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Slack Potassium Channels Modulate TRPA1-Mediated Nociception in Sensory Neurons - PubMed

  • ️Sat Jan 01 2022

Slack Potassium Channels Modulate TRPA1-Mediated Nociception in Sensory Neurons

Fangyuan Zhou et al. Cells. 2022.

Abstract

The transient receptor potential (TRP) ankyrin type 1 (TRPA1) channel is highly expressed in a subset of sensory neurons where it acts as an essential detector of painful stimuli. However, the mechanisms that control the activity of sensory neurons upon TRPA1 activation remain poorly understood. Here, using in situ hybridization and immunostaining, we found TRPA1 to be extensively co-localized with the potassium channel Slack (KNa1.1, Slo2.2, or Kcnt1) in sensory neurons. Mice lacking Slack globally (Slack-/-) or conditionally in sensory neurons (SNS-Slack-/-) demonstrated increased pain behavior after intraplantar injection of the TRPA1 activator allyl isothiocyanate. By contrast, pain behavior induced by the TRP vanilloid 1 (TRPV1) activator capsaicin was normal in Slack-deficient mice. Patch-clamp recordings in sensory neurons and in a HEK cell line transfected with TRPA1 and Slack revealed that Slack-dependent potassium currents (IKS) are modulated in a TRPA1-dependent manner. Taken together, our findings highlight Slack as a modulator of TRPA1-mediated, but not TRPV1-mediated, activation of sensory neurons.

Keywords: TRPA1; dorsal root ganglia; mice; pain; slack.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

Slack−/− mice display increased allyl isothiocyanate (AITC)-evoked but normal capsaicin-evoked pain behavior. (A) Time course of paw licking and biting (left) and the sum of licking and biting time over 30 min (right; p = 0.0394; n = 7 mice per group) after intraplantar AITC injection in wildtype (WT) and Slack−/− littermates. (B) Time course of mechanical hypersensitivity after intraplantar AITC injection. Two-way analysis of variance (ANOVA), effect of genotype (p = 0.0007) with Sidak’s multiple comparison test (p values represent comparisons between genotypes for each time point: 3 h, p = 0.0421; 24 h, p = 0.0076; 48 h, p = 0.0064); n = 8 mice per group. Note that both licking/biting and mechanical hypersensitivity are significantly increased in Slack−/− mice after AITC injection. (C) Quantitative RT-PCR in DRGs of WT and Slack−/− mice revealed that the transient receptor potential (TRP) ankyrin 1 (TRPA1) mRNA expression is not compensatorily regulated in the absence of Slack (p = 0.3727; n = 3 mice per group). (D) Time course of paw licking and biting (left) and the sum of licking and biting time over 10 min (right; p = 0.9621; n = 7–8 mice per group) after intraplantar capsaicin injection. (E) Time course of mechanical hypersensitivity after intraplantar capsaicin injection. Two-way ANOVA, effect of genotype (p = 0.5893; n = 7–8 mice per group). Note that the capsaicin-induced pain behavior was unaltered in Slack−/− mice. (F) Quantitative RT-PCR in DRGs of WT and Slack−/− mice revealed that TRP vanilloid 1 (TRPV1) mRNA expression is not compensatory regulated in the absence of Slack (p = 0.4874; n = 3 mice per group) (G) Double in situ hybridization of Slack mRNA and TRPA1 mRNA in DRGs. Scale bar, 50 µm. (H) Double-labeling immunostaining of Slack and TRPV1 in DRGs. A quantitative summary of co-expression in G (180 Slack-positive cells from 3 mice were counted) and H (204 Slack-positive cells from 4 mice were counted) is shown on the right. (I) Expression of TRPA1, TRPV1, and Slack (gene Kcnt1) across sensory neuron subsets from published scRNA-seq data. Data are presented as mean ± SEM. * p < 0.05.

Figure 2
Figure 2

SNS-Slack−/− mice display increased AITC-evoked pain behavior. (A) Quantitative RT-PCR in lumbar DRGs, lumbar spinal cord, cerebellum and prefrontal cortex revealed that Slack mRNA is selectively reduced in DRGs of SNS-Slack−/− mice (p = 0.0002; n = 3 mice per group). (B,C) Expression pattern and percentages of DRG neurons binding IB4 (2207 cells from 4 mice per group were counted), or immunoreactive for CGRP (2110 cells from 4 mice per group were counted), TH (839 cells from 4 mice per group were counted), NF200 (2751 cells from 4 mice per group were counted), are similar in SNS-Slack−/− and control mice. Scale bar, 50 µm. (D) The distribution of central terminals of primary afferents immunoreactive for CGRP or binding IB4 in the dorsal horn appears normal in SNS-Slack−/− mice. Scale bar, 50 µm. (E) Quantitative RT-PCR revealed that TRPA1 mRNA expression in lumbar DRGs is similar in control and SNS-Slack−/− mice (p = 0.8638; n = 3 mice per group). (F) Time course of paw licking and biting (left; p = 0.0148 for the 0–5 min period) and the sum of licking and biting time over 30 min (right; p = 0.0238; n = 8 mice per group) after intraplantar injection of AITC in control and SNS-Slack−/− littermates. (G) Time course of mechanical hypersensitivity after intraplantar AITC injection. Two-way ANOVA, effect of genotype (p = 0.0126) with Sidak’s multiple comparisons test (p = 0.0346, representing comparisons between genotypes for the 24 h time point); n = 8 mice per group. Data are presented as mean ± SEM. * p < 0.05.

Figure 3
Figure 3

Lbx1-Slack−/− mice display normal AITC-evoked pain behavior. (A) Quantitative RT-PCR in lumbar DRGs, lumbar spinal cord, cerebellum, and prefrontal cortex revealed that Slack mRNA levels are selectively reduced in the spinal cord of Lbx1-Slack−/− mice (p < 0.0001; n = 6 mice per group). (B) The distribution of GAD67+ inhibitory interneurons and PKCγ+ excitatory interneurons in the dorsal horn appears normal in Lbx1-Slack−/− mice. Scale bar, 50 µm. (C) Time course of paw licking and biting (left) and the sum of licking and biting time over 30 min (right; p = 0.7319; n = 6 mice per group) after intraplantar AITC injection in Lbx1-Slack−/− and control littermates. Data are presented as mean ± SEM. * p < 0.05.

Figure 4
Figure 4

AITC-mediated calcium influx is normal in sensory neurons of Slack−/− mice. (A) Representative examples of Fura-2-ratiometric calcium traces evoked by AITC and KCl in cultured lumbar DRG neurons of WT and Slack−/− mice. (B) Magnitude of the calcium response to AITC stimulation (WT, n = 452 neurons in 3 mice; Slack−/−, n = 476 neurons in 3 mice; p = 0.7000). (C) Percentage of responsive neurons to AITC stimulation (p = 0.9739). These data show that AITC-evoked calcium responses are normal in DRG neurons from Slack−/− mice. Data in B and C are presented as mean ± SEM.

Figure 5
Figure 5

AITC-mediated modulation of potassium currents in IB4+ sensory neurons from WT and Slack−/− mice. (A) IV relations of outward potassium currents (IK) obtained in whole-cell patch-clamp recordings in IB4+ sensory neurons from 4 WT (n = 18 cells) and 4 Slack−/− mice (n = 13 cells) before and after AITC (200 µM) application in the physiological extracellular buffer. Note that in this experimental setting (which includes 2 mM Ca2+ and 140 mM Na+ in the external solution), TRPA1 activation led to a significant reduction in IK in sensory neurons from WT but not Slack−/− mice. (B) IV relations of IK in sensory neurons from 3 WT (n = 13 cells) and 3 Slack−/− mice (n = 12 cells) before and after application of the TRPA1 antagonist A-967079 (10 µM) in the physiological extracellular buffer. The TRPA1 antagonist significantly reduced IK in sensory neurons from both WT and Slack−/− mice. (C) IV relations of IK in sensory neurons from 3 WT (n = 9 cells) and 3 Slack−/− mice (n = 8 cells) before and after AITC (200 µM) application in a Na+ free extracellular buffer. In this experimental setting (which includes 2 mM Ca2+ but no Na+ in the external solution), TRPA1 activation did not alter IK in sensory neurons from both WT and Slack−/− mice. Data are presented as mean ± SEM. * p < 0.05.

Figure 6
Figure 6

AITC-mediated modulation of potassium currents in transfected HEK293 cells. (A,B) IV relations of IK in HEK-Slack cells ((A); n = 5 cells) and HEK-Slack-TRPA1 cells ((B); n = 14 cells) obtained in whole-cell patch-clamp recordings using a Ca2+-free external solution before and after TRPA1 activation by 200 µM AITC. (C) IV relations of IK in HEK-Slack-TRPA1 cells before and after TRPA1 inhibition by 10 µM A-967079 (n = 9 cells) in whole-cell patch-clamp recordings using a Ca2+-free external solution. (D) IV relations of IK in HEK-Slack-TRPA1 cells before and after 200 µM AITC application (n = 9 cells) in whole-cell patch-clamp recordings in physiological extracellular buffer Note that in a Ca2+-free external solution, Slack-mediated IK is increased after TRPA1 activation. Data are presented as mean ± SEM. Paired t test, * p < 0.05.

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This research was funded by the Deutsche Forschungsgemeinschaft (LU 2514/1-1 to R.Lu.) and the Else Kröner-Fresenius-Stiftung (2018_A95 to A.S.)

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