ment/ Cap 0 0.014215126 0.002383963 3

ATP/ Hist/ Ment 0 0

ATP/ Hist/ Cap 0 0

ATP/ Ment/ Cap 0.127838734 0 1 0.851903695 0 1

Hist/ Ment/ Cap 0 0.262916356 0.115755496 5

ACh/ AITC/ ATP/ Hist 0 0

ACh/ AITC/ ATP/ Ment 1.008015187 0 1 0.017371312 0 1

ACh/ AITC/ ATP/ Cap 0.439306705 0.15326929 2 0

ACh/ AITC/ Hist/ Ment 0 0

ACh/ AITC/ Hist/ Cap 0 0

ACh/ AITC/ Ment/ Cap 0 0

ACh/ ATP/ Hist/ Ment 0 0

ACh/ ATP/ Hist/ Cap 0 0

ACh/ ATP/ Ment/ Cap 0 0

ACh/ Hist/ Ment/ Cap 0 0

AITC/ ATP/ Hist/ Ment 0 0

AITC/ ATP/ Hist/ Cap 0.789248021 0 1 0

AITC/ ATP/ Ment/ Cap 0 0

AITC/ Hist/ Ment/ Cap 0 0

ATP/ Hist/ Ment/ Cap 0 0

ACh/ AITC/ ATP/ Hist/

Ment

0 0.231060606 0 1

Cap

0 0

ACh/ AITC/ ATP/ Ment/

Cap

0 0

ACh/ AITC/ Hist/ Ment/

Cap

0 0

ACh/ ATP/ Hist/ Ment/

Cap

0.161029783 0 1 0

AITC/ ATP/ Hist/ Ment/

Cap

0 0

The numbers are from four separate experiments for each treatment condition. Abbreviations: ATP, adenosine triphosphate; AITC, Allyl Isothiocyanate (mustard oil); KCl, potassium chloride.

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Percent of DRG neurons responding to constellation pharmacology triggers

Percentage of cells Number of cells

0.002% DMSO 10 µM (S)-LCM 0.002% DMSO 10 µM (S)-LCM

responder Ach 30.90452261 10.48951049 246 90

responder AITC 15.07537688 5.244755245 120 45

responder ATP 10.92964824 5.944055944 87 51

responder Histamine 0.753768844 3.146853147 6 27

responder Menthol 5.653266332 26.10722611 45 224

responder Capsaicin 39.69849246 35.54778555 316 305

responder kcl 100 100 796 858

KCL only 26.25628141 33.68298368 209 289

all triggers 0 0 0 0

ACh 19.97487437 5.361305361 159 46

AITC 5.778894472 1.165501166 46 10

ATP 1.884422111 3.72960373 15 32

Hist 0.251256281 0.699300699 2 6

Ment 1.633165829 16.08391608 13 138

Cap 24.49748744 22.96037296 195 197

ACh/ AITC 0.753768844 0.116550117 6 1

ACh/ ATP 1.130653266 0.34965035 9 3

ACh/ Hist 0.125628141 0.116550117 1 1

ACh/ Ment 0.502512563 1.515151515 4 13

ACh/ Cap 6.030150754 2.331002331 48 20

AITC/ ATP 1.005025126 0.233100233 8 2

AITC/ Hist 0 0.233100233 0 2

AITC/ Ment 0.879396985 0.233100233 7 2

AITC/ Cap 1.884422111 2.331002331 15 20

ATP/ Hist 0 0.116550117 0 1

ATP/ Ment 0.125628141 0.466200466 1 4

ATP/ Cap 2.512562814 0.34965035 20 3

Hist/ Ment 0 1.048951049 0 9

Hist/ Cap 0 0.116550117 0 1

Ment/ Cap 0.502512563 5.011655012 4 43

ACh/ AITC/ ATP 0.125628141 0 1 0

ACh/ AITC/ Hist 0.125628141 0 1 0

ACh/ AITC/ Ment 0.125628141 0 1 0

ACh/ AITC/ Cap 0.125628141 0.116550117 1 1

ACh/ ATP/ Hist 0 0 0 0

ACh/ ATP/ Ment 0 0 0 0

ACh/ ATP/ Cap 0.879396985 0 7 0

ACh/ Hist/ Ment 0 0.116550117 0 1

ACh/ Hist/ Cap 0 0 0 0

ACh/ Ment/ Cap 0.251256281 0 2 0

AITC/ ATP/ Hist 0 0 0 0

AITC/ ATP/ Ment 1.130653266 0 9 0

AITC/ ATP/ Cap 0.753768844 0.233100233 6 2

AITC/ Hist/ Ment 0 0 0 0

AITC/ Hist/ Cap 0 0 0 0

AITC/ Ment/ Cap 0 0.34965035 0 3

ATP/ Hist/ Ment 0 0 0 0

ATP/ Hist/ Cap 0 0 0 0

ATP/ Ment/ Cap 0.125628141 0.116550117 1 1

Hist/ Ment/ Cap 0 0.582750583 0 5

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ACh/ AITC/ ATP/

Hist

0 0 0 0

ACh/ AITC/ ATP/

Ment

0.125628141 0.116550117 1 1

ACh/ AITC/ ATP/

Cap

0.251256281 0 2 0

ACh/ AITC/ Hist/

Ment 0 0 0 0

ACh/ AITC/ Hist/

Cap

0 0 0 0

ACh/ AITC/ Ment/

Cap

0 0 0 0

ACh/ ATP/ Hist/

Ment

0 0 0 0

ACh/ ATP/ Hist/ Cap 0 0 0 0

ACh/ ATP/ Ment/

Cap 0 0 0 0

ACh/ Hist/ Ment/ Cap 0 0 0 0

AITC/ ATP/ Hist/

Ment 0 0 0 0

AITC/ ATP/ Hist/

Cap

0.125628141 0 1 0

AITC/ ATP/ Ment/

Cap

0 0 0 0

AITC/ Hist/ Ment/

Cap

0 0 0 0

ATP/ Hist/ Ment/ Cap 0 0 0 0

ACh/ AITC/ ATP/

Hist/ Ment 0 0.116550117 0 1

ACh/ AITC/ ATP/

Hist/ Cap

0 0 0 0

ACh/ AITC/ ATP/

Ment/ Cap

0 0 0 0

ACh/ AITC/ Hist/

Ment/ Cap

0 0 0 0

ACh/ ATP/ Hist/

Ment/ Cap

0.125628141 0 1 0

AITC/ ATP/ Hist/

Ment/ Cap

0 0 0 0

The numbers are from four separate experiments for each treatment condition. Abbreviations: ATP, adenosine triphosphate; AITC, Allyl Isothiocyanate (mustard oil); KCl, potassium chloride.

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Frequency of DRG neurons per size responding to constellation pharmacology triggers

Trigger(s) 0-399 400-499 500-599 600-649 650-699 700-849 850-1000 >1000 0-399 400-499 500-599 600-649 650-699 700-849 850-1000 >1000

responder Ach 5 20 23 21 30 57 42 48 4 8 8 10 10 25 6 19

responder AITC 11 14 29 9 16 23 10 8 11 14 6 3 0 8 1 2

responder ATP 10 12 17 9 11 10 7 11 5 8 8 6 3 13 1 7

responder Histamine 1 0 0 1 1 2 0 1 5 9 5 2 1 1 1 3

responder Menthol 3 5 5 5 8 6 2 11 11 32 25 13 14 37 21 71

responder Capsaicin 20 45 57 34 30 63 32 35 19 47 50 26 32 63 28 40 responder kcl 42 93 108 66 81 178 92 136 49 99 118 74 69 178 77 194

KCL only 10 24 22 12 13 53 28 47 14 9 44 25 21 63 31 82

all triggers 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh 3 11 11 12 21 43 24 34 2 6 3 7 4 13 2 9

AITC 3 4 11 4 5 8 6 5 3 2 2 1 0 1 0 1

ATP 3 3 2 1 1 1 0 4 2 5 5 4 1 9 1 5

Hist 0 0 0 1 0 1 0 0 1 1 1 1 0 0 1 1

Ment 0 1 0 1 3 4 0 4 4 19 10 7 8 24 14 52

Cap 13 30 32 18 19 46 12 25 10 33 31 19 23 41 19 21

ACh/ AITC 0 1 0 0 1 3 1 0 0 0 0 1 0 0 0 0

ACh/ ATP 0 1 1 1 1 1 2 2 0 0 0 0 1 0 0 2

ACh/ Hist 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0

ACh/ Ment 0 1 0 0 0 0 0 3 0 1 1 1 0 4 1 5

ACh/ Cap 2 6 8 7 3 6 11 5 0 0 3 1 5 7 2 2

AITC/ ATP 1 1 1 0 2 2 0 1 0 1 0 0 0 1 0 0

AITC/ Hist 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0

AITC/ Ment 0 0 2 0 2 1 0 2 0 1 1 0 0 0 0 0

AITC/ Cap 2 2 5 1 2 2 1 0 4 7 2 1 0 4 1 1

ATP/ Hist 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0

ATP/ Ment 0 0 0 0 1 0 0 0 1 0 0 1 1 1 0 0

ATP/ Cap 0 4 6 3 2 3 1 1 0 0 1 1 0 1 0 0

Hist/ Ment 0 0 0 0 0 0 0 0 1 5 1 0 1 0 0 1

Hist/ Cap 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0

Ment/ Cap 0 1 0 1 0 0 2 0 1 4 9 3 4 6 5 11

ACh/ AITC/ ATP 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0

ACh/ AITC/ Hist 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0

ACh/ AITC/ Ment 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0

ACh/ AITC/ Cap 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0

ACh/ ATP/ Hist 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ ATP/ Ment 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ ATP/ Cap 0 0 1 0 1 1 3 1 0 0 0 0 0 0 0 0

ACh/ Hist/ Ment 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0

ACh/ Hist/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ Ment/ Cap 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0

AITC/ ATP/ Hist 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AITC/ ATP/ Ment 2 1 3 2 1 0 0 0 0 0 0 0 0 0 0 0

AITC/ ATP/ Cap 1 2 1 1 1 0 0 0 1 1 0 0 0 0 0 0

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AITC/ Hist/ Ment 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AITC/ Hist/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AITC/ Ment/ Cap 0 0 0 0 0 0 0 0 2 0 0 0 0 1 0 0

ATP/ Hist/ Ment 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ATP/ Hist/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ATP/ Ment/ Cap 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0

Hist/ Ment/ Cap 0 0 0 0 0 0 0 0 1 0 2 0 0 1 0 1

ACh/ AITC/ ATP/ Hist 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ AITC/ ATP/ Ment 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0

ACh/ AITC/ ATP/ Cap 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0

ACh/ AITC/ Hist/ Ment 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ AITC/ Hist/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ AITC/ Ment/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ ATP/ Hist/ Ment 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ ATP/ Hist/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ ATP/ Ment/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ Hist/ Ment/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AITC/ ATP/ Hist/ Ment 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AITC/ ATP/ Hist/ Cap 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AITC/ ATP/ Ment/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AITC/ Hist/ Ment/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ATP/ Hist/ Ment/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Ment

0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0

ACh/ AITC/ ATP/ Hist/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ AITC/ ATP/ Ment/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ AITC/ Hist/ Ment/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ACh/ ATP/ Hist/ Ment/ Cap 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0

AITC/ ATP/ Hist/ Ment/ Cap 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 The numbers are from four separate experiments for each treatment condition. Abbreviations: ATP, adenosine triphosphate; AITC, Allyl Isothiocyanate (mustard oil); KCl, potassium chloride. The size bins (vertical text) are in µm².

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Percent of DRG neurons responding to cold

Percentage of cells Number of cells

0.002% DMSO 10 µM (S)-LCM 0.002% DMSO 10 µM (S)-LCM

17°C 4.545455 4.787234 22 27

4°C 6.198347 12.76596 30 72

The numbers are from two separate experiments for each treatment condition