PMC

a. TMEM16C knockout rats display a significant reduction in paw withdrawal latency in the hot plate test at temperatures above 50°C (P<0.0001, two way ANOVA followed by Bonferroni test, *** P<0.001, * P<0.05, n=13). b. Compared to their wild type controls, TMEM16C knockout rats also withdraw their paws at a less intense mechanical stimulus (electronic Von Frey; Student’s t-test, ** P=0.0011, n=13). c. Representative traces of action potentials recorded in IB4 positive DRG neurons from wild type and TMEM16C knockout rats.

a–c. Slack and TMEM16C are co-expressed in small DRG neurons. Arrow points to neurons that are doubly stained for TMEM16C and Slack; arrowhead points to a larger neuron immunostained only for TMEM16C. Staining has been repeated at 3 times.
–f. Overlapping Slack and TMEM16C nerve terminal immunoreactivity in the superficial dorsal horn. The bright green nuclear staining represents non-specific background signal (arrow) characteristic of the TMEM16C antibody. Staining has been repeated at 3 times.
g–h. Greatly reduced Slack immunostaining in the DRG of TMEM16C knockout (h) vs wild type (g) rats. i. Western blot of Slack in the spinal cord and DRG from wild type and TMEM16C knockout rats. Full-length gels/blots are shown in . j. Quantification of immunostaining intensity of Slack positive neurons in the wild type and knockout DRG (P<0.0001, Student’s t test, n=71 for wild type and n=80 for knockout).
k. Plot of the normalized ratio of the density of the Slack band relative to the density of the alpha–tubulin band (P=0.0008, Student’s t–test, n=4).

a. Representative traces demonstrate Cd²⁺ sensitive outward currents in wild type and knockout neurons, obtained by subtraction of currents before and after Cd²⁺ application. Recordings are made with the bath containing (mM): 140 NaCl, 5 KCl, 1 MgCl₂, 2 EGTA, 10 HEPES, 10 Glucose and the internal solution in the pipette (mM): 140 K-gluconate, 10 KCl, 10 HEPES, 0.2 EGTA, 4 Mg-ATP, 0.4 Na-GTP, and 5 BAPTA.
b. Sample current traces with Li⁺ replacement recorded from wild type and knockout DRG neurons. Right, currents recorded with NaCl in the bath. Middle, currents recorded with LiCl. Left, KNa currents obtained by current subtraction. Recordings are made with the solution containing (mM): 140 NaCl, 5 KCl, 1 MgCl₂, 2.5 CaCl₂, 10 HEPES, 10 Glucose, and then the bath is switched to LiCl based solution (mM): 140 LiCl, 5 KCl, 1 MgCl₂, 2.5 CaCl₂, 10 HEPES, 10 Glucose. The pipette solution includes (mM): 140 K-gluconate, 10 KCl, 10 HEPES, 0.2 EGTA, 4 Mg-ATP, 0.4 Na-GTP
c. The I–V curve of the current density of the Cd²⁺ sensitive outward currents in the wild type and knockout DRG neurons (paired t-test, P=0.0040, n=7).
d. The I–V relationship of KNa current density in Li⁺ replacement experiments from wild type and knockout neurons (paired t-test, p<0.0233, n=7 and 9).

a .The left panel shows representative traces of inside-out excised patch recordings at –40 mV from HEK293 cells expressing Slack in the solution containing 0 mM Na⁺ (with 140 mM [K⁺]_o / 60 mM [K⁺]_i, 0 mM [Na⁺], 80 mM [Choline]), 10 mM Na (140 mM [K⁺]_o / 60 mM [K⁺]_i, 10 mM [Na]_i, 70 mM [Choline]_i) and 40 mM Na⁺ (140 mM [K⁺_o/ 60 mM [K]_i, 40 mM [Choline]_i); The right panel shows the all point histograms. The subconductance is indicated by arrows. Similar results were obtained in 12 cells with Slack expression.
b. Representative traces and all point histogram from excised patch recordings at –40 mV from HEK293 cells expressingTMEM16C and Slack in solutions containing 0 mM, 10 mM or 40 mM Na⁺. The two dashed red lines in the all point histogram indicate the closed (C) and full open state (O). Note the full conductance peak at 0 mM and 10 mM Na⁺. Similar results were obtained in 12 cells.
c. I–V curve of KNa single channels in inside-out patches excised from cells with or without TMEM16C co-expression, exposed to 0 mM Na⁺ solution and at two different sets of intracellular solutions: 140 mM [K⁺] and 140 mM [K⁺ _o]with E_rev 0 mV and 140 mM [K⁺]

a. Immunostaining for TMEM16C in wild type rat DRG. b. IB4-FITC staining in the wild type DRG. c, Overlay of a and b. (Arrow points to a double-labeled neuron and arrowhead marks one cell stained only for TMEM16C). d. Quantification of the percentage of TMEM16C positive neurons co–stained with IB4 (total of 170 TMEM16C positive neurons) and/or NF200 (total of 166 TMEM16C positive neurons). e. Absence of TMEM16C immunoreactivity in the DRG of a TMEM16C knockout rat; the bright red dots represent non-specific staining of neuronal nuclei (example pointed out by an arrow). f. IB4-FITC staining in the knockout DRG. g. Overlay of c and d.
h. Quantification of the percentage of IB4 positive neurons co-stained with TMEM16C, shows that at least 88% of IB4-positive neurons express TMEM16C (total of 125 IB4 positive neurons). i. Immunostaining of TMEM16C in the superficial dorsal horn of the spinal cord of wild type rats. j. IB4-FITC staining in the same region of spinal cord. k. Colocalization of TMEM16C in the IB4 positive nerve terminals in the spinal cord. Insert is a higher magnification image from the superficial dorsal horn. l–n. Absence of TMEM16C immunoreactivity in the IB4 positive nerve terminals in the spinal cord of TMEM16C knockout rats. Note the non-specific staining of the nuclei (bright red dots) with TMEM16C antibodies. o–r: Triple immunostaining of the DRG shows that approximately 50% of TrpV1 positive neurons express TMEM16C and those neurons are all IB4 positive. White arrow marks a neuron triple-labeled for 16C, IB4 and TrpV1; blue arrow points to a neuron stained with TrpV1 only; and yellow arrow points to a neuron stained with 16C and IB4, but not TrpV1; white arrowhead points to an example of non-specific staining of the nuclei with the TMEM16C antibody.
WT: wild type; KO: knockout. Each staining has been repeated at least 3 times.

a. Slack protein co-immunoprecipitates with TMEM16C using antibodies against HA for pull-down of associated proteins in lysates from cotransfected HEK293 cells. Note the absence of a band in the control IP from cells transfected only with Slack. Experiments were repeated 4 times. Full-length gels/blots are shown in .
b. Slack protein co-immunoprecipitates with TMEM16C; antibodies against TMEM16C were used for pull-down of associated proteins in lysates from brain tissue. Note the much fainter
band of Slack in the IP from TMEM16C KO rat tissue with the same amount of input in WT and KO. Experiments were repeated 3 times. Full-length gels/blots are shown in .
c. Slack esiRNA successfully knocks down Slack protein expression when co-transfected in HEK293 cells. Experiments were repeated 3 times. Full-length gels/blots are shown in .
d and e. Intrathecal injection of Slack esiRNA exhibit enhanced thermal pain sensitivity at 50°C in the hot plate test, but not at 52.5°C (P=0.0006, Two Way ANOVA followed by Bonferroni's post hoc test, n=7 for control and n=8 for esiRNA) and increased mechanical sensitivity in the electronic Von Frey test (P=0.0247, Student's test, n=7 and 8).
f. Quantification of Slack immunostaining intensity in IB4 positive neurons in L4 and L5 DRGs from rats with control esiRNA and Slack esiRNA intrathecal injection (65.77 ± 1.54, n=185 vs 40.11 ± 1.35, n=171, P<0.0001, Student's t-test).