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. 2021 Feb 18;106(8):2246–2250. doi: 10.3324/haematol.2020.274951

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.