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Letters to the Editor
The TRPV2 channel mediates Ca2+ influx and the Δ9-THC-dependent decrease in osmotic fragility in red blood cells
Water and ionic homeostasis of red blood cells (RBC) is regulated by various active and passive transport mecha- nisms in the RBC membrane, including channels like aqua- porins,1 the mechanically activated non-selective cation channel Piezo12 and the Ca2+-activated potassium channel KCa3.1.3 The human genome contains 27 genes that code for transient receptor potential (TRP) channels. The only TRP channel protein that has been detected in circulating mouse RBC is TRPC6,4 which might be associated with basal Ca2+ leakage and stress-stimulated Ca2+ entry.4 TRPC2 and TRPC3 are expressed by murine erythroid pre- cursors and splenic erythroblasts, and in these cells, ery- thropoietin stimulates an increase in intracellular calcium concentration via TRPC2 and TRPC3.5 In this study we identified the TRP vanilloid (TRPV) 2 channel protein in mouse and human RBC by specific antibodies and mass spectrometry. TRPV2-dependent currents and Ca2+ entry were activated by the TRPV2 agonists cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC)6 resulting in a left- shift of the hypotonicity-dependent hemolysis curve. This effect was reversed in the presence of the KCa3.1 inhibitor TRAM-34, whereas the knockout of Trpv2 right-shifted the hemolysis curve to higher tonicities.
We separated mouse RBC from other blood cells by cen- trifugation and analyzed protein lysates by nanoflow liq- uid chromatography tandem mass spectrometry (nano- LC-MS/MS). The identified proteins included TRPV2 (Online Supplementary Figure S1A). To enrich the TRPV2 protein we generated an antibody which recognizes the TRPV2 protein in RBC from wild-type (WT) animals but not in RBC from Trpv2 gene-deficient (KO) mice (Figure 1A). As an additional control, we used anti-TRPC6 anti- body and identified TRPC6 in RBC (Figure 1B). Total elu- ates of anti-mTRPV2 affinity purifications from RBC mem- branes of WT mice were analyzed by nano-LC-MS/MS, which retrieved peptides covering 54% of the accessible TRPV2 primary sequence (Online Supplementary Figure S1B).
To obtain a more comprehensive protein profile, we lysed WT and Trpv2-KO RBC, extracted the proteins, and measured the resulting tryptic peptides by nano-LC- MS/MS. A total of 1,450 proteins were identified (Online Supplementary Figure S1E), with TRPV2 present in all WT samples. Eighty-seven of the identified proteins were detected exclusively or with more than a 2-fold increase in WT RBC, while 13 proteins were detected with more than a 2-fold increase in Trpv2-KO RBC (Figure 1C, Online Supplementary Figure S1F) by semiquantitative exponen- tially modified protein abundance index (emPAI) analysis. Next, we evaluated the frequency of the identified pro- teins by spectral counting and normalized the data to band 3 (Figure 1D). TRPV2 ranked at position 560, about 0.4% of band 3, 50% and 84% less than ferroportin and Piezo1, respectively. In addition to TRPV2 and Piezo1, other chan- nels such as aquaporin1 and transmembrane channel like 8 (Online Supplementary Figure S1D) were identified. The KCa3.1 protein, on the other hand, seemed to be much less abundant, as we could identify only one KCa3.1 pep- tide in our experiments, which was below the threshold for unambiguous protein identification.
According to the proteomic profiling, Piezo1 and aqua- porin1 proteins were present in equal amounts in murine RBC from Trpv2-KO and WT animals. In contrast, several proteins that affect ion and fluid homeostasis were signif-
icantly less abundant in Trpv2-KO RBC, including the STE20-like- and the WNK1-serine/threonine protein kinases SLK (in humans also dubbed SPAK) and WNK1 (Figure 1C). Both kinases regulate the Na+-K+-Cl– sym- porter NKCC1 present in the erythrocyte membrane, resulting in the flux of NaCl and KCl into the cell with sub- sequent rehydration.7 This mechanism would be attenuat- ed in Trpv2-KO RBC with decreased WNK1, similar to renal cells that are also equipped with NKCC1 and WNK1 and in which WNK1 is inhibited under hypotonic condi- tions. Likewise, the significantly reduced amount of the casein kinase II (CKII)-α subunit (CSK21) in Trpv2-KO RBC (Figure 1C) could be part of mechanisms that com- pensate for the absence of TRPV2, as pharmacological inhibition of CKII-α causes shrinkage of RBC.8
Hematologic parameters from blood of Trpv2-KO and WT animals (Figure 1E), were not significantly different. However, when RBC were exposed to hypotonic solu- tions, keeping extracellular [Ca2+] at 76 mM, hemolysis of Trpv2-KO RBC occurred at higher tonicity (Figure 1F). The relative tonicity at half maximal lysis (C50) (Figure 1G) was 49.19±0.62 (WT, n=5) and 53.7±0.68 (Trpv2-KO, n=5; P<0.0001).
To isolate TRPV2 currents from murine RBC we applied the non-specific TRPV2 agonist 2-APB. Inward and out- ward currents with the outwardly rectifying current-volt- age (IV) relationship typical of TRPV2 currents were recorded by whole cell patch-clamping (Figure 2A, B) or by a miniaturized patch system (Figure 2C, D). A fraction of these 2-APB-induced currents was blocked by ruthenium red. Similar, but much larger currents were recorded from COS-7 cells, which overexpress the murine Trpv2 cDNA (Online Supplementary Figure S2E). Upon application of 2- APB, cytoplasmic [Ca2+] increased in WT RBC (Online Supplementary Figure S2A). The Ca2+ increase was blocked in the presence of ruthenium red but could also be induced in Trpv2-KO RBC (Online Supplementary Figure S2B-D). 2- APB blocks TRPC6 and KCa3.1 present in RBC and acts on additional targets.9 Thereby it may affect the RBC membrane potential and Ca2+-signaling pathways inde- pendently of TRPV2 during monitoring cytoplasmic Ca2+. As shown in COS-7 cells (Online Supplementary Figures S2E-I and S3A), which do not endogenously express TRPC6 or KCa3.1, the 2-APB-induced increase in cytosolic Ca2+ and plasma membrane currents required the presence of overexpressed mouse or human TRPV2. We therefore applied the more specific TRPV2 agonist Δ9-THC, which elicited Ca2+ influx in WT RBC; this influx was significant- ly reduced in Trpv2-KO RBC (Figure 2E, F) indicating that part of the Ca2+ increase was mediated by TRPV2. The antagonists of the G protein-coupled cannabinoid recep- tors type 1 (CB1) and type 2 (CB2), AM251 (AM) and JTE907 (JTE), had no effect on the Δ9-THC-elicited Ca2+- response in WT RBC (Figure 2G, H), demonstrating that TRPV2 mediates a significant fraction of THC-elicited Ca2+ influx and that the action of THC on TRPV2 is direct, and not mediated by CB1 or CB2 receptors.
The primary sequences of human and mouse TRPV2 are 80.4% identical, but the antibody against mTRPV2 does not recognize the hTRPV2 protein. We therefore generat- ed an antibody that recognizes the hTRPV2 protein by western blot (Figure 3A). Next, total eluates of anti- hTRPV2 affinity purifications from RBC membranes were analyzed by nano-LC-MS/MS that retrieved peptides cov- ering 54.8% of the hTRPV2 primary sequence (Online Supplementary Figure S1G). In similar experiments but with an antibody for hTRPC6, TRPC6 was not detectable in human RBC by either western blot or nano-LC-MS/MS.
The cannabinoid TRPV2 agonists CBD and Δ9-THC
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haematologica | 2021; 106(8)