Identification of SLC45A4 as a pain gene encoding a neuronal polyamine transporter

Abstract

Polyamines are regulatory metabolites with key roles in transcription, translation, cell signalling and autophagy¹. They are implicated in multiple neurological disorders including stroke, epilepsy and neurodegeneration and can regulate neuronal excitability through interactions with ion channels². Polyamines have been linked to pain showing altered levels in human persistent pain states and modulation of pain behaviour in animal models³. However, the systems governing polyamine transport within the nervous system remain unclear. In undertaking a Genome Wide Association Study (GWAS) of chronic pain intensity in the UK-Biobank we found significant association with variants mapping to the SLC45A4 gene locus. In the mouse nervous system SLC45A4 expression is enriched in all sensory neuron sub-types within the dorsal root ganglion including nociceptors. Cell-based assays show that SLC45A4 is a selective plasma membrane polyamine transporter, whilst the cryo-EM structure reveals a novel regulatory domain and basis for polyamine recognition. Mice lacking SLC45A4 show normal mechanosensitivity but reduced sensitivity to noxious heat and algogen induced tonic pain that is associated with reduced excitability of peptidergic nociceptors. Our findings thus establish a role for neuronal polyamine transport in pain perception and identify a new target for therapeutic intervention in pain treatment.

Figure 1.. UKB pain intensity (most bothersome) GWAS identifies novel signals.

a, A histogram illustrates the frequency distribution of pain intensity scores among participants, measured using the Numerical Rating Scale. Pain scores range from 0 (no pain) to 10 (worst possible pain). b, In a Manhattan plot from the UKB pain intensity GWAS, the genome-wide significant threshold is demarcated by a horizontal red line at 5 × 10⁻⁸. c, A regional plot centred on rs10625280, the principal SNP within the SLC45A4 gene, elucidates associations within this genomic region. SNPs are differentiated by a colour gradient based on their linkage disequilibrium (r² values) with the top independent significant SNP. The top lead SNPs in genomic risk loci, lead SNPs and independent significant SNPs are distinctly marked—encircled in black and highlighted in dark-purple, purple and red, respectively.

Figure 2.. SLC45A4 is a polyamine transporter with a novel plug domain.

a, Thermal stabilisation of SLC45A4 in the presence of metabolites in the AOP pathway using thermal stabilisation. n = 3 independent experiments, calculated mean and s.d. values are shown. b, Time course of ¹⁴C-SPD uptake in neuronal N2 cells overexpressing either human (Hs) or mouse (Mm) SLC45A4 in comparison to an empty vector control. n = 3 independent experiments, calculated mean and s.d. values are shown. c, Comparison of SLC45A4 activity in neuronal N2 cell under different external pH values. n = 3 independent experiments, calculated mean and s.d. values are shown. d, Polyamine competition of ¹⁴C-SPD uptake into neuronal N2 cells. The calculated mean (from 5 independent experiments) half-maximal inhibitory concentration values are shown ± s.d. e, Cryo-EM density of human SLC45A4 in LMNG:CHS detergent, contoured at a threshold level of 0.25. f, Cartoon representation of SLC45A4. The two 6TM bundles of the MFS fold are coloured by domain and the plug domain, which inserts in between the two is shown in purple. g, Topology diagram of SLC45A4, coloured blue to red from the amino terminus. h, Schematic of interactions between Lys450 and Arg453 in the plug domain and the polyamine binding site in SLC45A4. Hydrogen bonds and salt bridges are shown as black dashed bonds, cation-π bonds as green dashes and the charge interaction between Arg453 and Glu176, only observed in the nanodisc structure, as orange dashes. Residues are coloured by domain, as in panels e and f. i, ¹⁴C-SPD uptake in neuronal N2 cells overexpressing SLC45A4 and mutants of residues shown in h. n > 4 independent experiments.

Figure 3.. SLC45A4 is important for motor endurance, heat sensitivity and tonic pain.

a, qPCR analysis of SLC45A4 mRNA in tissues along the sensory neuraxis. From left to right, Sciatic nerve (n = 4 mice), Dorsal root ganglion (n = 5 mice), Spinal cord (n = 4 mice), Brain (n = 4 mice). b, SLC45A4 mRNA is expressed in all NeuN positive mouse DRG neurons (n = 3 mice, 1777 cells). c, Human SLC45A4-eGFP transfected in to mouse sensory neurons localises to the plasma membrane. Black arrows in the profile plot indicate the increased intensity of huSLC45A4-eGFP at the membrane. d, Schematic illustrating the SLC45A4 KO strategy using CRISPR/Cas9 deletion of exons 3–8. e, RNAscope ISH of SLC45A4 mRNA in WT and KO DRGs. SLC45A4 mRNA is absent in SLC45A4 KO mice. f, SLC45A4 KO mice show a longer latency to fall and a higher final speed, when challenged with a rotarod that gradually increases in speed (* P = 0.013, ** P = 0.0085). g, SLC45A4 HET and KO mice show a normal latency to withdraw from a noxious pin-prick (P > 0.05). h, SLC45A4 KO mice take longer to withdraw from a 48°C hotplate compared to WT and HET mice (** P = 0.002 and P = 0.0039 respectively, 20 s is the cut-off). i, SLC45A4 KO mice have an increased latency to withdraw from a 50°C hotplate compared to WT mice (* P = 0.039, 20 s is the cut-off). j, Mice were given free roam of a thermal gradient apparatus where HET and KO mice had a rightward shift in their thermal gradient profile, towards warmer temperatures (two-way ANOVA, with post hoc Holm-Sidak’s test, WT vs HET ** P = 0.009, WT vs KO * P = 0.027). Data points and non-linear fit gaussian curves shown. k, The non-linear fitted gaussian curves for the thermal gradient profiles of WT, HET, KO mice. There is a rightward shit in the HET and KO curves. A single curve cannot explain all data sets, a different curve for one data set, WT. (Extra sum-of-squares F test, * P =0.024). l, SLC45A4 KO mice display less nocifensive behaviours to a formalin injection compared to WT and HET mice (RM two-way ANOVA, post hoc Holm-Sidak’s test, 5 min: WT vs KO * P = 0.011, Het vs KO * P = 0.027. 10 min: HET vs KO * P = 0.018). m, The formalin test subclassed into Phase 1 (0–15 mins) and Phase 2 (15–60 mins). SLC45A4 KO mice show less nocifensive behaviours in Phase 1 compared to WT mice (Kruskal-Wallis test, * P = 0.02). Phase 2 is normal in all groups. All data mean ± s.e.m. f-m: WT n = 15 mice, HET n = 14 mice, KO n = 7 mice. F-i and m: one way ANOVA, with Tukey post-hoc test. Scale bars 100 μm.

Figure 4.. SLC45A4 regulates the excitability of peptidergic nociceptors.

a, Examples of sensory neuron subpopulation markers in WT and KO mice. b, Quantification of each subpopulation marker as a percentage of total DRG neurons, highlights no developmental difference in DRG subpopulations between WT and KO mice. WT n = 4 mice (no. cells: 1073 CGRP, 747 IB4, 742 NF200, 167 TH), KO n = 3 mice (no. cells: 761 GCRP, 524 IB4, 402 NF200, 151 TH, two-way ANOVA with post-hoc Holm-Sidak test, P > 0.05.) Scale bars 100 μm. c, Example images of intra-epidermal nerve fibre density (IENFD) in WT and KO mice, scale bars 20 μm. d, Quantification of IENFD highlights no loss of nociceptive nerve terminals in glabrous skin of KO mice, compared to WT (WT n = 3 mice, 303 fibres, 9 sections, KO n = 3 mice, 312 fibres, 9 sections, unpaired t-test, P > 0.05). e, Example images of an IB4-positive and IB4-negative small sensory neuron, distinguished by the presence or absence of IB4–488 live binding, scale 10 μm. Bottom: example traces of action potentials elicited by a threshold current injection, from IB4-postive and IB4-negative neurons from WT and KO mice. f, Threshold excitability (rheobase – minimum current required to elicit and action potential) is similar between WT and KO small sensory neurons that are IB4-postive or IB4-negative (IB4-pos: WT n = 9 cells, KO n = 10 cells, IB4-neg: WT n = 11 cells, KO n = 13 cells, Kruskal-Wallis test with Dunn’s multiple comparisons test, P > 0.05). g, IB4-postive nociceptors from KO mice show normal repetitive firing patterns to step-current injections, compared to WT. (RM two-way ANOVA, post hoc Holm-Sidak’s test, P > 0.05). h, IB4-postive nociceptors from WT and KO mice are comparable when challenged with ramp-current injections (WT n = 7 cells, KO n = 10 cells, RM two-way ANOVA, post hoc Holm-Sidak’s test, P > 0.05). i, IB4-negative nociceptors (peptidergic nociceptors) from KO mice are hypo-excitable to suprathreshold step-current injections, compared to WT neurons (RM two-way ANOVA, post hoc Holm-Sidak’s test, 100 pA * P = 0.039, 150 pA ** P = 0.0027, 200 pA *** P = 0.0002, 250 pA *** P = 0.0006, 300 pA * P = 0.047). j, Peptidergic nociceptors from KO mice are hypo-excitable to ramp-current injections, compared to WT neurons (RM two-way ANOVA, post hoc Holm-Sidak’s test, 300–1000ms * P = 0.036, 0.021, 0.011, 0.013, 0.021, 0.015, 0.021, 0.036, respectively).