Localization of the PIP2 Sensor of TRPV1 Ion Channels

Abstract

Although a large number of ion channels are now believed to be regulated by phosphoinositides, particularly phosphoinositide 4,5-bisphosphate (PIP2), the mechanisms involved in phosphoinositide regulation are unclear. For the TRP superfamily of ion channels, the role and mechanism of PIP2 modulation has been especially difficult to resolve. Outstanding questions include: is PIP2 the endogenous regulatory lipid; does PIP2 potentiate all TRPs or are some TRPs inhibited by PIP2; where does PIP2 interact with TRP channels; and is the mechanism of modulation conserved among disparate subfamilies? We first addressed whether the PIP2 sensor resides within the primary sequence of the channel itself, or, as recently proposed, within an accessory integral membrane protein called Pirt. Here we show that Pirt does not alter the phosphoinositide sensitivity of TRPV1 in HEK-293 cells, that there is no FRET between TRPV1 and Pirt, and that dissociated dorsal root ganglion neurons from Pirt knock-out mice have an apparent affinity for PIP2 indistinguishable from that of their wild-type littermates. We followed by focusing on the role of the C terminus of TRPV1 in sensing PIP2. Here, we show that the distal C-terminal region is not required for PIP2 regulation, as PIP2 activation remains intact in channels in which the distal C-terminal has been truncated. Furthermore, we used a novel in vitro binding assay to demonstrate that the proximal C-terminal region of TRPV1 is sufficient for PIP2 binding. Together, our data suggest that the proximal C-terminal region of TRPV1 can interact directly with PIP2 and may play a key role in PIP2 regulation of the channel.TRPV1 ion channels are capsaicin-, heat-, and acid-activated non-selective cation channels expressed in nociceptors of the peripheral nervous system as well as in the neurons of the hippocampus and cortex (1). TRPV1 is an essential component of inflammatory hyperalgesia, as TRPV1 knock-out mice show essentially no hyperalgesia in response to thermal and chemical stimuli during inflammation (2). The role of TRPV1 in nociception has made it an attractive target for pain therapies. In addition, the large physical size of its pore has been shown to allow cationic local anesthetics to enter nociceptors (3), raising the possibility of identifying more specific analgesics that do not interfere with non-painful sensation.Like many ion channels, TRPV1 is regulated by G-proteincoupled receptors (GPCR) 3 and receptor-tyrosine kinases (RTK), both of which generally sensitize nociceptors (4 -5). Bradykinin, a GPCR ligand, and nerve growth factor, an RTK ligand, are released in response to tissue injury and sensitize TRPV1 to subsequent activation by noxious stimuli (6). Because both GPCRs and RTKs can activate phospholipase C (PLC), degradation of PI(4,5)P 2 (PIP2) by PLC was originally proposed to constitute the common mechanism for bradykinin-and nerve growth factor-mediated sensitization of TRPV1 (6). In this PLC model of hyperalgesia, PIP2 is tonically bound to TRPV1 and inhibits chann...

Carboxyl-terminal Domain of Transient Receptor Potential Vanilloid 1 Contains Distinct Segments Differentially Involved in Capsaicin- and Heat-induced Desensitization

“…The TRPV1 CTD is also implicated in cap-saicin-induced desensitization through its ability to bind calmodulin (CaM) (17)(18)(19). The "TRP domain" within the TRPV1 CTD was also suggested to be involved in capsaicin-induced desensitization (20,21). Given the fact that there are regions and residues of TRPV1 that are distinctly involved in activation by capsaicin and heat and that capsaicin and heat desensitize TRPV1 through apparently distinct mechanisms, we hypothesized that capsaicin and heat desensitize TRPV1 through distinct segments within the CTD.…”

Exploring Functional Roles of TRPV1 Intracellular Domains with Unstructured Peptide-Insertion Screening

“…Among them, Ca 2+ ions are important because not only the effects are substantial and rapid but also Ca 2+ influx through activated channels provides a powerful physiological negative feedback mechanism 10 . Modulation of channel activity by intracellular Ca 2+ is mediated by multiple mechanisms utilizing calmodulin 34,35,41,42 , Phosphorylation 43 , and PIP2 [44][45][46][47][48] . As F430_3aa and E693_8aa exhibit specific and substantial deficits in Ca 2+ -dependent desensitization, they may serve as useful tools to further investigate the allosteric coupling process between the intracellular domains and the transmembrane core domain.…”

“…Extended recordings, however, revealed that there was a substantial difference in the current decline phase. The current decline represents Ca 2+ -dependent acute desensitization, a process thought to be mediated by multiple mechanisms, including binding of Ca 2+ -calmodulin to the ankyrin-like repeat domain 34,35,41,42 , phosphorylation 43 , and binding of PIP2 to the C-terminus [44][45][46][47][48] , that allosterically modulate the stability of the open pore conformation. For the wild-type channel, as expected little desensitization was observed in the absence of Ca 2+ .…”

Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels

“…PI(4,5)P 2 has been proposed to inhibit TRPV1 by binding to amino acids 777-820 in the distal C-terminal region (Prescott and Julius, 2003) and to activate TRPV1 by binding to the proximal C-terminal region represented by amino acids 682-725 ( Fig. 1 A, magenta; Ufret-Vincenty et al, 2011). In addition, mutations in the ankyrin repeat domains appear to abrogate PI(4,5)P 2 depletion-mediated desensitization of TRPV1, although a direct interaction between PI(4,5)P 2 and a fragment corresponding to the ankyrin selectivity.…”

“…Based on this structure, it seems reasonable to predict that GIRK2 might display some phosphoinositide Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels the response of pain receptor neurons to noxious stimuli, disagreement about whether PI(4,5)P 2 activates or inhibits TRPV1 continues (Chuang et al, 2001;Prescott and Julius, 2003;Stein et al, 2006;Lukacs et al, 2007Lukacs et al, , 2013a; Ufret-Vincenty et al, 2011;Cao et al, 2013a,b;Senning et al, 2014). There is also disagreement about whether PI(4,5)P 2 interacts directly with TRPV1 or acts via the integral membrane protein Pirt (Kim et al, 2008;Ufret-Vincenty et al, 2011). Finally, several regions of TRPV1 have been proposed for mediating the interaction with PI(4,5)P 2 , some of which are illustrated in Fig.…”