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Inflammatory pain is thought to be mediated in part through the action of inflammatory mediators on membrane receptors of peripheral nerve terminals, however, the downstream signaling events which lead to pain are poorly understood. In this study we investigated the nociceptive pathways induced by activation of protease-activated receptor 2 (PAR-2)...
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... research has established that the majority of neurons in rat DRG (both small and large) express robust M-type potassium currents conducted by Kv7.2, Kv7.3 and Kv7.5 subunits ( Passmore et al., 2003), and some 90% of small diameter nociceptive DRG neurons express PAR-2 receptors ( Amadesi et al., 2004;Dai et al., 2004). We therefore reasoned there must be co-expression of PAR-2 and M channels in the majority of small nociceptive neurons. Our patch clamp experiments confirmed this assumption. Whole cell currents were measured from small diameter (mean whole cell capacitance of 20.8 ? 1.1 pF, n = 44), predominantly TRPV1 positive (73% or 51/70 of such cells responded to 1 ?M capsaicin) DRG neurons. M current was measured using a standard voltage protocol (Fig 1A inset). Application of the hyperpolarizing test pulse resulted in a slowly deactivating whole cell current, characteristic of M current (see supplementary Table 1 for time constants), which was partially inhibited by the specific M channel blocker XE991 (3-10 ?M; Fig. 1C; 57 ? 4% inhibition of deactivation current, n = 7). In subsequent voltage clamp experiments we termed I M the XE991-sensitive component of the slowly deactivating current produced by stepping the membrane voltage from -30 to -60 mV. Addition of a specific peptide agonist of PAR-2 (2f-LIGRLO-amide, 10 ?M; PAR2-AP) to the bath solution resulted in a dramatic and sustained reduction in the deactivation current from 4.3 ? 0.4 to 2.5 ? 0.3 pA/pF (24/27 neurons, p<0.0001; Fig. 1B, C) which amounted to 80.5 ? 10.6% of I M . When applied after XE991, PAR2-AP had no further effect on the deactivation current (deactivation current in the presence of XE991 alone was 1.52 ? 0.32 pA/pF; and after subsequent co-application of XE991 + PAR2-AP became 1.38 ? 0.30 pA/pF; n = 7, paired t-test, Fig. 1C). These data indicate that the fraction of the deactivation current inhibited by PAR2-AP is a classical M current. Inhibition of M current by PAR-2AP or XE991 was accompanied by a substantial depolarization of the membrane potential by 8.2 ? 1.9 mV (from -65.6 ? 1.6 mV to -57.4 ? 1.7 mV; n = 20, p < 0.001) and 12.8 ? 2.1 mV (from -61.5 ? 3.2 mV to from -48.8 ? 2.7 mV; n = 8, p < 0.001) respectively (Fig. 1D). In current clamp experiments we found that I M inhibition by PAR2-AP lowered the threshold for action potential firing in 5/12 tested neurons (data not shown). The effect on excitability of DRG neurons in culture was most likely underestimated due to a slight depolarization causing partial inactivation of voltage- gated Na + channels ( Zimmermann et al., 2007). As we shall see later, behavioural experiments support a strong excitatory effect of M channel ...
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... research has established that the majority of neurons in rat DRG (both small and large) express robust M-type potassium currents conducted by Kv7.2, Kv7.3 and Kv7.5 subunits ( Passmore et al., 2003), and some 90% of small diameter nociceptive DRG neurons express PAR-2 receptors ( Amadesi et al., 2004;Dai et al., 2004). We therefore reasoned there must be co-expression of PAR-2 and M channels in the majority of small nociceptive neurons. Our patch clamp experiments confirmed this assumption. Whole cell currents were measured from small diameter (mean whole cell capacitance of 20.8 ? 1.1 pF, n = 44), predominantly TRPV1 positive (73% or 51/70 of such cells responded to 1 ?M capsaicin) DRG neurons. M current was measured using a standard voltage protocol (Fig 1A inset). Application of the hyperpolarizing test pulse resulted in a slowly deactivating whole cell current, characteristic of M current (see supplementary Table 1 for time constants), which was partially inhibited by the specific M channel blocker XE991 (3-10 ?M; Fig. 1C; 57 ? 4% inhibition of deactivation current, n = 7). In subsequent voltage clamp experiments we termed I M the XE991-sensitive component of the slowly deactivating current produced by stepping the membrane voltage from -30 to -60 mV. Addition of a specific peptide agonist of PAR-2 (2f-LIGRLO-amide, 10 ?M; PAR2-AP) to the bath solution resulted in a dramatic and sustained reduction in the deactivation current from 4.3 ? 0.4 to 2.5 ? 0.3 pA/pF (24/27 neurons, p<0.0001; Fig. 1B, C) which amounted to 80.5 ? 10.6% of I M . When applied after XE991, PAR2-AP had no further effect on the deactivation current (deactivation current in the presence of XE991 alone was 1.52 ? 0.32 pA/pF; and after subsequent co-application of XE991 + PAR2-AP became 1.38 ? 0.30 pA/pF; n = 7, paired t-test, Fig. 1C). These data indicate that the fraction of the deactivation current inhibited by PAR2-AP is a classical M current. Inhibition of M current by PAR-2AP or XE991 was accompanied by a substantial depolarization of the membrane potential by 8.2 ? 1.9 mV (from -65.6 ? 1.6 mV to -57.4 ? 1.7 mV; n = 20, p < 0.001) and 12.8 ? 2.1 mV (from -61.5 ? 3.2 mV to from -48.8 ? 2.7 mV; n = 8, p < 0.001) respectively (Fig. 1D). In current clamp experiments we found that I M inhibition by PAR2-AP lowered the threshold for action potential firing in 5/12 tested neurons (data not shown). The effect on excitability of DRG neurons in culture was most likely underestimated due to a slight depolarization causing partial inactivation of voltage- gated Na + channels ( Zimmermann et al., 2007). As we shall see later, behavioural experiments support a strong excitatory effect of M channel ...
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... research has established that the majority of neurons in rat DRG (both small and large) express robust M-type potassium currents conducted by Kv7.2, Kv7.3 and Kv7.5 subunits ( Passmore et al., 2003), and some 90% of small diameter nociceptive DRG neurons express PAR-2 receptors ( Amadesi et al., 2004;Dai et al., 2004). We therefore reasoned there must be co-expression of PAR-2 and M channels in the majority of small nociceptive neurons. Our patch clamp experiments confirmed this assumption. Whole cell currents were measured from small diameter (mean whole cell capacitance of 20.8 ? 1.1 pF, n = 44), predominantly TRPV1 positive (73% or 51/70 of such cells responded to 1 ?M capsaicin) DRG neurons. M current was measured using a standard voltage protocol (Fig 1A inset). Application of the hyperpolarizing test pulse resulted in a slowly deactivating whole cell current, characteristic of M current (see supplementary Table 1 for time constants), which was partially inhibited by the specific M channel blocker XE991 (3-10 ?M; Fig. 1C; 57 ? 4% inhibition of deactivation current, n = 7). In subsequent voltage clamp experiments we termed I M the XE991-sensitive component of the slowly deactivating current produced by stepping the membrane voltage from -30 to -60 mV. Addition of a specific peptide agonist of PAR-2 (2f-LIGRLO-amide, 10 ?M; PAR2-AP) to the bath solution resulted in a dramatic and sustained reduction in the deactivation current from 4.3 ? 0.4 to 2.5 ? 0.3 pA/pF (24/27 neurons, p<0.0001; Fig. 1B, C) which amounted to 80.5 ? 10.6% of I M . When applied after XE991, PAR2-AP had no further effect on the deactivation current (deactivation current in the presence of XE991 alone was 1.52 ? 0.32 pA/pF; and after subsequent co-application of XE991 + PAR2-AP became 1.38 ? 0.30 pA/pF; n = 7, paired t-test, Fig. 1C). These data indicate that the fraction of the deactivation current inhibited by PAR2-AP is a classical M current. Inhibition of M current by PAR-2AP or XE991 was accompanied by a substantial depolarization of the membrane potential by 8.2 ? 1.9 mV (from -65.6 ? 1.6 mV to -57.4 ? 1.7 mV; n = 20, p < 0.001) and 12.8 ? 2.1 mV (from -61.5 ? 3.2 mV to from -48.8 ? 2.7 mV; n = 8, p < 0.001) respectively (Fig. 1D). In current clamp experiments we found that I M inhibition by PAR2-AP lowered the threshold for action potential firing in 5/12 tested neurons (data not shown). The effect on excitability of DRG neurons in culture was most likely underestimated due to a slight depolarization causing partial inactivation of voltage- gated Na + channels ( Zimmermann et al., 2007). As we shall see later, behavioural experiments support a strong excitatory effect of M channel ...
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... research has established that the majority of neurons in rat DRG (both small and large) express robust M-type potassium currents conducted by Kv7.2, Kv7.3 and Kv7.5 subunits ( Passmore et al., 2003), and some 90% of small diameter nociceptive DRG neurons express PAR-2 receptors ( Amadesi et al., 2004;Dai et al., 2004). We therefore reasoned there must be co-expression of PAR-2 and M channels in the majority of small nociceptive neurons. Our patch clamp experiments confirmed this assumption. Whole cell currents were measured from small diameter (mean whole cell capacitance of 20.8 ? 1.1 pF, n = 44), predominantly TRPV1 positive (73% or 51/70 of such cells responded to 1 ?M capsaicin) DRG neurons. M current was measured using a standard voltage protocol (Fig 1A inset). Application of the hyperpolarizing test pulse resulted in a slowly deactivating whole cell current, characteristic of M current (see supplementary Table 1 for time constants), which was partially inhibited by the specific M channel blocker XE991 (3-10 ?M; Fig. 1C; 57 ? 4% inhibition of deactivation current, n = 7). In subsequent voltage clamp experiments we termed I M the XE991-sensitive component of the slowly deactivating current produced by stepping the membrane voltage from -30 to -60 mV. Addition of a specific peptide agonist of PAR-2 (2f-LIGRLO-amide, 10 ?M; PAR2-AP) to the bath solution resulted in a dramatic and sustained reduction in the deactivation current from 4.3 ? 0.4 to 2.5 ? 0.3 pA/pF (24/27 neurons, p<0.0001; Fig. 1B, C) which amounted to 80.5 ? 10.6% of I M . When applied after XE991, PAR2-AP had no further effect on the deactivation current (deactivation current in the presence of XE991 alone was 1.52 ? 0.32 pA/pF; and after subsequent co-application of XE991 + PAR2-AP became 1.38 ? 0.30 pA/pF; n = 7, paired t-test, Fig. 1C). These data indicate that the fraction of the deactivation current inhibited by PAR2-AP is a classical M current. Inhibition of M current by PAR-2AP or XE991 was accompanied by a substantial depolarization of the membrane potential by 8.2 ? 1.9 mV (from -65.6 ? 1.6 mV to -57.4 ? 1.7 mV; n = 20, p < 0.001) and 12.8 ? 2.1 mV (from -61.5 ? 3.2 mV to from -48.8 ? 2.7 mV; n = 8, p < 0.001) respectively (Fig. 1D). In current clamp experiments we found that I M inhibition by PAR2-AP lowered the threshold for action potential firing in 5/12 tested neurons (data not shown). The effect on excitability of DRG neurons in culture was most likely underestimated due to a slight depolarization causing partial inactivation of voltage- gated Na + channels ( Zimmermann et al., 2007). As we shall see later, behavioural experiments support a strong excitatory effect of M channel ...
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... research has established that the majority of neurons in rat DRG (both small and large) express robust M-type potassium currents conducted by Kv7.2, Kv7.3 and Kv7.5 subunits ( Passmore et al., 2003), and some 90% of small diameter nociceptive DRG neurons express PAR-2 receptors ( Amadesi et al., 2004;Dai et al., 2004). We therefore reasoned there must be co-expression of PAR-2 and M channels in the majority of small nociceptive neurons. Our patch clamp experiments confirmed this assumption. Whole cell currents were measured from small diameter (mean whole cell capacitance of 20.8 ? 1.1 pF, n = 44), predominantly TRPV1 positive (73% or 51/70 of such cells responded to 1 ?M capsaicin) DRG neurons. M current was measured using a standard voltage protocol (Fig 1A inset). Application of the hyperpolarizing test pulse resulted in a slowly deactivating whole cell current, characteristic of M current (see supplementary Table 1 for time constants), which was partially inhibited by the specific M channel blocker XE991 (3-10 ?M; Fig. 1C; 57 ? 4% inhibition of deactivation current, n = 7). In subsequent voltage clamp experiments we termed I M the XE991-sensitive component of the slowly deactivating current produced by stepping the membrane voltage from -30 to -60 mV. Addition of a specific peptide agonist of PAR-2 (2f-LIGRLO-amide, 10 ?M; PAR2-AP) to the bath solution resulted in a dramatic and sustained reduction in the deactivation current from 4.3 ? 0.4 to 2.5 ? 0.3 pA/pF (24/27 neurons, p<0.0001; Fig. 1B, C) which amounted to 80.5 ? 10.6% of I M . When applied after XE991, PAR2-AP had no further effect on the deactivation current (deactivation current in the presence of XE991 alone was 1.52 ? 0.32 pA/pF; and after subsequent co-application of XE991 + PAR2-AP became 1.38 ? 0.30 pA/pF; n = 7, paired t-test, Fig. 1C). These data indicate that the fraction of the deactivation current inhibited by PAR2-AP is a classical M current. Inhibition of M current by PAR-2AP or XE991 was accompanied by a substantial depolarization of the membrane potential by 8.2 ? 1.9 mV (from -65.6 ? 1.6 mV to -57.4 ? 1.7 mV; n = 20, p < 0.001) and 12.8 ? 2.1 mV (from -61.5 ? 3.2 mV to from -48.8 ? 2.7 mV; n = 8, p < 0.001) respectively (Fig. 1D). In current clamp experiments we found that I M inhibition by PAR2-AP lowered the threshold for action potential firing in 5/12 tested neurons (data not shown). The effect on excitability of DRG neurons in culture was most likely underestimated due to a slight depolarization causing partial inactivation of voltage- gated Na + channels ( Zimmermann et al., 2007). As we shall see later, behavioural experiments support a strong excitatory effect of M channel ...
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... order to verify that the observed effects of PAR-2 on M current were not an artefact of culture conditions we developed a method of perforated patch recording from acute DRG 'slices' (See Supplementary Methods). These recordings ( Supplementary Fig. 1) were consistent with the experiments on cultured neurons validating both the expression of M current in native DRG neurons and its functional coupling to PAR-2 ...
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... to Web version on PubMed Central for supplementary material. M current was recorded in small diameter DRG neurons using the perforated patch technique. (A) Exemplar experiment showing whole-cell current elicited by the voltage protocol depicted in the inset above (values in mV). Drugs were added to the bath solution sequentially, protease activated receptor-2 activating peptide (PAR2-AP) (10 ?M), XE991 (XE, 10 ?M). Zero current is indicated by the dotted line. (B) Representative time course of M current inhibition by PAR2-AP. Plotted is the deactivation current measured as the difference between the current 10 ms into the voltage pulse from -30 mV to -60 mV and the steady state current at -60 mV and normalised to cell capacitance (I deac density). Drugs were applied during the periods indicated by the bars. (C & D) Summary of the effects of PAR2- AP and XE991 on M current (C) and resting membrane potential (V m ) (D). Bars are mean ? SEM; number of experiments given above bars in parentheses. Significant difference from basal (*) and from PAR2-AP ( ?); NS -no significant difference. (A) Reconstitution of PAR-2 inhibition of M current in CHO cells. Cells were transfected with hKv7.2, hKv7.3 and hPAR-2 cDNA and current was recorded in perforated patch mode. Deactivation current when stepping the membrane voltage from 0 mV to -60 mV is normalized to cell capacitance. Drugs (PAR2-AP, 10 ?M; XE991, 3 ?M) were applied during the periods indicated by the bars. Inset shows current traces taken at the time points indicated by coloured triangles. (B) Recovery of M current in DRG neurons. Recording conditions as in Figure 1A-B. (C) recording similar to that shown in B but trypsin (20 nM) is applied instead of PAR2-AP. (D) Summary of recovery of Kv7.2/7.3 current (CHO) or native M current (DRG) after inhibition by PAR2-AP or trypsin. Data represented as % recovery of the XE991 sensitive deactivation current fraction (I M ). (A) DRG neurons were transfected with the optical probe for PIP 2 /IP 3 (PLC?-PH-GFP) and imaged using a swept field confocal microscope. Time course of changes in cytosolic fluorescence intensity (F) normalised to basal fluourescence levels (F 0 ) (mean ? SEM, n = 7). Only cells which displayed translocation of the probe were included (7/12). PAR2-AP was added to the bath solution as indicated by the black bar. (B) An exemplar cell imaged as in A. Images before application (basal), during application (PAR-2) and after the wash-out of PAR2-AP (wash) are shown on the left. White arrow indicates line used for intensity profiles shown on the right. Note the two fluorescence intensity peaks under basal conditions which correspond to the plasma membrane and are lost upon PAR-2 activation. (C, D) Calcium imaging from individual DRG neurons loaded with Fluo4-AM. Lower bars indicate [Ca 2+ ] o (mM). Drugs (PAR2-AP, 10 ?M; capsaicin, 1 ?M) were applied during the periods indicated by the bars. (A) Buffering of the cytosolic Ca 2+ and membrane PIP 2 through the whole-cell pipette. Representative recordings from DRG neurons temporally aligned for the onset of the PAR2- AP application. Data are normalised to the basal level of I M (I 0 ). PAR2-AP was added to the bath solution 60s after establishing a stable current level as indicated by the black bar. Pipette solutions contain: 0.2 mM BAPTA, no added Ca 2+ (control, black circles); 0.2 mM BAPTA, 100 ?M diC8-PIP 2 (PIP 2 , red circles); 10 mM BAPTA, free [Ca 2+ ] i buffered to 75 nM with CaCl 2 (BAPTA, blue circles); 10 mM BAPTA, 100 ?M diC8-PIP 2 (BAPTA+PIP 2 , green circles). Recordings were performed in whole-cell mode. Individual traces were fit with a generic sigmoid function for better comparison. (B) Mean data of the experiments shown in A. Number of experiments is given within bars. (*) difference from control, ( ?) difference from PIP 2 . (C) Perforated patch experiments similar to those in A. Black circles depict control experiments similar to that of Fig. 1B; red circles -PLC inhibitor edelfosine (10 ?M) added 10 minutes before commencing experiment and remained throughout; blue circles -as before but specific IP 3 receptor blocker Xestosspongin C (1 ?M) is used instead of edelfosine. (D) Mean data of the experiments shown in C. (*) difference from control, ( ?) difference from edelfosine. (A) XE991 induces acute pain in a concentration-dependent manner. 50 ?l solutions of different concentrations (as indicated below the bars) were injected into the hind paw of adult rats. Nocifensive behaviour was quantified by observing time spent grooming and flinching during 45 min, n = 6 for each experiment. (*) difference with the vehicle control; ( ?) difference from XE991 2 ?M; ( ?) difference from XE991 200 ?M. (B) Pre-application of XE991 does not significantly increase nocifensive behaviour induced by PA2-AP. Drugs were injected as following: Vh1 -vehicle (50 ?l) control; PAR2-AP -PAR2-AP, 50 ...
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... to Web version on PubMed Central for supplementary material. M current was recorded in small diameter DRG neurons using the perforated patch technique. (A) Exemplar experiment showing whole-cell current elicited by the voltage protocol depicted in the inset above (values in mV). Drugs were added to the bath solution sequentially, protease activated receptor-2 activating peptide (PAR2-AP) (10 ?M), XE991 (XE, 10 ?M). Zero current is indicated by the dotted line. (B) Representative time course of M current inhibition by PAR2-AP. Plotted is the deactivation current measured as the difference between the current 10 ms into the voltage pulse from -30 mV to -60 mV and the steady state current at -60 mV and normalised to cell capacitance (I deac density). Drugs were applied during the periods indicated by the bars. (C & D) Summary of the effects of PAR2- AP and XE991 on M current (C) and resting membrane potential (V m ) (D). Bars are mean ? SEM; number of experiments given above bars in parentheses. Significant difference from basal (*) and from PAR2-AP ( ?); NS -no significant difference. (A) Reconstitution of PAR-2 inhibition of M current in CHO cells. Cells were transfected with hKv7.2, hKv7.3 and hPAR-2 cDNA and current was recorded in perforated patch mode. Deactivation current when stepping the membrane voltage from 0 mV to -60 mV is normalized to cell capacitance. Drugs (PAR2-AP, 10 ?M; XE991, 3 ?M) were applied during the periods indicated by the bars. Inset shows current traces taken at the time points indicated by coloured triangles. (B) Recovery of M current in DRG neurons. Recording conditions as in Figure 1A-B. (C) recording similar to that shown in B but trypsin (20 nM) is applied instead of PAR2-AP. (D) Summary of recovery of Kv7.2/7.3 current (CHO) or native M current (DRG) after inhibition by PAR2-AP or trypsin. Data represented as % recovery of the XE991 sensitive deactivation current fraction (I M ). (A) DRG neurons were transfected with the optical probe for PIP 2 /IP 3 (PLC?-PH-GFP) and imaged using a swept field confocal microscope. Time course of changes in cytosolic fluorescence intensity (F) normalised to basal fluourescence levels (F 0 ) (mean ? SEM, n = 7). Only cells which displayed translocation of the probe were included (7/12). PAR2-AP was added to the bath solution as indicated by the black bar. (B) An exemplar cell imaged as in A. Images before application (basal), during application (PAR-2) and after the wash-out of PAR2-AP (wash) are shown on the left. White arrow indicates line used for intensity profiles shown on the right. Note the two fluorescence intensity peaks under basal conditions which correspond to the plasma membrane and are lost upon PAR-2 activation. (C, D) Calcium imaging from individual DRG neurons loaded with Fluo4-AM. Lower bars indicate [Ca 2+ ] o (mM). Drugs (PAR2-AP, 10 ?M; capsaicin, 1 ?M) were applied during the periods indicated by the bars. (A) Buffering of the cytosolic Ca 2+ and membrane PIP 2 through the whole-cell pipette. Representative recordings from DRG neurons temporally aligned for the onset of the PAR2- AP application. Data are normalised to the basal level of I M (I 0 ). PAR2-AP was added to the bath solution 60s after establishing a stable current level as indicated by the black bar. Pipette solutions contain: 0.2 mM BAPTA, no added Ca 2+ (control, black circles); 0.2 mM BAPTA, 100 ?M diC8-PIP 2 (PIP 2 , red circles); 10 mM BAPTA, free [Ca 2+ ] i buffered to 75 nM with CaCl 2 (BAPTA, blue circles); 10 mM BAPTA, 100 ?M diC8-PIP 2 (BAPTA+PIP 2 , green circles). Recordings were performed in whole-cell mode. Individual traces were fit with a generic sigmoid function for better comparison. (B) Mean data of the experiments shown in A. Number of experiments is given within bars. (*) difference from control, ( ?) difference from PIP 2 . (C) Perforated patch experiments similar to those in A. Black circles depict control experiments similar to that of Fig. 1B; red circles -PLC inhibitor edelfosine (10 ?M) added 10 minutes before commencing experiment and remained throughout; blue circles -as before but specific IP 3 receptor blocker Xestosspongin C (1 ?M) is used instead of edelfosine. (D) Mean data of the experiments shown in C. (*) difference from control, ( ?) difference from edelfosine. (A) XE991 induces acute pain in a concentration-dependent manner. 50 ?l solutions of different concentrations (as indicated below the bars) were injected into the hind paw of adult rats. Nocifensive behaviour was quantified by observing time spent grooming and flinching during 45 min, n = 6 for each experiment. (*) difference with the vehicle control; ( ?) difference from XE991 2 ?M; ( ?) difference from XE991 200 ?M. (B) Pre-application of XE991 does not significantly increase nocifensive behaviour induced by PA2-AP. Drugs were injected as following: Vh1 -vehicle (50 ?l) control; PAR2-AP -PAR2-AP, 50 ...
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... One hallmark feature of M-channels is their susceptibility to the modulation by an array of neurotransmitters and inflammatory mediators. Stimulation of Gq-coupled B 2 receptor by bradykinin, protease-activated receptor 2 by proteases and P2Y1 receptor by ATP inhibits I M in nociceptive sensory neurons of dorsal root ganglia (DRG) (Linley et al., 2008;Liu et al., 2010;Yousuf et al., 2011). Nerve injury, tissue inflammation or prolonged exposure to the mixture of inflammatory mediators has been shown to downregulate the expression of Kcnq2 and Kcnq3 in DRG neurons (Mucha et al., 2010;Rose et al., 2011;Zhang et al., 2019a;Zhang et al., 2019b). ...
Heightened excitability of vagal sensory neurons in inflammatory visceral diseases contributes to unproductive and difficult-to-treat neuronally based symptoms such as visceral pain and dysfunction. Identification of targets and modulators capable of regulating the excitability of vagal sensory neurons may lead to novel therapeutic options. KCNQ1-5 genes encode KV7.1-7.5 potassium channel α-subunits. Homotetrameric or heterotetrameric KV7.2-7.5 channels can generate the so-called M-current (IM) known to decrease the excitability of neurons including visceral sensory neurons. This study aimed to address the hypothesis that KV7.2/7.3 channels are key regulators of vagal sensory neuron excitability by evaluating the effects of KCNQ2/3-selective activator, ICA-069673, on IM in mouse nodose neurons and determining its effects on excitability and action potential firings using patch clamp technique. The results showed that ICA-069673 enhanced IM density, accelerated the activation and delayed the deactivation of M-channels in a concentration-dependent manner. ICA-069673 negatively shifted the voltage-dependent activation of IM and increased the maximal conductance. Consistent with its effects on IM, ICA-069673 induced a marked hyperpolarization of resting potential and reduced the input resistance. The hyperpolarizing effect was more pronounced in partially depolarized neurons. Moreover, ICA-069673 caused a 3-fold increase in the minimal amount of depolarizing current needed to evoke an action potential, and significantly limited the action potential firings in response to sustained suprathreshold stimulations. ICA-069673 had no effect on membrane currents when Kcnq2 and Kcnq3 were deleted. These results indicate that opening KCNQ2/3-mediated M-channels is sufficient to suppress the excitability and enhance spike accommodation in vagal visceral sensory neurons. Significance Statement This study supports the hypothesis that selectively activating KCNQ2/3-mediated M-channels is sufficient to suppress the excitability and action potential firings in vagal sensory neurons. These results provide evidence in support of further investigations into the treatment of various visceral disorders that involve nociceptor hyperexcitability with selective KCNQ2/3 M-channel openers.
... Multiple signaling molecules are implicated in the regulation of resting membrane potentials in nociceptive sensory neurons. Resting membrane potentials in nociceptive sensory neurons are depolarized by pro-inflammatory mediators, like nerve growth factor [40], serotonin [43], proteases [44], and osteoarthritic synovial fluid [45]. In animals with spinal cord injury, it was reported that elevation of resting membrane potentials in nociceptive sensory neurons is maintained by cAMP via PKA [38]. ...
Patients with systemic lupus erythematosus (SLE) often suffer from chronic pain. Little is known about the peripheral mechanisms underlying the genesis of chronic pain induced by SLE. The aim of this study was to investigate whether and how membrane properties in nociceptive neurons in the dorsal root ganglions (DRGs) are altered by SLE. We found elevation of resting membrane potentials, smaller capacitances, lower action potential thresholds and rheobases in nociceptive neurons in the DRGs from MRL/lpr mice (an SLE mouse model) with thermal hyperalgesia. DRGs from MRL/lpr mice had increased protein expressions in TNFα, IL-1β, and phosphorylated ERK but suppressed AMPK activity, and no changes in sodium channel 1.7 protein expression. We showed that intraplantar injection of Compound C (an AMPK inhibitor) induced thermal hyperalgesia in normal mice while intraplantar injection of AICAR (an AMPK activator) reduced thermal hyperalgesia in MRL/Lpr mice. Upon inhibition of AMPK membrane properties in nociceptive neurons from normal control mice could be rapidly switched to those found in SLE mice with thermal hyperalgesia. Our study indicates that increased excitability in peripheral nociceptive sensory neurons contributes to the genesis of thermal hyperalgesia in mice with SLE, and AMPK regulates membrane properties in nociceptive sensory neurons as well as thermal hyperalgesia in mice with SLE. Our study provides a basis for targeting signaling pathways regulating membrane properties of peripheral nociceptive neurons as a means for conquering chronic pain caused by SLE.
... Both ATP and bradykinin evoke action potential firing through multiple mechanisms, including the inhibition of KV7 channels in a phospholipase C-(PLC) dependent manner (Stemkowski et al., 2002;Liu et al., 2010). It is likely that many PLC-coupled receptors, such as protease activated receptor 2 (PAR2) (Linley et al., 2008) and Mas-related G-protein-coupled receptor member D (MrgD) (Crozier et al., 2007), stimulate action potential discharge through the inhibition of KV7 channels. Suppression of KV7 channel activity triggers membrane depolarisation necessary for the activation of voltage-gated Na + (NaV) channels required to further amplify depolarisations (e.g., NaV1.9) and trigger action potential generation (e.g., NaV1.8). ...
In numerous subtypes of central and peripheral neurons, small and intermediate conductance Ca2+-activated K+ (SK and IK, respectively) channels are important regulators of neuronal excitability. Transcripts encoding SK channel subunits, as well as the closely related IK subunit, are co-expressed in the soma of colonic afferent neurons with receptors for the algogenic mediators adenosine triphosphate (ATP) and bradykinin, P2X3 and B2, highlighting the potential utility of these channels as drug targets for the treatment of abdominal pain in gastrointestinal diseases such as irritable bowel syndrome. Despite this, pre-treatment with the dual SK/IK channel opener SKA-31 had no effect on the colonic afferent response to ATP, bradykinin or noxious ramp distention of the colon. Inhibition of SK or IK channels with apamin or TRAM-34, respectively, yielded no change in spontaneous baseline afferent activity, indicating these channels are not tonically active. In contrast to its lack of effect in electrophysiological experiments, comparable concentrations of SKA-31 abolished ongoing peristaltic activity in the colon ex vivo. Treatment with the KV7 channel opener retigabine blunted the colonic afferent response to all applied stimuli. Our data therefore highlight the potential utility of KV7, but not SK/IK, channel openers as analgesic agents for the treatment of abdominal pain.
... In contrast, potassium channels underlying the M-current-K V 7.2/3-have been identified as inhibitors of DRG activity and promoters of pain resilience (9,10). Further, these classes of channels have been shown to be pathologically regulated in manners consistent with their physiological roles; expression of Na V 1.7 is up-regulated, while K V 7.2 is down-regulated in models of inflammatory pain and neuropathy (11)(12)(13)(14)(15)(16)(17). Finally, both Na V 1.7 and K V 7 channels are localized in the distal axons of peripheral sensory neurons (18,19). ...
Inflammation causes pain by shifting the balance of ionic currents in nociceptors toward depolarization, leading to hyperexcitability. The ensemble of ion channels within the plasma membrane is regulated by processes including biogenesis, transport, and degradation. Thus, alterations in ion channel trafficking may influence excitability. Sodium channel NaV1.7 and potassium channel KV7.2 promote and oppose excitability in nociceptors, respectively. We used live-cell imaging to investigate mechanisms by which inflammatory mediators (IM) modulate the abundance of these channels at axonal surfaces through transcription, vesicular loading, axonal transport, exocytosis, and endocytosis. Inflammatory mediators induced a NaV1.7-dependent increase in activity in distal axons. Further, inflammation increased the abundance of NaV1.7, but not of KV7.2, at axonal surfaces by selectively increasing channel loading into anterograde transport vesicles and insertion at the membrane, without affecting retrograde transport. These results uncover a cell biological mechanism for inflammatory pain and suggest NaV1.7 trafficking as a potential therapeutic target.
... The transcription and translation of Na V and K V channels are dysregulated oppositely in pain states. For example, Na V 1.7 and Na V 1.8 expression is upregulated in sensory neurons in models of inflammation and chemotherapy-induced neuropathy (Black et al., 2004;Gould et al., 2004;Strickland et al., 2008;Liang et al., 2013;Li et al., 2018), whereas K V 7.2 is downregulated in models of nerve injury and inflammation (Linley et al., 2008;Rose et al., 2011). ...
Neuronal excitability relies on coordinated action of functionally distinct ion channels. Voltage-gated sodium (NaV) and potassium (KV) channels have distinct but complementary roles in firing action potentials: NaV channels provide depolarizing current while KV channels provide hyperpolarizing current. Mutations and dysfunction of multiple NaV and KV channels underlie disorders of excitability, including pain and epilepsy. Modulating ion channel trafficking may offer a potential therapeutic strategy for these diseases. A fundamental question, however, is whether these channels with distinct functional roles are transported independently or packaged together in the same vesicles in sensory axons. We have used Optical Pulse-Chase Axonal Long-distance (OPAL) imaging to investigate trafficking of NaV and KV channels and other axonal proteins from distinct functional classes in live rodent sensory neurons (from male and female rats). We show that, similar to NaV1.7 channels, NaV1.8 and KV7.2 channels are transported in Rab6a-positive vesicles, and that each of the NaV channel isoforms expressed in healthy, mature sensory neurons - NaV1.6, NaV1.7, NaV1.8, and NaV1.9 - are co-transported in the same vesicles. Further, we show that multiple axonal membrane proteins with different physiological functions - NaV1.7, KV7.2, and TNFR1 - are co-transported in the same vesicles. However, vesicular packaging of axonal membrane proteins is not indiscriminate, since another axonal membrane protein - NCX2 - is transported in separate vesicles. These results shed new light on the development and organization of sensory neuron membranes, revealing complex sorting of axonal proteins with diverse physiological functions into specific transport vesicles.Significance StatementNormal neuronal excitability is dependent on precise regulation of membrane proteins including NaV and KV channels, and imbalance in the level of these channels at the plasma membrane could lead to excitability disorders. Ion channel trafficking could potentially be targeted therapeutically, which would require better understanding of the mechanisms underlying trafficking of functionally diverse channels. Optical Pulse-chase Axonal Long-distance (OPAL) imaging in live neurons permitted examination of the specificity of ion channel trafficking, revealing co-packaging of axonal proteins with opposing physiological functions into the same transport vesicles. This suggests that additional trafficking mechanisms are necessary to regulate levels of surface channels and reveals an important consideration for therapeutic strategies that target ion channel trafficking for the treatment of excitability disorders.
... Neuronal cultures. DRG neurons were dissociated and cultured as described previously (Linley et al., 2008;Liu et al., 2010). Briefly, adult rats were humanely euthanized by cervical dislocation under isoflurane anesthesia. ...
GABA is a major inhibitory neurotransmitter in the mammalian central nervous system (CNS). Inhibitory GABAA channel circuits in the dorsal spinal cord are the gatekeepers of the nociceptive input from the periphery to the CNS. Weakening of these spinal inhibitory mechanisms is a hallmark of chronic pain. Yet, recent studies have suggested the existence of an earlier GABAergic “gate” within the peripheral sensory ganglia. In this study, we performed systematic investigation of plastic changes of the GABA-related proteins in the dorsal root ganglion (DRG) in the process of neuropathic pain development. We found that chronic constriction injury (CCI) induced general downregulation of most GABAA channel subunits and the GABA-producing enzyme, glutamate decarboxylase, consistent with the weakening of the GABAergic inhibition at the periphery. Strikingly, the α5 GABAA subunit was consistently upregulated. Knock-down of the α5 subunit in vivo moderately alleviated neuropathic hyperalgesia. Our findings suggest that while the development of neuropathic pain is generally accompanied by weakening of the peripheral GABAergic system, the α5 GABAA subunit may have a unique pro-algesic role and, hence, might represent a new therapeutic target.
... Dorsal root ganglion, peripheral neurons, sensory neurons, and nociceptor neurons express all the three mAHP channels (Akins and McCleskey, 1993;Villière and McLachlan, 1996;Amir and Devor, 1997;Boettger et al., 2002;Passmore et al., 2003;Linley et al., 2008;Hou et al., 2015). These channels regulate the firing activity of the aforementioned neurons, thereby modulating pain responses. ...
SK, HCN, and M channels are medium afterhyperpolarization (mAHP)-mediating ion channels. The three channels co-express in various brain regions, and their collective action strongly influences cellular excitability. However, significant diversity exists in the expression of channel isoforms in distinct brain regions and various subcellular compartments, which contributes to an equally diverse set of specific neuronal functions. The current review emphasizes the collective behavior of the three classes of mAHP channels and discusses how these channels function together although they play specialized roles. We discuss the biophysical properties of these channels, signaling pathways that influence the activity of the three mAHP channels, various chemical modulators that alter channel activity and their therapeutic potential in treating various neurological anomalies. Additionally, we discuss the role of mAHP channels in the pathophysiology of various neurological diseases and how their modulation can alleviate some of the symptoms.
... The activation of PAR2 in DRG neurons stimulates PLC and induces PK activation that modulates the activity of TRPV1, TRPV4, TRPA1 [16,24,[29][30][31], voltage-gated Kv7 potassium and calcium-activated Cl − channels [17,32,33]. This regulates the excitability of these primary nociceptive neurons, neuropeptide releases [5] and synaptic transmissions at the first nociceptive synapse in the spinal cord dorsal horn and leads to hypersensitivity in vivo [34,35]. ...
The mechanisms of inflammatory pain need to be identified in order to find new superior treatments. Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) are highly co-expressed in dorsal root ganglion neurons and implicated in pain development. Here, we examined the role of spinal PAR2 in hyperalgesia and the modulation of synaptic transmission in carrageenan-induced peripheral inflammation, using intrathecal (i.t.) treatment in the behavioral experiments and recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs and eEPSCs) in spinal cord slices. Intrathecal PAR2-activating peptide (AP) administration aggravated the carrageenan-induced thermal hyperalgesia, and this was prevented by a TRPV1 antagonist (SB 366791) and staurosporine i.t. pretreatment. Additionally, the frequency of the mEPSC and sEPSC and the amplitude of the eEPSC recorded from the superficial dorsal horn neurons were enhanced after acute PAR2 AP application, while prevented with SB 366791 or staurosporine pretreatment. PAR2 antagonist application reduced the thermal hyperalgesia and decreased the frequency of mEPSC and sEPSC and the amplitude of eEPSC. Our findings highlight the contribution of spinal PAR2 activation to carrageenan-induced hyperalgesia and the importance of dorsal horn PAR2 and TRPV1 receptor interactions in the modulation of nociceptive synaptic transmission.
... Kcnq channels encode K + channels that represent the M-current that regulates nociceptor signaling, especially at peripheral terminals 46,63 , and also plays an important role in generation of spontaneous activity after nerve injury 64 . The M current is also critical in mediating hyperalgesic actions of many G q -protein receptor activators such as bradykinin 65 and protease activated receptors 66 . Orai1 as a key component of storeoperated calcium channels that regulate nociception via modulation of neuronal excitability and A-type potassium channels 67 . ...
Many chronic pain conditions show sex differences in their epidemiology. This could be attributed to sex-dependent differential expression of genes (DEGs) involved in nociceptive pathways, including sensory neurons. This study aimed to identify sex-dependent DEGs in estrous female versus male sensory neurons, which were prepared by using different approaches and ganglion types. RNA-seq on non-purified sensory neuronal preparations, such as whole dorsal root ganglion (DRG) and hindpaw tissues, revealed only a few sex-dependent DEGs. Sensory neuron purification increased numbers of sex-dependent DEGs. These DEG sets were substantially influenced by preparation approaches and ganglion types [DRG vs trigeminal ganglia (TG)]. Percoll-gradient enriched DRG and TG neuronal fractions produced distinct sex-dependent DEG groups. We next isolated a subset of sensory neurons by sorting DRG neurons back-labeled from paw and thigh muscle. These neurons have a unique sex-dependent DEG set, yet there is similarity in biological processes linked to these different groups of sex-dependent DEGs. Female-predominant DEGs in sensory neurons relate to inflammatory, synaptic transmission and extracellular matrix reorganization processes that could exacerbate neuro-inflammation severity, especially in TG. Male-selective DEGs were linked to oxidative phosphorylation and protein/molecule metabolism and production. Our findings catalog preparation-dependent sex differences in neuronal gene expressions in sensory ganglia.
... These channels fall into the broad category of delayed rectifiers but are frequently discussed separately as they display limited structural similarity to other K v channel types . Unlike some other K v channels, M-channels are modulated by G-protein coupled agonists (Selyanko et al., 1990;Suh et al., 2004), inflammatory mediators (Linley et al., 2008), including bradykinin (Cruzblanca et al., 1998) and ATP (Ford et al., 2003), src-tyrosine kinases , Ca 2+ acting via calmodulin Li et al., 2005) and by the membrane phospholipid phosphatidylinositol 4,5bisphosphate (PIP 2 ) (Ford et al., 2003;Li et al., 2005;Du et al., 2018). G q coupled agonists suppress M-channel conductance via phospholipase C mediated depletion of PIP 2 (Ford et al., 2003;Suh et al., 2004). ...
Sensory abnormalities generated by nerve injury, peripheral neuropathy or disease are often expressed as neuropathic pain. This type of pain is frequently resistant to therapeutic intervention and may be intractable. Numerous studies have revealed the importance of enduring increases in primary afferent excitability and persistent spontaneous activity in the onset and maintenance of peripherally induced neuropathic pain. Some of this activity results from modulation, increased activity and /or expression of voltage-gated Na⁺ channels and hyperpolarization-activated cyclic nucleotide–gated (HCN) channels. K⁺ channels expressed in dorsal root ganglia (DRG) include delayed rectifiers (Kv1.1, 1.2), A-channels (Kv1.4, 3.3, 3.4, 4.1, 4.2, and 4.3), KCNQ or M-channels (Kv7.2, 7.3, 7.4, and 7.5), ATP-sensitive channels (KIR6.2), Ca²⁺-activated K⁺ channels (KCa1.1, 2.1, 2.2, 2.3, and 3.1), Na⁺-activated K⁺ channels (KCa4.1 and 4.2) and two pore domain leak channels (K2p; TWIK related channels). Function of all K⁺ channel types is reduced via a multiplicity of processes leading to altered expression and/or post-translational modification. This also increases excitability of DRG cell bodies and nociceptive free nerve endings, alters axonal conduction and increases neurotransmitter release from primary afferent terminals in the spinal dorsal horn. Correlation of these cellular changes with behavioral studies provides almost indisputable evidence for K⁺ channel dysfunction in the onset and maintenance of neuropathic pain. This idea is underlined by the observation that selective impairment of just one subtype of DRG K⁺ channel can produce signs of pain in vivo. Whilst it is established that various mediators, including cytokines and growth factors bring about injury-induced changes in DRG function and excitability, evidence presently available points to a seminal role for interleukin 1β (IL-1β) in control of K⁺ channel function. Despite the current state of knowledge, attempts to target K⁺ channels for therapeutic pain management have met with limited success. This situation may change with the advent of personalized medicine. Identification of specific sensory abnormalities and genetic profiling of individual patients may predict therapeutic benefit of K⁺ channel activators.
