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Knockout of Slo2.2 enhances itch, abolishes KNa current, and increases action potential firing frequency in DRG neurons.

Martinez-Espinosa PL, Wu J, Yang C, Gonzalez-Perez V, Zhou H, Liang H, Xia XM, Lingle CJ - Elife (2015)

Bottom Line: Noting the prevalence of Slo2.2 in dorsal root ganglion, we find that KO of Slo2.2, but not Slo2.1, results in enhanced itch and pain responses.In dissociated small diameter DRG neurons, KO of Slo2.2, but not Slo2.1, abolishes KNa current.Activation of KNa acts as a brake to initiation of the first depolarization-elicited AP with no discernible effect on afterhyperpolarizations.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States.

ABSTRACT
Two mammalian genes, Kcnt1 and Kcnt2, encode pore-forming subunits of Na(+)-dependent K(+) (KNa) channels. Progress in understanding KNa channels has been hampered by the absence of specific tools and methods for rigorous KNa identification in native cells. Here, we report the genetic disruption of both Kcnt1 and Kcnt2, confirm the loss of Slo2.2 and Slo2.1 protein, respectively, in KO animals, and define tissues enriched in Slo2 expression. Noting the prevalence of Slo2.2 in dorsal root ganglion, we find that KO of Slo2.2, but not Slo2.1, results in enhanced itch and pain responses. In dissociated small diameter DRG neurons, KO of Slo2.2, but not Slo2.1, abolishes KNa current. Utilizing isolectin B4+ neurons, the absence of KNa current results in an increase in action potential (AP) firing and a decrease in AP threshold. Activation of KNa acts as a brake to initiation of the first depolarization-elicited AP with no discernible effect on afterhyperpolarizations.

No MeSH data available.


Related in: MedlinePlus

Confirmation of properties of single KNa channels that are deleted by Slo2 dKO.(A) The cytosolic face of an excised inside-out patch from a DRG neuron was exposed to 70 mM Na+ solution and channel activity was monitored over a range of voltages. Average activity exhibited only weak voltage-dependence. (B) Single channel amplitude was measured at four voltages for a set of four patches, yielding a single channel conductance of 127 pS.DOI:http://dx.doi.org/10.7554/eLife.10013.014
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fig6s3: Confirmation of properties of single KNa channels that are deleted by Slo2 dKO.(A) The cytosolic face of an excised inside-out patch from a DRG neuron was exposed to 70 mM Na+ solution and channel activity was monitored over a range of voltages. Average activity exhibited only weak voltage-dependence. (B) Single channel amplitude was measured at four voltages for a set of four patches, yielding a single channel conductance of 127 pS.DOI:http://dx.doi.org/10.7554/eLife.10013.014

Mentions: Sensory neurons contain a rich variety of K+ currents (Vydyanathan et al., 2005; Dobler et al., 2007; Li et al., 2007; Cho et al., 2009; Zhang et al., 2010b; Liu et al., 2013) that complicate unambiguous definition of KNa current, for which selective pharmacological tools are lacking. We have not had reliable success with subtractive methods involving Na+ current inhibition or Na+ replacement. To test for the presence of KNa current in small diameter DRG neurons, we used a method previously applied to rat DRG neurons (Bischoff et al., 1998): a K+ background current arising from defined pipette Na+ is measured using hyperpolarizing voltage-steps during the first 5 min following formation of the whole-cell recording configuration. With 0 mM pipette Na+, little background current is observed with voltage-steps from −80 to −120 mV (Figure 6A,C). With 70 mM pipette Na+, net current elicited by the same voltage-step gradually increases over 3 min reaching a plateau near 1 nA (Figure 6A,C). At longer times following whole-cell access, current activated by 70 mM pipette Na+ gradually diminishes (Figure 6—figure supplement 1) despite no change in voltage-dependent Na+ current. As in rat DRG neurons (Bischoff et al., 1998), the KNa current is blocked by extracellular 20 mM Cs+, with stronger inhibition at −120 mV than −80 mV reflecting the voltage-dependence of Cs+ inhibition (Figure 6A, Figure 6—figure supplement 2). The average amplitude of KNa current was similar for WT and Slo2.1 KO DRG neurons (Figure 6B,C), while there was no KNa current in Slo2.2 KO or Slo2 dKO neurons (Figure 6B,C). Despite considerable variability in total KNa current among neurons from either WT or Slo2.1 KO animals (Figure 6D), the total current always exceeds that observed in WT cells with 0 Na+, or in Slo2 dKO or Slo2.2 KO cells with 70 mM Na+ (Figure 6D). Excised inside-out patches confirmed that Slo2 dKO removed a Na–dependent K+ channel (Figure 6E) which exhibited little voltage-dependence over the range of −80 through −20 mV (Figure 6E, Figure 6—figure supplement 3A) with a single channel conductance of about 127 pS (Figure 6E, Figure 6—figure supplement 3B). Finally, we compared the whole-cell steady-state current–voltage (I–V) relationship between WT and Slo2 dKO cells over the range of −125 to −25 mV, with 70 mM pipette Na+ along with the steady-state IV relationship persisting in WT cells after 30 min with 70 mM pipette Na+ (Figure 6F). This shows the relatively voltage-independent nature of the background KNa conductance (reversal at EK) when the cytosolic Na+ concentration is constant.10.7554/eLife.10013.011Figure 6.The absence of Slo2.2 reduces Na+-dependent leak current in acutely dissociated mouse DRG neurons.


Knockout of Slo2.2 enhances itch, abolishes KNa current, and increases action potential firing frequency in DRG neurons.

Martinez-Espinosa PL, Wu J, Yang C, Gonzalez-Perez V, Zhou H, Liang H, Xia XM, Lingle CJ - Elife (2015)

Confirmation of properties of single KNa channels that are deleted by Slo2 dKO.(A) The cytosolic face of an excised inside-out patch from a DRG neuron was exposed to 70 mM Na+ solution and channel activity was monitored over a range of voltages. Average activity exhibited only weak voltage-dependence. (B) Single channel amplitude was measured at four voltages for a set of four patches, yielding a single channel conductance of 127 pS.DOI:http://dx.doi.org/10.7554/eLife.10013.014
© Copyright Policy
Related In: Results  -  Collection

License
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fig6s3: Confirmation of properties of single KNa channels that are deleted by Slo2 dKO.(A) The cytosolic face of an excised inside-out patch from a DRG neuron was exposed to 70 mM Na+ solution and channel activity was monitored over a range of voltages. Average activity exhibited only weak voltage-dependence. (B) Single channel amplitude was measured at four voltages for a set of four patches, yielding a single channel conductance of 127 pS.DOI:http://dx.doi.org/10.7554/eLife.10013.014
Mentions: Sensory neurons contain a rich variety of K+ currents (Vydyanathan et al., 2005; Dobler et al., 2007; Li et al., 2007; Cho et al., 2009; Zhang et al., 2010b; Liu et al., 2013) that complicate unambiguous definition of KNa current, for which selective pharmacological tools are lacking. We have not had reliable success with subtractive methods involving Na+ current inhibition or Na+ replacement. To test for the presence of KNa current in small diameter DRG neurons, we used a method previously applied to rat DRG neurons (Bischoff et al., 1998): a K+ background current arising from defined pipette Na+ is measured using hyperpolarizing voltage-steps during the first 5 min following formation of the whole-cell recording configuration. With 0 mM pipette Na+, little background current is observed with voltage-steps from −80 to −120 mV (Figure 6A,C). With 70 mM pipette Na+, net current elicited by the same voltage-step gradually increases over 3 min reaching a plateau near 1 nA (Figure 6A,C). At longer times following whole-cell access, current activated by 70 mM pipette Na+ gradually diminishes (Figure 6—figure supplement 1) despite no change in voltage-dependent Na+ current. As in rat DRG neurons (Bischoff et al., 1998), the KNa current is blocked by extracellular 20 mM Cs+, with stronger inhibition at −120 mV than −80 mV reflecting the voltage-dependence of Cs+ inhibition (Figure 6A, Figure 6—figure supplement 2). The average amplitude of KNa current was similar for WT and Slo2.1 KO DRG neurons (Figure 6B,C), while there was no KNa current in Slo2.2 KO or Slo2 dKO neurons (Figure 6B,C). Despite considerable variability in total KNa current among neurons from either WT or Slo2.1 KO animals (Figure 6D), the total current always exceeds that observed in WT cells with 0 Na+, or in Slo2 dKO or Slo2.2 KO cells with 70 mM Na+ (Figure 6D). Excised inside-out patches confirmed that Slo2 dKO removed a Na–dependent K+ channel (Figure 6E) which exhibited little voltage-dependence over the range of −80 through −20 mV (Figure 6E, Figure 6—figure supplement 3A) with a single channel conductance of about 127 pS (Figure 6E, Figure 6—figure supplement 3B). Finally, we compared the whole-cell steady-state current–voltage (I–V) relationship between WT and Slo2 dKO cells over the range of −125 to −25 mV, with 70 mM pipette Na+ along with the steady-state IV relationship persisting in WT cells after 30 min with 70 mM pipette Na+ (Figure 6F). This shows the relatively voltage-independent nature of the background KNa conductance (reversal at EK) when the cytosolic Na+ concentration is constant.10.7554/eLife.10013.011Figure 6.The absence of Slo2.2 reduces Na+-dependent leak current in acutely dissociated mouse DRG neurons.

Bottom Line: Noting the prevalence of Slo2.2 in dorsal root ganglion, we find that KO of Slo2.2, but not Slo2.1, results in enhanced itch and pain responses.In dissociated small diameter DRG neurons, KO of Slo2.2, but not Slo2.1, abolishes KNa current.Activation of KNa acts as a brake to initiation of the first depolarization-elicited AP with no discernible effect on afterhyperpolarizations.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Washington University School of Medicine, St. Louis, United States.

ABSTRACT
Two mammalian genes, Kcnt1 and Kcnt2, encode pore-forming subunits of Na(+)-dependent K(+) (KNa) channels. Progress in understanding KNa channels has been hampered by the absence of specific tools and methods for rigorous KNa identification in native cells. Here, we report the genetic disruption of both Kcnt1 and Kcnt2, confirm the loss of Slo2.2 and Slo2.1 protein, respectively, in KO animals, and define tissues enriched in Slo2 expression. Noting the prevalence of Slo2.2 in dorsal root ganglion, we find that KO of Slo2.2, but not Slo2.1, results in enhanced itch and pain responses. In dissociated small diameter DRG neurons, KO of Slo2.2, but not Slo2.1, abolishes KNa current. Utilizing isolectin B4+ neurons, the absence of KNa current results in an increase in action potential (AP) firing and a decrease in AP threshold. Activation of KNa acts as a brake to initiation of the first depolarization-elicited AP with no discernible effect on afterhyperpolarizations.

No MeSH data available.


Related in: MedlinePlus