Fig1_Activity: --- Dorsal root ganglion neurons were transfected with Axon-jGCaMP8m and cultured in microfluidic chambers for 5-6 days. Both the axon and soma compartments were treated with inflammatory mediators or normal media for four hours. Then, the treatment was removed by thoroughly washing both chambers with DRG neuronal imaging saline. Multiple fields of view containing GCaMP-expressing axonal ends were selected and green time-lapse movies were acquired at 10 Hz. Then, DRG NIS containing 10 nM ProTx-II (Tocris) and 0.1% BSA was added to both chambers by fluid exchange over approximately five minutes. After another five minutes of incubation, the same fields of view were imaged again. GCaMP-expressing axon endings which were clearly separate from other axons were selected for analysis. Calcium time-course data was extracted from the distal most 30 µm of axons. Regions of Interest 6 pixels wide were generated over this final 30 µm of the axon and the average fluorescence intensity within this region was measured at each timepoint. The percent change in fluorescence at each timepoint was calculated relative to a least-squares regression line that had been fit to the entire time-course. Calcium events were defined as any instance where the fluorescence intensity increased by > 8% between two consecutive frames. ### Description of the Data and file structure The data are measurements of fluorescence intensity within individual axon endings over time. Each column represents a given timepoint and each row represents a given axon. Reading the data from left to right across a row provides the time course of fluorescence intensity within a given axon over the 2-minute observation. The right-most column is the number of calcium events which occurred in each axon during the observational period (as calculated by the method above). --- Fig2_Current: --- #### Voltage-clamp recordings Macroscopic currents were recorded in voltage-clamp mode using an EPC-10 amplifier and the PatchMaster Next program (HEKA Electronik). Series resistance compensation of 80-90% was applied to reduce voltage error. Recordings were sampled at 50 kHz through a low-pass Bessel filter of 2.9 kHz. After achieving the whole-cell configuration, a 5-min delay was applied to allow adequate time for the pipette solution and cytoplasmic milieu to equilibrate. Current density was measured by normalizing peak currents with cell capacitance. Recordings from cells in the IM treatment condition were acquired after 4 hours of incubation with the previously described IM cocktail. ##### NaV1.7 DRGs from adult (4-6 week old) NaV1.8-KO mice were harvested and dissociated. After trituration, neurons were transfected with 2.0 µg of eGFP-2A-hNaV1.7 (with the Y362S substitution to render the channel TTX-resistant) using a Nucleofector IIS (Lonza) and Amaxa Basic Neuron SCN Nucleofector Kit. Cells were plated into 24-well plates containing cover slips coated with poly-L-lysine and laminin and maintained in DRG media at 37º C for 24 hours after transfection before acquiring patch-clamp recordings. To measure NaV1.7 currents, small diameter (<25 µM) DRG neurons with green fluorescence were selected for whole-cell voltage clamp recording. Patch pipettes were fabricated from borosilicate glass (World Precision Instruments) using a P-97 puller (Sutter Instruments) and fire-polished for a resistance of 0.8-1.2 megaohms when filled with internal solution. The pipette internal solution contained (in mM): 140 CsF, 10 NaCl, 1.1 EGTA, 10 HEPES, 20 Dextrose (pH 7.3 with CsOH, adjusted to 310 mOsm/L with dextrose). External bath solution contained (in mM): 140 NaCl, 20 TEA-Cl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 Sucrose, 0.1 CdCl2, 0.001 TTX (pH 7.3 with NaOH, adjusted to 320 mOsm/L with sucrose). Currents were evoked by 100 ms depolarizing voltage steps from -80 to +40 mV in 5-mV increments from a holding potential of -100 mV. ##### KV7 (M-current) Neonatal rat DRG neurons were harvested and dissociated and plated onto 24-well plates containing cover slips coated with poly-L-lysine and laminin and maintained in DRG media for 2 hours before acquiring patch-clamp recordings. To measure endogenous M currents, small diameter (<25 µM) DRG neurons were selected for whole-cell voltage clamp recording. Patch pipettes were fabricated from borosilicate glass (World Precision Instruments) using a P-97 puller (Sutter Instruments) and fire-polished for a resistance of 4.0-5.0 megaohms when filled with internal solution. The pipette internal solution contained (in mM): 80 K-acetate, 30 KCl, 1 CaCl2, 3 EGTA, 40 HEPES, 3 MgCl2 (pH 7.3 w/ KOH, adjusted to 310 mOsm/L with dextrose). External bath solution contained (in mM): 144 NaCl, 2.5 KCl, 2 CaCl2, 0.5 MgCl2, 5 HEPES, 10 Dextrose, 0.001 TTX, 0.1 CdCl2, 0.02 ZD-7288 (pH 7.3 w/ NaOH, adjusted to 320 mOsm/L with dextrose). TTX, CdCl2, and ZD-7288 were included in the bath to block voltage-gated Na+, voltage-gated Ca2+, and HCN channels, respectively. A standard voltage protocol was applied to evoke M-current. Cells were held at -20mV. A series of 500ms hyperpolarizing pulses from -20 to -100 mV were applied in 5 mV increments. M-current currents were determined using the methods described by Tzour et al. Briefly, voltage steps induced slow current relaxations which represent slow M-current deactivation. Current relaxations were fit by exponential curves (beginning after the capacitance artefact) and were extrapolated to the beginning of the command pulse. M-current amplitudes were assessed as the difference between the peak current at command onset and the steady state current at command offset. ### Description of the Data and file structure #### NaV1.7 The excel files containing data for NaV1.7 recordings includes columns which correspond to the following: the name of the cell recorded, the value of the compensated slow capacitance transient, the maximal inward current, and the value of the peak current normalized to the capacitance of the cell. #### KV7 The excel files containing data for KV7 contains a set of current measurements for each cell (identified in row 1). Current relaxations were fit with exponential curves, which allowed for the determination of a “steady-state” current value. “Peak” current (i.e. most positive current value in the recording following the capacitance artefact) was determined for each cell. Maximal M-currents occurred between -35 and -55 mV voltage steps for all cells, so the difference between Peak and Steady-state currents was determined for voltage steps at -35, -40, -45, -50, and -55 mV. This was indicated as “Processed-value” and corresponds to the peak M-current in that cell. The value of the compensated slow capacitance transient is included (Row titled: “Capacitance”) and “Current Density” is defined as the peak current normalized to the capacitance. --- Fig2_Surface Expression: --- Dorsal root ganglion neurons were transfected with Halo-NaV1.7 or KV7.2-Halo and cultured in microfluidic chambers for 5 days. Both soma and axon chambers were then treated for 4 hours with either inflammatory mediators or normal media. Axonal surface channels were then labeled with cell-impermeable JF635i-Halo-tag Ligand and unbound ligand was washed away. After labeling, the dishes were placed in a stage-top incubator and multiple axons (in ~3 fields of view) were imaged every ~4 seconds over the course of an hour. NaV1.7 surface expression was quantified as the average fluorescence intensity of the most distal 30 µm of labeled axons in the first frame of the movie, before significant endocytosis has occurred. ### Description of the Data and file structure Each cell contains a background-subtracted measure of fluorescence intensity for an individual axon. The columns contain independent measurements from either control or IM-treated axons. --- Fig3_Anterograde_Retrograde: --- Dorsal root ganglion neurons were transfected with Halo-NaV1.7 or KV7.2-Halo and cultured in microfluidic chambers for 5 days. Both soma and axon chambers were then treated for 4 hours with either inflammatory mediators or normal media. Channels in the soma compartment were labeled with cell-permeable, red JF549-Halo-tag ligand while channels in the axonal compartment were labeled with cell-permeable, far-red JF646-Halo-tag Ligand. After labeling, anterograde vesicles were visualized using red imaging and separately, retrograde vesicles were visualized using far-red imaging as they were transported between the two chambers. Resulting movies were opened in ImageJ and the KymographClear toolset was used to create kymographs of selected axons. Specifically, axons containing trafficked vesicles were traced manually using a segmented line, and KymographClear extracts the signal under that line and converts it into a two-dimensional image with distance along the axon on the x-axis and time on the y-axis. Two kymographs were generated for each axon, one from the red movie showing anterograde trafficking and one from the far-red movie showing retrograde trafficking. Kymographs were analyzed using the automated kymograph analysis software KymoButler, which uses a machine learning algorithm to trace vesicle tracks. Vesicle flux was determined by counting the number of vesicles which crossed the midline of the kymograph in either the anterograde or retrograde direction. Vesicle intensity was determined as the average of fluorescence values of pixels along the vesicle track, minus the background signal of the kymograph (defined as the modal value for that kymograph). Vesicle velocity was calculated as the average over the duration of the track, including pauses and stops. The fluorescence intensities and velocities of multiple vesicles within an axon were averaged. Only axons that were separate from other axons were analyzed. ### Description of the Data and file structure The kymographs which were created by KymographClear and analyzed by KymoButler are included as single color 32-bit tif files. The kymograph dimensions are 0.201 um/pixel (x-axis) and 0.33 s/pixel (y-axis). For each set of kymographs (Nav1.7 or Kv7.2, Anterograde(Ant) or Retrograde(Ret)) there is a spreadsheet which contains the processed outputs of KymoButler analysis. Each cell contains the vesicular flux, average intensity, or average velocity of the vesicles within a given axon. --- Fig4_Insertion_Removal: --- Dorsal root ganglion neurons were transfected with Halo-NaV1.7 or KV7.2-Halo and cultured in microfluidic chambers for 5 days. NaV1.7 channels at the surface of axons were labeled with cell-impermeable, red JF549i-Halo-tag Ligand (100 nM) for 15 minutes and excess ligand was thoroughly washed away. Then, axons were exposed to cell-impermeable, far-red JF635i-Halo-tag Ligand (10 nM), which was maintained throughout the experiment. JF635i-Halo-tag ligand was used at a lower concentration (10 instead of 100 nM) to minimize fluorescent background while still allowing for rapid labeling of newly inserted proteins. Labeled axons were identified and imaged using red and far-red imaging in a stage-top incubator for 6 hours in serum-free media with or without inflammatory mediator cocktail. For each time point and field of view, confocal z-stacks were acquired in both red and far-red imaging channels. For analysis, z-stacks were processed by maximum-intensity projection and the mean fluorescence intensity of each color was measured within the distal most 30 µm of the axon. The background of the maximum intensity projection (modal intensity value) was determined for each color, timepoint, and field of view and was subtracted from each intensity measurement. Original surface NaV1.7 (JF549i) signal was normalized to the value at time 0 for each axon. ### Description of the Data and file structure Each value is a fluorescence intensity measure from a given axon. Each column represents an axon, and each row is a given time-point after the start of the experiment. Reading a column from top to bottom gives the time course for that axon over the 6 hour experiment. "Null" values indicate that an axon was not measured at a particular time point (either because it was not in the focal plane or grew out of the field of view). --- Fig5_Co-trafficking: --- Dorsal root ganglion neurons were transfected with SNAP-NaV1.7 and KV7.2-Halo and cultured in microfluidic chambers for 5-6 days. Both soma and axon chambers were then treated for 4 hours with either an inflammatory mediator cocktail (IM) or normal media (Ctl). JF646-Halo-tag ligand and JF552-cpSNAP-tag ligand (100 nM each) were added to the soma chamber for 30 minutes before imaging. Axons within the axonal chamber were then imaged in red and far-red by rapid laser and color filter switching. Axons were selected for analysis if there was at least one moving vesicle containing each protein visible in the axon, indicating that the neuron had been transfected with both constructs. Kymographs of these axons were created, and the fluorescence intensity in both colors was measured for each vesicle manually. The background was measured for each color and kymograph, and subtracted from the vesicle measurements. A cutoff of 100 A.U. was used to categorize vesicles as positive or negative for each protein. To compare the amount of NaV and KV channels in individual double positive vesicles, the KV7.2 fluorescence intensity was subtracted from the NaV1.7 intensity. The values for all of the vesicles within a given axon were averaged. The distributions were then normalized to the control condition by subtracting the mean of the control from each data point. ### Description of the Data and file structure Two-color kymographs from control or IM-treated axons are included in 32-bit tif format. For each kymograph, channel 1 contains red JF552-cpSNAP-tag (NaV1.7) signal and channel 2 contains far-red JF646-Halo-tag (KV7.2) signal. The kymograph dimensions are 0.302 um/pixel (x-axis) and 0.70 s/pixel (y-axis). The processed data file contains the vesicle counts from each experimental replicate, classified as Nav single positive, Kv single positive, or double positive down columns, and separated by replicate across rows. This file also contains the double positive vesicle intensity, where each value represents the average Nav-Kv fluorescence intensity for the double positive vesicles in a given axon, normalized to the average control value. --- FigS1_RT-qPCR: --- Dorsal root ganglion neurons were isolated and plated into 6-well cell culture plates (Corning) which had been pre-coated with poly-D-lysine for 1 hour at 37°C and subsequently with laminin for 2 hours at 37°C. The cells were then treated with IM cocktail for 4 or 24 hours. After the cells had been in culture for 48 hours, RNA was extracted using the RNeasy® kit (Qiagen) according to the manufacturer’s protocol. Complementary DNA was generated from 100 ng of DRG RNA using Bio-Rad iScript™ Reverse Transcription Supermix. The following Prime-PCR probe Bio-Rad assays were used: Actb (qRnoCIP0050804), Gapdh (qRnoCIP0050838), NaV1.7 (qRnoCIP0023430). KV7.2 RNA was measured using Rat Kcnq2 TaqMan® Gene Expression Assay (Rn00591249¬_m1, Fisher Scientific). 10 µl PCR reactions were run with Bio-Rad SsoAdvanced™ Universal Probes Supermix in technical triplicate for each sample using the Bio-Rad CFX96 Touch System with the following thermal cycling procedure; 95°C for 30 s followed by 40 cycles of 95°C for 10 s and 60°C for 20 s. Three independent cultures (biological replicates) were used for each condition and three technical replicates were averaged for each biological replicate. The target genes were normalized to Actin and Gapdh expression. No reverse transcriptase controls (NRT) and no template controls (NTC) were used for each condition, and if any control reaction passed threshold with a Cq less than 35, data from that sample was excluded. The relative quantity and normalized expression data (ΔΔCq) were processed using Bio-Rad CFX Maestro. ### Description of the Data and file structure In the data excel file, Cq values for three technical replicates for each sample are listed across rows. Three independent biological replicates are arranged vertically. The relative normalized expression results (calculated by the ΔΔCq method) are also listed with upper and lower limits. --- FigS2_Endocytosis: --- Dorsal root ganglion neurons were transfected with Halo-NaV1.7 or KV7.2-Halo and cultured in microfluidic chambers for 5 days. Both soma and axon chambers were then treated for 4 hours with either inflammatory mediators or normal media. Axonal surface channels were then labeled with cell-impermeable JF635i-Halo-tag Ligand and unbound ligand was washed away. After labeling, the dishes were placed in a stage-top incubator and multiple axons (in ~3 fields of view) were imaged every ~4 seconds over the course of an hour. Because these movies were acquired at a lower frame rate (~0.25 Hz), vesicle tracks along kymographs were discontinuous, which interfered with automated analysis. Thus, kymographs were generated using KymographClear, but endosome tracks were traced and analyzed manually: fluorescence intensity of each endosome was measured and background subtracted; each endosome within a kymograph was counted, and the total was normalized to the length of the movie to calculate endosome flux per 60 minutes; velocity (instantaneous, excluding stops) was extracted using KymographDirect. ### Description of the Data and file structure The kymographs which were created by KymographClear are included as single color 32-bit tif files. For each set of kymographs (Nav1.7 or Kv7.2) there are processed data spreadsheets which contain the processed outputs. Each cell contains the vesicular flux, average intensity, or average velocity of the vesicles within a given axon.