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. 2021 Aug 1;106(8):2246-2250.
doi: 10.3324/haematol.2020.274951.

The TRPV2 channel mediates Ca2+ influx and the Δ9-THC-dependent decrease in osmotic fragility in red blood cells

Anouar Belkacemi  1 , Claudia Fecher Trost  1 , René Tinschert  1 , Daniel Flormann  2 , Mahsa Malihpour  1 , Christian Wagner  3 , Markus R Meyer  4 , Andreas Beck  1 , Veit Flockerzi  5
Affiliations

The TRPV2 channel mediates Ca2+ influx and the Δ9-THC-dependent decrease in osmotic fragility in red blood cells

Anouar Belkacemi et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
TRPV2 protein in mouse red blood cells. (A, B) Western blot of wild-type, Trpv2 knockout (KO) (A) and Trpc6 KO (B) red blood cell (RBC) proteins, using antibodies against mouse TRPV2, TRPC6 and b-actin. (C) Semi-quantitative analysis of differentially expressed proteins identified by mass spectrometry in wildtype and Trpv2 KO RBC lysates. Up- and down-regulated proteins were identified based on at least 2-fold changes with a P-value <0.05, calculated by an unpaired two-tailed Student t test. The heatmap shows the Z-scores of the exponentially modified protein abundance index (emPAI) values of mass spectrometry measurements of five independent wild-type and four independent Trpv2 KO samples. (D) Relative abundance of the TRPV2 protein compared to that of the 1,450 proteins identified in mouse erythrocyte membrane fractions. Rank represents the order of the identified protein obtained by spectral counting with the P-value calculated by an unpaired Student t test. NA, not applicable. (E) Hematologic parameters of the blood from wild-type and Trpv2 KO. RBC: red blood cell; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; RDW: red cell distribution width with P-value calculated by an unpaired two-tailed Student t test. (F) Hemolysis (%) of RBC collected from wild-type (black) and Trpv2 KO mice (red) in buffer A (149 mM NaCl, 2 mM CaCl2, 4 mM KCl, 2 mM HEPES, pH7.4), diluted 26-fold in buffer B (0-149 mM NaCl, 2 mM HEPES, pH 7.4) as indicated; extracellular [Ca2+] was kept at ~76 mM. (G) Tonicity at which 50% lysis occurred (C50), calculated by sigmoidal fitting from experiments in (F). Single values and mean ± standard error of mean from five independent experiments performed in triplicate are shown with the P-value calculated by an unpaired twotailed Student t test.
Figure 2.
Figure 2.
TRPV2 function in mouse red blood cells. (A-D) In- and outward currents at -80 and +80 mV shown as mean ± standard error of mean (SEM), recorded from mouse red blood cells (RBC) using a patch pipette (A) or a miniaturized patch clamp system (port-a-patch) (C) plotted versus time (number of cells in brackets). TRPV2 currents were activated by the application of 2-APB (black line) in the absence and presence of 10 mM ruthenium red (RR, blue line) with the corresponding current-voltage relationships (IV) at the peak net currents (Imax net), shown as mean ± SEM in (B) and (D). Patch pipette resistances were 10-15 MΩ when filled with standard internal solution (in mM): 120 Cs-glutamate, 8 NaCl, 1 MgCl2, 10 HEPES, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetracesium salt (Cs-BAPTA), 3.1 CaCl2 (100 nM free Ca2+, calculated with WebMaxC), pH7.2 with CsOH. Standard external solution contained (in mM): 140 NaCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 10 glucose, pH 7.2 with NaOH. For experiments with the miniaturized patch system, the intracellular solution contained (in mM): 60 Cs-methansulfonate, 8 NaCl, 1 MgCl2, 3.1 CaCl2, 60 CsF, 10 HEPES, 10 BAPTA (100 nM free Ca2+, calculated with WebMaxC), 10 glucose, pH 7.2 with CsOH and the extracellular solution contained (in mM): 140 NaCl, 2 MgCl2, 1.35 CaCl2, 10 HEPES, 10 glucose, pH 7.2 with NaOH. (E, G) Mean Fluo-4 fluorescence (F/F0) traces showing changes in the cytosolic [Ca2+] of RBC isolated from wild-type (black) and Trpv2 KO mice (red) in the absence (E) and presence (G, blue) of the CB1/CB2-receptor antagonists AM251 and JTE907 (100 nM each), challenged by the application of 30 mM Δ9-tetrahydrocannabinol (Δ9-THC, line). Ca2+-imaging measurements were performed in the presence of a Tyrode solution (in mM): 135 NaCl, 5.4 KCl, 1 MgCl2, 10 HEPES, 10 glucose, and 1.8 CaCl2, pH 7.35; RBC were loaded with 5 μM Fluo-4 and the fluorescence was excited at 488 nm every 3 seconds with the emitted fluorescence detected at >515 nm. (F, H) Summary of peak amplitudes from (E) and (G) shown as mean ± SEM with P-values calculated by the unpaired two-tailed Student t test (ns, not significant). Numbers of measured cells (x) within (y) independent experiments are indicated in brackets and bars.
Figure 3.
Figure 3.
TRPV2 protein and function in human red blood cells. (A) Western blot of protein lysates of COS-7 cells transfected with human TRPV2 cDNA, the cDNA of green fluorescent protein (GFP) as a control, and human red blood cells (RBC) using anti-human TRPV2. (B, C) Representative traces of cytosolic Ca2+ changes, detected as Fluo-4 fluorescence (F/F0), in human RBC challenged by the application of 100 mM cannabidiol (CBD) (B) or 30 mM Δ9-tetrahydrocannabinol (Δ9- THC) (C). (D) Summary of the peak amplitudes in (B) and (C) as mean ± standard error of mean (SEM) (78 and 82 cells measured in 3 independent experiments each). (E) In- and outward currents at -80 and +80 mV in the absence and presence of Δ9-THC (black line) recorded from human RBC and plotted versus time. The corresponding current-voltage relationships (IV) of the basic current (Imin) and the peak net current in Δ9-THC (Imax net) are depicted in (F) and (G). Data are shown as mean ± SEM (number of cells indicated in brackets). (H) Confocal microscopic images of human RBC not treated (control, left) or treated with 100 mM cannabidiol (CBD, middle) or 30 mM Δ9-THC (right). (I) Bar graphs showing the percentage of discocytes (black), stomatocytes (red) and spherocytes (blue) as mean ± SEM, from three independent healthy donors, treated as in (H), with P-values calculated by one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparison. The classification was done with 3-D stacks of confocal images. (J) Hemolysis (%) of human RBC treated with the vehicle (control, black), 30 mM Δ9-THC (gray open circle) or 30 mM Δ9-THC in the presence of 100 nM CB1- and CB2-receptor antagonists AM251 and JTE907 (Δ9-THC + AM + JTE, blue) plotted versus the extracellular NaCl concentration (mM) and respective tonicity (%), with extracellular [Ca2+] kept at 76 mM as described in Figure 1F. (K) Tonicity at which 50% lysis occurred (C50), calculated by sigmoidal fitting of the individual experiments in (J). (L) Hemolysis (%) of human RBC in buffer A (149 mM NaCl, 2 mM CaCl2, 4 mM KCl, 2 mM HEPES, pH 7.4), treated with vehicle (control, black), 30 mM Δ9-THC (gray open circle), 2 mM TRAM-34 (green) or 30 mM Δ9-THC plus TRAM-34 (red) for 30 min, after 26-fold dilution in buffer B (0-149 mM NaCl, 2 mM CaCl2, 4 mM KCl, 2 mM HEPES, pH 7.4); extracellular [Ca2+] was kept at 2 mM. (M) Tonicity at which 50% lysis occurred (C50), calculated by sigmoidal fitting of the individual experiments in (L). Data in (K) and (M) are shown as means ± SEM from two independent experiments performed in triplicate with the P-value calculated by one-way ANOVA, followed by the Bonferroni multiple comparison. (N) Working model for how TRPV2, activated by Δ9-THC, modulates the TRAM-34 sensitive KCa3.1 activity in a human RBC. Note that TRPC6 was not detectable in human RBC.
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References

    1. Agre P, Preston GM, Smith BL, et al. . Aquaporin CHIP: the archetypal molecular water channel. Am J Physiol. 1993;265(4 Pt 2):F463-476. - PubMed
    1. Cahalan SM, Lukacs V, Ranade SS, Chien S, Bandell M, Patapoutian A. Piezo1 links mechanical forces to red blood cell volume. Elife. 2015;4:e07370. - PMC - PubMed
    1. Maher AD, Kuchel PW. The Gardos channel: a review of the Ca2+- activated K+ channel in human erythrocytes. Int J Biochem Cell Biol. 2003;35(8):1182-1197. - PubMed
    1. Foller M, Kasinathan RS, Koka S, et al. . TRPC6 contributes to the Ca2+ leak of human erythrocytes. Cell Physiol Biochem. 2008;21(1-3):183-192. - PubMed
    1. Hirschler-Laszkiewicz I, Zhang W, Keefer K, et al. . Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis. Exp Hematol. 2012;40(1):71-83. - PMC - PubMed

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