Deletion of annexin 2 light chain p11 in nociceptors causes deficits in somatosensory coding and pain behavior

Abstract

The S100 family protein p11 (S100A10, annexin 2 light chain) is involved in the trafficking of the voltage-gated sodium channel Na(V)1.8, TWIK-related acid-sensitive K+ channel (TASK-1), the ligand-gated ion channels acid-sensing ion channel 1a (ASIC1a) and transient receptor potential vanilloid 5/6 (TRPV5/V6), as well as 5-hydroxytryptamine receptor 1B (5-HT1B), a G-protein-coupled receptor. To evaluate the role of p11 in peripheral pain pathways, we generated a loxP-flanked (floxed) p11 mouse and used the Cre-loxP recombinase system to delete p11 exclusively from nociceptive primary sensory neurons in mice. p11-null neurons showed deficits in the expression of Na(V)1.8, but not of annexin 2. Damage-sensing primary neurons from these animals show a reduced tetrodotoxin-resistant sodium current density, consistent with a loss of membrane-associated Na(V)1.8. Noxious coding in wide-dynamic-range neurons in the dorsal horn was markedly compromised. Acute pain behavior was attenuated in certain models, but no deficits in inflammatory pain were observed. A significant deficit in neuropathic pain behavior was also apparent in the conditional-null mice. These results confirm an important role for p11 in nociceptor function.

Figure 1.

Generation and analysis of p11 floxed mice. a, Structure of the targeting construct, wild-type locus, and recombination event. loxP sites are represented by white arrows. X, The sites of homologous recombination; B, BamHI restriction sites; Neo, a neomycin resistance cassette; TK, the thymidine kinase-negative selection marker. Exons are represented by numbered black blocks. b, Southern blot with a BamHI fragment to confirm correct targeting of the p11 locus. Insertion of loxP sites introduces an additional BamHI restriction site and thus a smaller DNA fragment. DNA from wild-type, heterozygous, and homozygous floxed animals is shown. c, PCR showing deletion of p11 exon 2 in DNA from DRG, but not from spinal cord (SC) or brain. d, RT-PCR from DRG mRNA, demonstrating deletion of p11 exon 2 from p11 conditional-null (cKO), but not littermate control (WT), DRG. RNA was used as a negative control in the PCR. e, Western blot showing deletion of p11 in global-null animals (KO), but not in controls (WT). Protein (40 ng) was taken from total cell lysate. Anti-glyceraldehyde phosphate dehydrogenase was used as a loading control.

Figure 2.

Effects of p11 deletion on cell survival and membrane trafficking of p11-associated proteins. a, Acutely cultured DRG neurons from p11 conditional-null (cKO) and littermate control animals (Wild-type) stained with antibody to p11. Locations of unstained cells in the conditional-null are shown by white arrows, replicated on the adjacent bright-field image. b, Acutely cultured DRG neurons (as in a) stained with antibodies to p11 (green) and Na_V1.8 (red). Overlap of p11 and Na_V1.8 staining can be seen in cells from littermate control; p11 and Na_V1.8 expression appears mutually exclusive in cells from the cKO animals. c, DRG sections from floxed p11 littermate control (Wild-type) and p11 conditional-null mice (cKO) stained with anti-peripherin (green) and N200 (red). No difference in ratio was observed between littermate control (n = 11) and p11 conditional-null (n = 24) sections. Peripherin control (Peri), 67 ± 3%; p11 conditional-null, 64 ± 4%; N-200 control, 37 ± 3%; p11 conditional-null, 38 ± 4%. Error bars represent mean ± SEM. d, Acutely cultured DRG neurons (Wild-type, littermate control; cKO, p11 conditional-null) stained with anti-annexin 2. Membrane localization is evident but appears to be unaffected by p11 deletion. e, Western blots performed on crude DRG plasma membrane preparations from global p11-null (KO) and control (WT) animals, using anti-annexin 2 (e1). No differences were evident (WT 20, 40 μg; KO 20, 40 μg). e2, Anti-Na_V1.8, using crude plasma membrane preparations. A reduction in membrane levels was observed (WT 20, 40 μg; KO 20, 40 μg). e3, Anti-Na_V1.8, using total cell lysate. No differences were observed (WT and KO, 400 μg). e4, Anti-5-HT_1B. No differences in membrane levels were observed (WT and KO, 40 μg). e5, Anti-ASIC1. No differences in membrane levels were observed (WT and KO, 40 μg).

Figure 3.

Electrophysiology of p11-null mice. a, Whole-cell voltage clamp of DRG neurons. a1, TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) peak sodium current densities in floxed p11 (white; n = 24) and p11-null (black; n = 42) mice. A significant decrease in TTX-R sodium current density was observed in the p11-null mice. p11-null, 0.037 ± 0.0056 nA/pF and n = 42; floxed p11, 0.071 ± 0.016 nA/pF and n = 26 (p = 0.016; Mann–Whitney Rank Sum test). No significant difference was observed in TTX-S current density. p11-null, 0.14 ± 0.017 nA/pF and n = 42; floxed p11, 0.11 ± 0.024 nA/pF and n = 22 (p = 0.19). a2, Example TTX-R and TTX-S current traces from representative neurons. a3, Current–voltage relationship for TTX-R currents from p11-null (black boxes; n = 7) and littermate control (white boxes; n = 7) neurons. No difference was observed. b, p11 conditional-null mice (black boxes) display marked deficits in mechanically evoked dorsal horn neuronal activity compared with littermate controls (white boxes). WDR neurons recorded from p11-null mice (n = 11) show significantly reduced activity (p < 0.05) to punctate mechanical von Frey stimuli. Data are shown during stimulus application (b1) and for total evoked activity to the stimulus (b2) compared with littermate control mice (n = 11). c, The same neurons show significantly reduced response (p < 0.05) to thermal stimuli (water jet). Data are shown during stimulus application (c1) and for total evoked activity to the stimulus (c2). d, These neurons also show significantly reduced activity to noxious pinch stimuli (p < 0.05), but not to brush or noxious cold (1°C) when compared with controls for the duration of the stimuli (d1) and for total evoked activity to the stimulus (d2). Stimuli were applied for 10 s to the peripheral receptive field of the neuron on the hindpaw. Data are expressed as mean ± SEM; *p < 0.05.

Figure 4.

Acute pain behavior in p11 conditional-null and control (floxed p11) mice. a, Response to mechanical stimulation using von Frey hairs was not significantly different between littermate control floxed p11 (white; n = 5; 0.059 ± 0.008 g) and p11 conditional-null (black; n = 7; 0.067 ± 0.010 g) mice. b, p11 conditional-null mice (black; n = 13) showed partial analgesia to noxious mechanical pressure applied to the tail using the Randall–Selitto apparatus compared with littermate controls (white; n = 13) (p11 conditional-null, 0.0019 ± 0.00012 N and n = 13; floxed p11 littermate control, 0.0026 ± 0.00025 N and n = 13) (p = 0.013; Student's t test). c, Noxious thermal stimulation using Hargreaves' apparatus. No significant difference in latency of hindpaw withdrawal was observed between littermate control (white; n = 11; 7.41 ± 0.35 s) and p11 conditional-null (black; n = 12; 8.28 ± 0.50 s) mice. d, Response to noxious thermal stimulation using the hotplate apparatus was not significantly different between littermate control (white; n = 14) and p11 conditional-null (black; n = 15) mice. At 50°C, littermate control, 22.4 ± 1.9 s; conditional-null, 25.1 ± 2.4 s (p = 0.38; Student's t test). At 55°C, littermate control, 10.9 ± 0.6 s; conditional-null, 13.1 ± 1.3 s (p = 0.12; Student's t test). All results are shown as mean ± SEM; *p < 0.05.

Figure 5.

Inflammatory and neuropathic pain behavior in p11 conditional-null mice. a, Thermal hyperalgesia, tested using Hargreaves' apparatus, after intraplantar injection of 2% carrageenan (20 μl). Both floxed p11 littermate control (white; n = 6) and p11 conditional-null (black; n = 6) mice developed pronounced hyperalgesia, with no significant difference between genotypes. b, NGF-induced hyperalgesia. Both littermate control (white; n = 5) and p11 conditional-null (black; n = 7) mice developed profound thermal hyperalgesia. c1, Pain behavior after intraplantar injection of 20 μl of 5% formalin. Time spent licking/biting the injected hindpaw was recorded in 5 min sections. No significant reduction in time of pain behavior was seen at Phase 1 (0–10 min) or at Phase 2 (10–55 min) between littermate control (white; n = 7) and p11 conditional-null (black; n = 5) mice. Phase 1 littermate control, 87.4 ± 5.3 s; conditional-null, 100.6 ± 22.2 s. Phase 2 littermate control, 286.8 ± 33.9 s; conditional-null, 283.2 ± 43.2 s. c2, Time course of the formalin test. A characteristic biphasic response was observed. d, Mechanical allodynia, measured by using von Frey hairs, resulting from the Chung model of neuropathic pain. A significant reduction in mechanical allodynia (p = 0.031; two-way repeated measures ANOVA) was observed overall in the p11 conditional-null (black; n = 7) when compared with the littermate control (white; n = 5) mice. Data are expressed as mean ± SEM; *p < 0.05 (individual points).