Water and ionic homeostasis of red blood cells (RBC) is regulated by various active and passive transport mechanisms in the RBC membrane, including channels like aquaporins, 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 precursors and splenic erythroblasts, and in these cells, erythropoietin 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 leftshift 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 centrifugation and analyzed protein lysates by nanoflow liquid 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 antibody and identified TRPC6 in RBC (Figure 1B). Total eluates of anti-mTRPV2 affinity purifications from RBC membranes 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-LCMS/ 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 exponentially modified protein abundance index (emPAI) analysis. Next, we evaluated the frequency of the identified proteins 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 channels 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 peptide in our experiments, which was below the threshold for unambiguous protein identification.

According to the proteomic profiling, Piezo1 and aquaporin1 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 significantly 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^– symporter NKCC1 present in the erythrocyte membrane, resulting in the flux of NaCl and KCl into the cell with subsequent rehydration.⁷ This mechanism would be attenuated 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 conditions. 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 compensate 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 solutions, 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 outward currents with the outwardly rectifying current-voltage (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 independently 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 significantly 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 receptors 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 generated an antibody that recognizes the hTRPV2 protein by western blot (Figure 3A). Next, total eluates of antihTRPV2 affinity purifications from RBC membranes were analyzed by nano-LC-MS/MS that retrieved peptides covering 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 elicited Ca²⁺ influx in human RBC (Figure 3B-D). Inward and outward currents with the outward rectifying IV relation were obtained by patch clamp recordings from human RBC after application of Δ9-THC (Figure 3E-G). Although the currents obtained from human RBC have a small amplitude, their IV match the TRPV2 current signature obtained from COS-7 overexpressing human TRPV2 cDNA upon application of Δ9-THC or CBD (Online Supplementary Figure S3B, C).

Assessment by confocal microscopy revealed that 95.3±2.4% of the human RBC had a biconcave discshaped form. Adding CBD or Δ9-THC shifted the morphology of these biconcave discocytes to concave RBC, the stomatocytes, which in the presence of CBD and Δ9-THC make up 92.7±1.3% (CBD) and 66.3±17.1% (THC) of the total RBC, the remaining cells being discocytes and more spherical-shaped spherocytes (Figure 3H, I). The TRPV2 agonist-induced shape change of the RBC was maintained in the presence of the CB1 and CB2 antagonists (Online Supplementary Figure S3D, E), indicating that the major fraction of the cannabinoids’ effect on RBC morphology is mediated by TRPV2. After addition of Δ9-THC, human RBC showed reduced osmotic fragility, as demonstrated by the left-shifted hemolysis curve in response to the hypotonicity challenge, independently of whether cannabinoid receptor antagonists were absent or present (Figure 3J, K). Similarly, but to a lesser extent, Δ9-THC shifts the C⁵⁰ value after treating WT murine RBC to lower tonicities (C⁵⁰, in the absence, 49.05±1.53, and in the presence of Δ9-THC, 46.08±1.55). This effect was reversed by pretreatment with the KCa3.1 antagonist TRAM-34, in the presence of 76 mM (Online Supplementary Figure S3F, G) or 2 mM extracellular Ca²⁺ (Figure 3L, M). The data indicate that TRPV2, like Piezo1 and, maybe TRPC6, enables an influx of cations including Ca²⁺. The increase of intracellular Ca²⁺ by TRPV2 activates KCa3.1 which allows K⁺ efflux, resulting in the shift of the hemolysis curve. This shift does not occur in the presence of the KCa3.1 antagonist TRAM-34 (Figure 3L-N).

Stabilization of the RBC membrane against hypotonic hemolysis by Δ9-THC and CBD has been described¹⁰ and it has been shown that in the presence of Δ9-THC at concentrations of >15 mM almost all RBC assume a stomatocyte- like concave shape.¹¹ Some of those results were attributed to interactions between the hydrophobic, naturally occurring cannabinoids and the membrane lipids of the RBC. However, membrane partitioning experiments, electron spin resonance spectrometry and experiments with artificial liposomes of different compositions which were tested for the release of trapped markers in the presence of Δ9-THC, suggested additional mechanisms.^12-14

Stabilization of the RBC membrane against hypotonic hemolysis by Δ9-THC and CBD has been described¹⁰ and it has been shown that in the presence of Δ9-THC at concentrations of >15 mM almost all RBC assume a stomatocyte- like concave shape.¹¹ Some of those results were attributed to interactions between the hydrophobic, naturally occurring cannabinoids and the membrane lipids of the RBC. However, membrane partitioning experiments, electron spin resonance spectrometry and experiments with artificial liposomes of different compositions which were tested for the release of trapped markers in the presence of Δ9-THC, suggested additional mechanisms.^12-14

The data described in our study point to TRPV2 being a specific molecular target for Δ9-THC and CBD in RBC. Activation of the TRPV2 channel by the compounds present in the Cannabis sativa plant makes RBC more resistant to lysis in response to hypotonic solutions. Whether our data explain why hemp products have been used in folk medicine to treat malaria since ancient times¹⁵ needs to be shown by further studies.