Supplemental Figure S1
Screening of 33 Trp channels revealed expression of candidate TRP in the SCN of adult mouse brain. Autoradiograms show mRNA expression in the SCN of A, PKR2, B, TrpV2, C, TrpC1, D, TrpC2, E, TrpC4, F, TrpC5, G, PKDILI-1, H, TrpM7, I, PKD1, J, PKD2, K, TrpV3.
Figure 1
Colocalization of PKR2 mRNA and TrpV2 protein expression in SCN. A, PKR2 mRNA and B, TrpV2 protein in SCN neurons of WT mouse. C, Colocalization of TrpV2 and PKR2 in A and B. Scale bars=50 µm. D-F, Confocal images of colocalization in the SCN neurons, 100x magnification. TrpV2 protein (red); PKR2 mRNA (green).
Supplemental Figure S2
Absence of oscillation of TrpV2 mRNA in the SCN. A, PK2 mRNA oscillates over a 24 hr period in the SCN. B, TrpV2 mRNA remains unchanged over 24 hr period. C, Quantification by MCID analysis of PK2 and TrpV2 mRNA. Animals were subjected to constant darkness for 2 days. Each value is the mean of 4–6 animals. Solid squares, PK2; open squares, TrpV2. Scale bar=1 mm.
Figure 2
Oscillation of TrpV2 protein expression in SCN of WT and PK2 KO mice. A, Coexpression of TrpV2 protein and PKR2 mRNA in the SCN of WT mouse at CT4. TrpV2 protein expression is reduced in the SCN of WT mouse SCN at CT16 (B). TrpV2 level are similar in the SCN of PK2 KO mouse at CT4 (C), and CT16 (D). Images of left column are of TrpV2 protein (red) alone; images of right column are of TrpV2 protein and PKR2 mRNA (green). E, Graphical representation of TrpV2 protein level in the SCN of WT and PK2 KO mice at CT4 and CT16 ***p<0.0001. Scale bar=500 µm.
Figure 3
PK2-induced Ca2+ mobilization of PKR2:TrpV2- expressing CHO cells. A, PKR2 and B, PKR2:TrpV2 CHO cell Ca2+ mobilization in Ca2+ free HBSS. Initial injection at 1 sec of either HBSS and PK2 followed by 3 mM CaCl2 injection at 30 sec. C, Graphical representation of area under the curve (AUC) for calcium peak, ***p<0.0001. D, PKR2-expressing CHO were unaffected by 3 min pretreatment of increasing doses of ruthenium red (RR), Results are ± SEM from 5–6 separate experiments. E, RR dose-dependently decreased the calcium mobilization inPKR2:TrpV2-expressing CHO cells. F, Calcium mobilization was significantly increased by the expression of TrpV2, which was inhibited by RR.
Figure 4
PK2-evoked current in oocytes that coexpress PKR2:TrpV2. A, PK2 (50 nM)-evoked currents in PKR2- (left) and PKR2:TrpV2-expressing (right) oocytes. Graph inset displays the significance in peak amplitude, ***p<0.0001. PKR2 injected, n=15; PKR2:TrpV2 co-injection, n=11. B, Oocytes expressing PKR2:TrpV2 were pretreated with RR (3 min). Graph inset shows the decrease in peak amplitude with increasing doses of RR. **p<0.01, *p<0.05; n=20.
Figure 5
PK2-induced cell surface translocation and large cluster formation of TrpV2 in CHO cells. All cells were serum-starved and subsequently treated with PK2 or PK2 antagonist. Cells were then fixed at indicated time points and immunostained using a rabbit-anti-TrpV2 antibody. A, no PK2 added, B, 30 min, C, 60 min, D, 90 min, E, 120 min. Pretreatment with F, PKR antagonist (PKRA7). Scale bar=40 µm. G, Higher magnification images (63x) of individual CHO cells. Left to right equates to images A-F. H, Percentage of cells showing translocation of TrpV2 in response to PK2. ***p<0.001, #p<0.05.
Supplemental Figure S3
Z-stacking confocal imaging of TrpV2 translocation to surface membrane. A, no PK2 treatment. CHO Cells were treated with PK2 for 10 min B, 10 min, C, 30 min, D, 60 min, E, 90 min, F, 120 min. Note the apparent cell surface expression of TrpV2 in response to PK2 treatment. Cell surface expression of TrpV2 was detected by immunostaining with anti-TrpV2 antibody. Scale bar=40 µm.