Knockout of Slo2.2 enhances itch, abolishes KNa current, and increases action potential firing frequency in DRG neurons
- Washington University School of Medicine, United States
Figures
Figure 1
Construction and validation of Slo2.1 and Slo2.2 KO mice.
(A) Upper row: map of WT mouse Kcnt2 (encoding Slo2.1) gene locus within genomic DNA bracketing the targeted exon 22. Second row: map of the targeting vector, showing M1uI site for vector …
Figure 2
Slo2.1 and Slo2.2 subunits are absent in Kcnt2 and Kcnt1 KO mice, respectively, exhibit differential tissue distribution, and coassemble in some tissue.
(A) Top, brain membrane proteins from WT, Slo2.1 KO, and Slo2.2 KO mice were probed with N11/33 anti-Slo2.1 antibody (Antibodies Inc.). Middle, brain membrane proteins were separated and probed with …
Figure 3
Slo2 dKO shortens hotplate response latency, increases responses to hindpaw injections of capsaicin, but does not influence formalin responses.
(A) Latencies to aversive response following placement on a 55°C hotplate are plotted for the indicated genotypes, showing means, sem, and individual latencies. From left to right, n = 19, 19, 24, …
Figure 4 with 1 supplement
The absence of Slo2.2, but not Slo2.1, results in enhancement of chloroquine (CQ) and histamine (HA)-induced itch.
(A) Each point shows mean number of scratching bouts per 5 min bins for WT mice (n = 15, black circles) and Slo2 dKO mice (n = 19, red circles) after injection of 200 μg CQ. (B) Mean scratching …
Figure 4—figure supplement 1
Concentration-dependence of itch response to HA and compound 48–80.
(A) Scratching behavior in WT and Slo2 dKO mice is plotted following injection of 1 mg HA. (B) Scratching behavior after 0.3 mg HA is compared. (C) Scratching behavior after 0.1 mg HA is displayed. …
Figure 5
CQ enhances itch-type behavior following cheek injection, but not pain-type behavior.
(A) Total scratching bouts using the hindpaw to scratch the cheek was monitored following cheek injection of 200 μg CQ in WT and Slo2 dKO mice. During the first 5 min interval, distributions …
Figure 6 with 5 supplements
The absence of Slo2.2 reduces Na+-dependent leak current in acutely dissociated mouse DRG neurons.
(A) Traces on the top show currents (evoked by indicated voltage protocol) for four time points following formation of a whole-cell recording with 70 mM pipette Na+. Green: immediately following …
Figure 6—figure supplement 1
KNa current runs down during constant cytosolic 70 mM Na+.
(A) The indicated voltage protocol was used to elicit currents in a WT DRG neuron with 70 mM pipette Na+. Red trace indicates current activated by step to −5 mV. Bottom record in each case shows …
Figure 6—figure supplement 2
Cs+ inhibition of KNa current exhibits voltage-dependence.
(A) Steady-state current was measured over the indicated voltages with 70 mM pipette Na+. Application of 20 mM extracellular Cs+ markedly inhibits the KNa current, with stronger inhibition at more …
Figure 6—figure supplement 3
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 …
Figure 6—figure supplement 4
Na+-dependent leak current is present in both IB4+ and IB4− neurons and runs down with time in culture.
(A) Whole-cell current was activated by voltage-steps from −80 to −120 mV, with 70 mM Na+ in the pipette solution. Both IB4+ and IB4− neurons exhibit substantial KNa current. (B) Dissociated DRG …
Figure 6—figure supplement 5
The absence of Slo2.2 and, to a lesser extent, Slo2.1, reduces Na+-dependent leak current in mouse DRG neurons in DRG tissue slices.
(A) Traces on the left show currents evoked by the indicated voltage steps at three time points following formation of a whole-cell recording with 70 mM pipette Na+, an initial trace immediately …
Figure 7
Evoked action potential (AP) firing is increased in IB4+ DRG neurons from Slo2 dKO mice.
(A) 40, 60, 100, and 200 pA current injections (1 s) were used to elicit firing in WT (left) and dKO IB4+ DRG neurons from a holding potential of −60 mV. The pipette solution contained 10 mM Na+. (B)…
Figure 8
Slo2 dKO results in reduced AP threshold.
(A) A 20 ms current injection of different amplitudes applied with membrane potential adjusted to −60 mV was used to examine AP threshold for a WT (left) and a Slo2 dKO (right) DRG neuron with 10 mM …
Figure 9 with 3 supplements
Cs+ inhibition of outward current in WT, but not dKO, neurons recapitulates properties of Slo2 dKO.
(A) Traces show averaged currents activated by the indicated voltage-ramp protocol (top) for 10 WT and 10 dKO neurons. Number shows the voltage at which net current crosses the 0-current level …
Figure 9—figure supplement 1
Normalized ramp-activated currents reveals that application of Cs+ shifts the apparent range of inward current activation in a fashion similar to dKO of Slo2 currents.
Ramp-activated currents as in Figure 9 were normalized in each cell to the largest inward current and then averaged. The most negative inward current values of the averaged traces differ among each …
Figure 9—figure supplement 2
Comparison of step-activated inward and steady-state currents activated in WT and dKO cells for comparison of ramp-activated outward current.
(A) 20 ms voltage steps from −120 mV to +40 mV were applied from a holding potential of −60 mV, with pipette and extracellular solutions appropriate to allow normal cell firing. Figure plots peak …
Figure 9—figure supplement 3
Evaluating the potential impact of a small K+ conductance near resting potential.
(A) A modified GHK conductance equation (top) was used to calculate membrane potential. In the absence of explicit estimates of specific ion conductances at rest, we assume EK = −80 mV, ENa = 60 mV, …
Figure 10
AP trains in IB4+ small diameter DRG neurons do not develop slow AHPs.
(A) A cell was maintained at a resting potential of −50 mV and stimulated with either 1, 5, of 10 pulses of 10 ms duration and 200 pA amplitude, with a pulse frequency of 7.1 Hz. A brief …
Videos
Video 1
Response of a Slo2 dKO mouse to CQ injection (related to Figure 4A).
The nape of the neck of a Slo2 dKO mouse was injected with 10 μl 200 μM choroquine. Video recording was begun about 10 s after injection.
Video 2
Response of a WT mouse to CQ injection (related to Figure 4A).
The nape of the neck of a WT mouse was injected with 10 μl 200 μM choroquine. Acquisition of video was begun about 10 s after injection.
Tables
Table 1
Properties of IB4+ WT and Slo2 dKO DRG neurons (10 and 0 mM pipette Na+)
| Pipette Na+ | IB4+ WT | IB4+ dKO | p-values | ||||
|---|---|---|---|---|---|---|---|
| 10 mM Na+ | mean | sem | n | mean | sem | n | K-S statistic |
| Cm (pF) | 16.1 | 0.3 | 64 | 15.9 | 0.5 | 41 | 0.574 |
| m.p. (mV) | −54.2 | 0.6 | 57 | −50.8 | 0.9 | 41 | 0.001 |
| Rin (MΩ) | 1251.1 | 130.8 | 13 | 1212.9 | 148.8 | 13 | 0.828 |
| rheobase (pA) | 86.6 | 4.6 | 44 | 58.1 | 3.4 | 31 | 0.000 |
| dV/dt AP threshold (mV) | −25.31 | 0.64 | 14 | −27.89 | 0.65 | 10 | 0.032 |
| AP peak (mV) | 39.2 | 2.2 | 14 | 41.7 | 1.6 | 10 | 0.877 |
| AP half-width (ms) | 5.7 | 0.3 | 14 | 5.6 | 0.3 | 10 | 0.771 |
| AHP (mV) | −74.0 | 0.4 | 14 | −72.6 | 0.5 | 10 | 0.124 |
| 60 pA AP count | 2.3 | 0.5 | 64 | 9.7 | 1.9 | 41 | 0.000 |
| 100 pA AP count | 5.5 | 0.8 | 64 | 17.7 | 2.7 | 41 | 0.000 |
| 200 pA AP count | 11.4 | 1.5 | 64 | 28.9 | 5.1 | 41 | 0.000 |
| Pipette Na+ | IB4− WT | IB4− dKO | p-values | ||||
|---|---|---|---|---|---|---|---|
| 0 Na+ | mean | sem | n | mean | sem | n | K-S statistic |
| Cm (pF) | 16.7 | 0.9 | 12 | 16.7 | 0.8 | 12 | 0.991 |
| m.p. (mV) | −54.0 | 1.4 | 11 | −47.0 | 1.4 | 12 | 0.007 |
| Rin (MΩ) | 1381.0 | 194.5 | 12 | 1136.4 | 95.0 | 11 | 0.459 |
| rheobase (pA) | 92.5 | 8.7 | 12 | 60.8 | 5.1 | 12 | 0.0048 |
| dV/dt AP threshold (mV) | −22.7 | 0.7 | 10 | −25.8 | 0.5 | 10 | 0.001 |
| AP peak (mV) | 45.3 | 2.9 | 10 | 51.7 | 1.6 | 10 | 0.313 |
| AP half-width (ms) | 5.1 | 0.3 | 10 | 4.5 | 0.2 | 10 | 0.313 |
| AHP (mV) | −72.5 | 0.7 | 10 | −73.7 | 0.4 | 10 | 0.313 |
| 60 pA AP count | 1.2 | 0.5 | 12 | 5.8 | 1.2 | 12 | 0.005 |
| 100 pA AP count | 3.5 | 1.1 | 12 | 12.3 | 2.1 | 12 | 0.019 |
| 200 pA AP count | 6.8 | 2.4 | 11 | 19.9 | 3.0 | 10 | 0.005 |
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Cm, cell capacitance; m.p., resting potential; Rin, input resistance measured by current deflection arising from a 10 mV pulse from −60 to −70 mV; AP half-width, measured at half peak amplitude; AHP, measured following a single evoked AP; AP count, number of APs in 1 s of specified injected current. Rheobase, defined as smallest injected current which elicited an action potential during a 20 ms current injection.
-
AP, action potential.
Table 2
Primers used for Real-Time PCR
| Gene | Primer | Amplicon length |
|---|---|---|
| Kcnt2 | Forward: 5′-TCTATTTGAAACAATACTCCTTGG-3′ | 149 bp |
| Reverse: 5′-GAACAAATAGATTTCTTAAGGTGG-3′ | ||
| Kcnt1 | Forward: 5′-CTCACACACCCTTCCAACATGCGG-3′ | 161 bp |
| Reverse: 5′-ATGCTGATACTAAATACTCGACCA-3′ | ||
| Β-actin | Forward: 5′-TGGAGAAGAGCTATGAGCTGCCTG-3′ | 127 bp |
| Reverse: 5′-GTAGTTTCATGGATGCCACAGGAT-3′ |
