Article Figures & Data
Figures
- Figure 1.
Genetic ablation of the pore-forming exon of the murine Kcnt1 gene in ES cells. A, Murine WT Kcnt1 locus with exon 11 encoding the pore (P, red box) and surrounding exons 5–19 (black boxes) including relevant restriction sites (top). In ES cells, a single loxP recombination site (triangle) and a floxed neo/tk-cassette were introduced into the 3′-intronic and 5′-intronic regions flanking exon 11 (P) by homologous recombination resulting in the Slack L3-allele. L2 ES cells with floxed pore exons were generated by Cre recombinase-mediated excision of the selection cassette. L2 ES cells were injected into C57BL/6 blastocysts to obtain the L2/+ mouse line as described in Materials and Methods. Germline excision of the pore exon (L1) was conducted in mice by crossing Slack L2/+ animals to the Cre deleter mouse strain. Selected probes, restriction sites, and fragment sizes for the identification of the correct L3, L2, and L1 alleles by Southern blot in ES cells are shown. B, Verification of correct homologous and Cre recombination in ES cells by Southern blot analyses using the different probes. C, Representative example of a genotyping PCR using genomic DNA from mouse tail-tip biopsies as templates. D, The absence of the pore-forming exon was further verified by reverse transcription PCR analyses on total RNA isolated from cerebral cortex of Slack−/− mice using a pore exon-specific primer pair.
- Figure 2.
Expression of Slack channels in DRG neurons. A, Immunofluorescence of Slack in lumbar DRGs of WT and Slack−/− mice revealed specific Slack expression in 31.6 ± 1.7% (2571 cells counted, n = 3 mice) of DRG neurons. B, Western blot analysis of Slack (140 kDa) in DRG homogenates of WT and Slack−/− mice confirms the specificity of the anti-Slack antibody. GAPDH (36 kDa) was used as loading control. C, D, Typical examples of Slack immunoreactivity in nociceptor subpopulations positive for IB4 and CGRP. E, Quantitative summary of DRG neuron populations expressing Slack. Most Slack-positive cells bind IB4 (86.5 ± 1.6%, 2108 cells counted, n = 3 mice) and are therefore nonpeptidergic, whereas a few Slack-positive cells colocalize with CGRP in peptidergic DRG neurons (12.6 ± 0.33%, 1114 cells counted, n = 3 mice). F, Size distribution of Slack-positive DRG neurons compared with those expressing IB4, CGRP, and neurofilament 200 (NF200), a marker of large myelinated DRG neurons. The data demonstrate that Slack channels are nearly exclusively expressed in nociceptive DRG neurons of small and medium diameter. Scale bars, 50 μm.
- Figure 3.
Expression of Slack channels in the spinal cord. A, Immunofluorescence of Slack in the lumbar spinal cord of WT and Slack−/− mice reveals specific Slack expression in the superficial dorsal horn. B, Western blot analysis of Slack (140 kDa) in spinal cord homogenates of WT and Slack−/− mice confirms the specificity of the anti-Slack antibody. GAPDH (36 kDa) was used as loading control. C, Colocalization of Slack with IB4 shows that Slack channels are present in central terminals of sensory neurons entering the superficial dorsal horn. Scale bars, 100 μm.
- Figure 4.
Slack generates KNa and regulates AP firing in sensory neurons. A, B, Representative current traces from whole-cell voltage-clamp recordings on IB4-positive WT (n = 17) and Slack−/− (n = 16) DRG neurons monitoring outward K+ currents in presence of 140 mm NaCl (black traces) and after replacement of NaCl with 140 mm choline chloride (red traces). Currents were elicited by protocols consisting of 1000-ms-long test pulses ranging from −80 to +110 mV in steps of 10 mV. Holding potential was −70 mV. All neurons were first recorded in presence of NaCl. To visualize the KNa current, the current traces obtained with 140 mm NaCl and 140 mm choline chloride were digitally subtracted and depicted as green traces. Please note that the absence of extracellular Na+ was verified by the lack of Na+ inward currents (red traces). C, Representative current traces from whole-cell current-clamp recordings on IB4-positive WT (n = 21) and Slack−/− (n = 22) DRG neurons. APs were elicited by 1000 ms current injections corresponding to 2–3 times the threshold of a single AP. Pictured are three observed patterns of AP firing during the current injection, i.e., no accommodation (left), accommodation (middle), and strong accommodation without repetitive firing (right). D, Percentage of DRG neurons from WT and Slack−/− mice generating outward K+ currents (KNa). E, Mean KNa current amplitudes in DRG neurons from WT (n = 8) and Slack−/− (n = 2) mice. F, Percentage of DRG neurons from WT and Slack−/− mice generating APs without accommodation, i.e., cells with repetitive firing of APs. *p < 0.05.
- Figure 5.
Basal sensitivity is normal in Slack−/− mice. A, B, Mechanical pain sensitivity was measured using the dynamic plantar (A; n = 15–17/genotype) and the Randall-Selitto tests (B; n = 8/genotype). C, D, Thermal heat pain sensitivity was assessed by tail-flick (C; n = 8/genotype) and hot plate tests (D; n = 14/genotype). E, Cold pain sensitivity was investigated on a 0°C cold plate (n = 14/genotype). No significant differences were observed between Slack−/− and WT mice in any of these tests.
- Figure 6.
Increased neuropathic pain behavior in Slack−/− mice. A, B, Paw-withdrawal latencies of Slack−/− and WT mice after mechanical stimulation in the SNI model (n = 8/genotype; A) and the paclitaxel model (2 mg/kg, i.p., for 5 consecutive days; n = 8–9/genotype; B) of neuropathic pain. Slack−/− mice demonstrated increased mechanical hypersensitivity compared with WT littermates. *p < 0.05, WT versus Slack−/−, repeated-measures ANOVA with Bonferroni's post hoc test.
- Figure 7.
Increased neuropathic pain behavior after conditional deletion of Slack channels in sensory neurons. A, Double-labeling immunostaining revealed that virtually all Slack-positive DRG neurons are also positive for NaV1.8. B, Slack immunofluorescence was not detectable in DRGs of SNS-Slack−/− mice, in which the Cre/loxP-mediated recombination of the Slack encoding gene Kcnt1 occurred in NaV1.8-positive sensory neurons. C, Western blot analysis demonstrated that Slack expression was considerably reduced in the spinal cord but normal in the brain of SNS-Slack−/− mice. GAPDH was used as loading control. D, Paw-withdrawal latencies of SNS-Slack−/− and SNS-Ctrl mice after mechanical stimulation in the SNI model of neuropathic pain (n = 8/genotype). SNS-Slack−/− mice demonstrated increased mechanical hypersensitivity compared with SNS-Ctrl mice. *p < 0.05, WT versus Slack−/−, repeated-measures ANOVA with Bonferroni's post hoc test. Scale bars, 50 μm.
- Figure 8.
Intrathecal (i.t.) NAD+ ameliorates neuropathic pain in a Slack-dependent manner. Animals were subjected to SNI to induce neuropathic pain, and NAD+ was intrathecally administered 14 d thereafter. Time courses of paw-withdrawal latencies after mechanical stimulation (n = 7–8/genotype). *p < 0.05, before NAD+ injection (“after SNI”) versus after NAD+ injection in WT mice, repeated-measures ANOVA with Dunnett's post hoc test.
- Figure 9.
Loxapine ameliorates neuropathic pain in a Slack-dependent manner. A–C, Neuropathic pain was induced in WT and Slack−/− mice by SNI surgery. Fourteen days thereafter, animals were intraperitoneally injected with loxapine (A; n = 12–13/genotype), olanzapine (B; n = 7–8/genotype), or pregabalin (C; n = 7/genotype), and the mechanical paw-withdrawal latencies were assessed over 6 h. Note that loxapine inhibited the neuropathic pain behavior in WT but not in Slack−/− mice, whereas olanzapine did not affect the pain behavior in both genotypes. Pregabalin was used as a positive control. *#p < 0.05, before drug injection (“after SNI”) versus after drug injection in WT and Slack−/− mice, respectively; repeated-measures ANOVA with Bonferroni's post hoc test.
- Figure 10.
Inflammatory pain behavior is normal in Slack−/− mice. A, B, Formalin test. A, The time course of paw licking induced by 0.5% formalin injection was similar between Slack−/− and WT mice. B, Statistical analyses revealed no significant differences in the first (1–10 min) and the second (11–45 min) phase of paw licking (n = 6/genotype). C, Zymosan model. Paw-withdrawal latencies after mechanical stimulation after injection of zymosan into a hindpaw did not differ between genotypes (n = 7–8/genotype). D, The zymosan-induced mechanical hypersensitivity in WT mice was not affected by intraperitoneal administration of the Slack channel opener loxapine (0.175 mg/kg) 3 h after zymosan injection (n = 8/genotype).
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