1. Volume 93, Issue 4, Pages 806-821.e9 (February 2017)
FGF13 Selectively Regulates Heat Nociception by Interacting with Nav1.7 
Liu Yang, Fei Dong, Qing Yang, Pai-Feng Yang, Ruiqi Wu, Qing-Feng Wu, Dan Wu, Chang-Lin Li, Yan-Qing Zhong, Ying-Jin Lu, Xiaoyang Cheng, Fu-Qiang Xu, Limin Chen, Lan Bao, Xu Zhang 
Neuron 
Volume 93, Issue 4, Pages e9 (February 2017)
DOI: /j.neuron
Copyright © 2017 Elsevier Inc. Terms and Conditions

2. Figure 1 FGF13 in DRG Neurons Is Required for Heat Nociception
(A) Immunostaining shows that FGF13 is present in DRG neurons, afferent fibers in the laminae I-II of spinal cord (SC), and efferent fibers in the plantar glabrous skin of Fgf13F/Y (F/Y) mice. FGF13 staining is abolished in SNS-Cre/Fgf13F/Y (−/Y) mice. Scale bars, 50 μm for DRG, 100 μm for SC and skin.
(B) Immunoblotting shows that the FGF13 level decreases in both the DRG and SC of −/Y mice.
(C–F) −/Y mice do not respond to noxious heat. The tail-flick (C), Hargreaves (D), and hot plate (E) tests show that F/Y mice display nociceptive responses to noxious heat stimulation and that the response latency decreases as the stimuli temperature increases. However, −/Y mice do not respond to noxious heat stimulation (n = 14 in the tail-flick test; n = 10 in the Hargreaves test; n = 13 for F/Y and n = 14 for −/Y in the hot plate test). In the CFA-induced inflammatory pain model, F/Y mice show hyperalgesic responses to radiant noxious heat applied to the plantar skin. −/Y mice do not show hyperalgesia (n = 12 for F/Y and n = 14 for −/Y) (F). All the tests were stopped at the cutoff (C.O.) time if the animal did not respond.
(G) The two-temperature choice test shows that −/Y mice exhibit normal sensitivity to innocuous temperatures (n = 12–20 for F/Y and n = 8–15 for −/Y) (left). −/Y mice are less sensitive to noxious heat conditions than F/Y mice, but −/Y mice exhibit normal sensitivity to noxious cold (n = 9 for F/Y and n = 7 for −/Y) (right).
(H) The fMRI activation maps describe brain activity in response to noxious heat (47.5°C) stimulation applied to the left hindpaw of mice. The top and middle rows show fMRI activation maps of F/Y (n = 16) and −/Y (n = 15) mice, respectively. The bottom row shows the statistical difference map overlaid with a mouse atlas for the identification of brain regions with altered noxious heat responsiveness. Scale bar, t value range. L, left; R, right; d, dorsal; v, ventral; Amyg, amygdala; CPu, caudate nucleus and putamen (striatum); HPC, hippocampus; Ins, insular cortex; MD, mediodorsal nuclei of thalamus; PAG, periaqueductal gray; PF, parafascicular nuclei of thalamus; RSG, retrosplenial granular cortex; S1 and S2, the primary and secondary somatosensory cortices.
(I and J) Comparison of changes in the BOLD signal of fMRI between F/Y and −/Y mice in ten brain regions (I and J) and between the left (ipsilateral) and right (contralateral to the site of heat stimulation) hemispheres in six brain regions (I).
(K) Amplitude differences in percentage of fMRI signal changes between F/Y and −/Y mice in ten brain regions.
The data are shown as mean ± SEM; ∗∗p < 0.01 and ∗∗∗p < versus the indicated group. See also Figures S1–S4.
Neuron  , e9DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 2
Heat-Evoked APs Are Reduced in FGF13-Deficient Small DRG Neurons, and Functional Deficits Are Rescued by GST-FGF13B-TAT
(A) AP firing was recorded in small DRG neurons perfused with extracellular solution ranging from room temperature to 45°C–49°C. DRG neurons of F/Y mice fire APs with accelerating frequency and relatively uniform amplitude, but the DRG neurons of −/Y mice show rapidly reduced AP firing. The AP firing deficits are rescued by the cell-permeable protein GST-FGF13B-TAT.
(B) The number of DRG neurons responding to heat stimulation is not changed in −/Y mice, while the number of heat-sensitive neurons is decreased in Trpv1−/− mice.
(C–F) The AP firing frequency (C and D) and amplitude (E and F) is quantified. The AP firing distribution is shown by the percentage of AP spike numbers distributed at each degree from 38°C to 45°C (C). The percentage of APs firing at 45°C reveals the remaining APs at high temperature (D). The fitting curve for AP amplitudes from 38°C to 45°C in each group shows the change in amplitude with temperature (E). The ratio of the AP amplitude at 45°C to the first AP reveals the change induced by heat stimulation (F). As the temperature becomes higher than 42°C, AP frequency is accelerated and AP amplitude decreases to ∼66% in F/Y neurons, while AP tends to cease firing and AP amplitude drops rapidly to ∼9% in −/Y neurons. GST-FGF13B-TAT, but not GST-TAT, rescues the AP firing of −/Y neurons. Trpv1 knockout does not alter the AP firing of small DRG neurons during heat stimulation. In (C) and (E), n = 17 for F/Y, n = 13 for −/Y, n = 13 for −/Y + GST-FGF13B-TAT, n = 10 for −/Y + GST-TAT and n = 11 for Trpv1−/−; in (D) and (F), n = 16 for F/Y, n = 14 for −/Y, n = 13 for −/Y + GST-FGF13B-TAT, n = 10 for −/Y + GST-TAT, n = 11 for Trpv1−/−.
(G) GST-FGF13B-TAT (0.5 mg/kg, i.t.) was injected into mice. Immunoblotting shows that GST-FGF13B-TAT infiltrates into the DRG and SC and interacts with Nav1.7. The interaction remains more than 1 hr after injection (n = 3). The arrows point to the band of the exogenous FGF13 with a GST tag.
(H) The administration of GST-FGF13B-TAT (0.5 mg/kg or 1 mg/kg, i.t.) rescues the heat nociception of −/Y mice in the tail-flick test at 52°C. The best rescue effect appears at ∼1 hr after injection (n = 5 for a dose of 0.5 mg/kg and n = 4 for a dose of 1 mg/kg). The test was stopped at the cutoff (C.O.) time.
The data are shown as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < versus the indicated group. See also Figure S5.
Neuron  , e9DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

4. Figure 3 FGF13 Interacts with Nav1.7
(A) Dual fluorescent in situ hybridization shows the coexistence of FGF13 and Nav1.7 mRNA in DRG neurons (arrows). Scale bar, 50 μm.
(B) Co-IP shows that FGF13 interacts with Nav1.7 in the DRG and SC. The FGF13/Nav1.7 interaction level is higher than the interaction levels between FGF13 and other sodium or TRP channels (n = 3–6).
(C) A GST pull-down assay shows that GST-FGF13B protein interacts with Nav1.7. The interaction between GST-FGF13B and Nav1.8 is much weaker (n = 3).
(D) The C terminus of Nav1.7 (Nav1.7CT) was fused to myc-CD8α, which anchors Nav1.7CT to the cell membrane. Co-IP shows that FGF13 interacts with Nav1.7CT fused to myc-CD8α, but not with myc-CD8α.
(E) A schematic illustration of GST-flag-Nav1.7CT truncations. Nav1.7CT was truncated into nine fragments to search for the FGF13-binding site. The position of the major binding site f8 is marked in red.
(F) A GST pull-down assay with two purified proteins, GST-flag-Nav1.7CT and FGF13B, shows the direct interaction between FGF13 and Nav1.7. The major interaction site is f8 (1,876–1,896 aa) of Nav1.7CT (n = 4).
The data are shown as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < versus the indicated group. See also Figure S6.
Neuron  , e9DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

5. Figure 4 FGF13 Increases Sodium Currents
(A) FGF13B expression increases Nav1.7 current in HEK293 cells transfected with the plasmid expressing Nav1.7.
(B) The I-V curve shows the Nav1.7 current density in HEK293 cells is enhanced by FGF13B (n = 16 for Nav1.7 + vector and n = 17 for Nav1.7 + FGF13B).
(C) The voltage dependence of Nav1.7 activation and inactivation in HEK293 cells is not changed by FGF13B (activation: n = 16 for Nav1.7 + vector and n = 17 for Nav1.7 + FGF13B; inactivation: n = 15 for Nav1.7 + vector and n = 17 for Nav1.7 + FGF13B).
(D) The TTX-S sodium current is reduced in small DRG neurons of −/Y mice.
(E) The I-V curve shows that the TTX-S sodium current density is reduced in small DRG neurons of −/Y mice (n = 23 for F/Y and n = 20 for −/Y).
(F) The activation and inactivation voltage dependence of the TTX-S sodium channel is not altered in small DRG neurons of −/Y mice (activation: n = 23 for F/Y and n = 20 for −/Y; inactivation: n = 16 for F/Y and n = 17 for −/Y).
(G) The sodium current of small DRG neurons was recorded at 30°C and 45°C. The I-V curve shows that the sodium current is smaller in small DRG neurons of −/Y mice than in those of F/Y mice in both temperature conditions; in addition, treatment at 45°C reduces the sodium current of −/Y neurons, but not that of F/Y neurons (n = 17 for F/Y and n = 15 for −/Y).
The data are shown as mean ± SEM; ∗∗p < 0.01 and ∗∗∗p < versus the indicated group.
Neuron  , e9DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

6. Figure 5
FGF13 Maintains Nav1.7 Levels in Cell Membrane during Heat Stimulation
(A) The diagram shows the experimental procedure. The DRG neurons were dissociated and stimulated in 43°C water bath for 30 s or 1 min. Then, the membrane proteins were extracted by surface biotinylation and processed for immunoblotting.
(B) The amount of Nav1.7 in the plasma membrane at room temperature (RT) is not apparently changed by Fgf13 knockout. However, the Nav1.7 level in the membrane is decreased after stimulation at 43°C in −/Y neurons, but not in F/Y neurons (n = 4).
(C) The diagram shows the experimental procedure. The cells transfected with the plasmids expressing FGF13B and Nav1.7 were stimulated at 43°C. The DSS crosslinking agent was added before or after the heat stimulation. The membrane proteins were extracted by surface biotinylation and processed for immunoblotting.
(D) Immunoblotting shows the FGF13/Nav1.7 complex formation after DSS crosslinking in HEK293 cells expressing FGF13B and Nav1.7. The amount of the FGF13/Nav1.7 complex is increased after stimulation at 43°C. The increased complex formation is detected in both the plasma membrane (PM) and the cell lysate (n = 4).
(E) The FGF13/Nav1.7 complex and its increased formation after heat stimulation are also observed in DRG neurons (n = 4).
(F) Co-IP shows the heat-enhanced FGF13/Nav1.7 interaction in the enriched membrane component of FGF13B- and Nav1.7-expressing HEK293 cells (n = 4).
The data are shown as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < versus the indicated group. See also Figure S7.
Neuron  , e9DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

7. Figure 6
Nav1.7CT-TAT Blocks the FGF13/Nav1.7 Interaction in DRG Neurons and Impairs Heat-Evoked AP Firing
(A) The cell-permeable protein GST-flag-Nav1.7CT-TAT (Nav1.7CT-TAT) was purified and used to block the FGF13/Nav1.7 interaction. Coomassie brilliant blue (CBB) staining shows the protein from the first four elution units (upper). HEK293 cells were incubated with different concentrations of Nav1.7CT-TAT for 1 hr. Nav1.7CT-TAT was detected in the cell lysate by immunoblotting (lower).
(B) Co-IP shows that a 30 min treatment with Nav1.7CT-TAT (1 μM) disrupts the interaction between endogenous FGF13 and Nav1.7 in cultured DRG neurons (n = 3).
(C and D) Heat-induced AP firing is impaired in small DRG neurons by 1 μM Nav1.7CT-TAT treatment for 30 min. The impaired AP firing exhibits two patterns: APs with reduced amplitude and APs firing with very few spikes (C). The attenuated AP firing mimics the defects observed in small DRG neurons of −/Y mice (C). The ratio of the AP amplitude at 45°C to that of the first AP is used to quantify the change of AP firing induced by heat stimulation (D). Nav1.7CT-TAT did not alter the firing of small DRG neurons from −/Y mice (D). The heat-induced AP firing was reduced in small DRG neurons treated with 10 nM Nav1.7 inhibitor Protoxin-II (D). n = 11 for elution buffer, n = 16 for GST-flag-Nav1.7CT-TAT, n = 12 for GST-flag-TAT, n = 9 for GST-flag-Nav1.7CT-TAT treating −/Y neurons, and n = 11 for Protoxin-II.
The data are shown as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < versus the indicated group.
Neuron  , e9DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

8. Figure 7
Nav1.7CT-TAT Blocks Endogenous FGF13/Nav1.7 Interaction and Impairs Heat Nociception
(A) Co-IP shows that the Nav1.7CT-TAT (60 mg/kg, i.p., 4 injections, one injection per hour) blocks the FGF13/Nav1.7 interaction in afferent fibers to the SC, but not in the DRG (n = 4).
(B) The tail-flick test at 52°C shows that the Nav1.7CT-TAT (i.p.) dose-dependently impairs noxious heat sensation (n = 3).
(C) A proposed model of the mechanism for noxious heat sensation. During heat stimulation, thermosensor activation in MHNs leads to cation influxes and initiates AP firing. The heat-facilitated FGF13/Nav1.7 interaction stabilizes Nav1.7 in the plasma membrane (PM) to sustain AP firing. A lack of FGF13 reduces the number of Nav1.7 in the PM and the heat-induced AP firing and impairs heat nociception.
The data are shown as mean ± SEM; ∗p < 0.05 and ∗∗∗p < versus the indicated group.
Neuron  , e9DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions