Distinct Mechanisms Account for In Vitro Activation and Sensitization of TRPV1 by the Porphyrin Hemin

Abstract

TRPV1 mediates pain occurring during sickling episodes in sickle cell disease (SCD). We examined if hemin, a porphyrin released during intravascular hemolysis modulates TRPV1. Calcium imaging and patch clamp were employed to examine effects of hemin on mouse dorsal root ganglion (DRG) neurons and HEK293t cells expressing TRPV1 and TRPA1. Hemin induced a concentration-dependent calcium influx in DRG neurons which was abolished by the unspecific TRP-channel inhibitor ruthenium red. The selective TRPV1-inhibitor BCTC or genetic deletion of TRPV1 only marginally impaired hemin-induced calcium influx in DRG neurons. While hTRPV1 expressed in HEK293 cells mediated a hemin-induced calcium influx which was blocked by BCTC, patch clamp recordings only showed potentiated proton- and heat-evoked currents. This effect was abolished by the PKC-inhibitor chelerythrine chloride and in protein kinase C (PKC)-insensitive TRPV1-mutants. Hemin-induced calcium influx through TRPV1 was only partly PKC-sensitive, but it was abolished by the reducing agent dithiothreitol (DTT). In contrast, hemin-induced potentiation of inward currents was not reduced by DTT. Hemin also induced a redox-dependent calcium influx, but not inward currents on hTRPA1. Our data suggest that hemin induces a PKC-mediated sensitization of TRPV1. However, it also acts as a photosensitizer when exposed to UVA-light used for calcium imaging. The resulting activation of redox-sensitive ion channels such as TRPV1 and TRPA1 may be an in vitro artifact with limited physiological relevance.

Sickle cell disease patient plasma sensitizes iPSC-derived sensory neurons from sickle cell disease patients

“…This suggests a role of plasma factors in overcoming the desensitization of untreated SCD iSNs to TRPV1 agonism, perhaps through localization or sensitization of TRPV1 channels at the plasma membrane 83,84 . Increased ET1 73,74 and factors associated with hypoxia (H 2 O 2 , TNFα, IL6) 85,86 , hemolysis (hemin) 87,88 , and acidosis (H + ) 89,90 are proposed sensitizers of TRPV1 and are also increased during acute SCD pain 91–93 .…”

Molecular mechanisms of MrgprA3‐independent activation of the transient receptor potential ion channels TRPA1 and TRPV1 by chloroquine

“…cDNA of MrgprA3 was a kind gift from Dr. Xinzhong Dong (Baltimore, MD, USA). HEK 293 cells with a stable expression of TRPA1 and TRPV1 were cultured and used as previously described (Palmaers et al, 2021). CHO cells with a stable expression of mTRPA1 were kindly provided by Dr. Ardem Patapoutian (San Diego, CA, USA).…”

“…Dorsal root ganglion (DRG) neurons from C57Bl/6 wild type mice were employed as described previously (Palmaers et al, 2021). Mice were anaesthetized by isoflurane and killed by decapitation.…”

“…CHO cells with a stable expression of mTRPA1 were kindly provided by Dr. Ardem Patapoutian (San Diego, CA, USA). Dorsal root ganglion (DRG) neurons from C57Bl/6 wild type mice were employed as described previously(Palmaers et al, 2021). Mice were anaesthetized by isoflurane and killed by decapitation.…”