Molecular Cloning of an N-terminal Splice Variant of the Capsaicin Receptor

613

457

We have compared activation by heat of TRPV4 channels, heterogeneously expressed in HEK293 cells, and endogenous channels in mouse aorta endothelium (MAEC). Increasing the temperature above 25°C activated currents and increased [Ca 2؉ ] i in HEK293 cells transfected with TRPV4 and in MAEC. When compared with activation of TRPV4 currents by the selective ligand 4␣PDD (␣-phorbol 12,13-didecanoate), heat-activated currents in both systems showed the typical biophysical properties of currents through TRPV4, including their single channel conductance. Deletion of the three N-terminal ankyrin binding domains of TRPV4 abolished current activation cells by heat in HEK293. In inside-out patches, TRPV4 could not be activated by heat but still responded to the ligand 4␣PDD. In MAEC, the same channel is activated under identical conditions as in the HEK expression system. Our data indicate that TRPV4 is a functional temperature-sensing channel in native endothelium, that is likely involved in temperature-dependent Ca 2؉ signaling. The failure to activate TRPV4 channels by heat in inside-out patches, which responded to 4␣PDD, may indicate that heat activation depends on the presence of an endogenous ligand, which is missing in inside-out patches.Sensing of temperature in the body and the environment is one of the most essential mechanisms for controlling the homeostasis of several regulatory pathways in the mammalian body (1). In recent years, unraveling of thermosensing mechanisms has been very successful, because at least four members of the TRPV subfamily of transient receptor potential cation channels, TRPV 1 1, -2, -3, and -4 and a more distantly related protein TRPM8 have been identified (for a unified nomenclature see Refs. 2 and 3) as sensors of temperature. Proteins of this subfamily typically contain 3-6 ankyrin repeats in the N terminus, and six transmembrane segments with a pore region between segments 5 and 6. The first identified non-mammalian member of this subfamily, the Caenorhabditis elegans OSM-9 channel, is activated by changes in osmolarity (4). The second protein of the TRPV family that has been identified is the mammalian vanilloid receptor channel VR1 (TRPV1), which is activated by vanilloid compounds such as capsaicin, pepper, hot chili, moderate heat, or protons (5). Unlike TRPV1, another close relative of this channel, TRPV2, is constitutively activated by growth factors (6) or by noxious heat (7). TRPV1 and TRPV2 are activated by temperatures above 43 and 52°C, respectively (5, 7). Currents through TRPV3 exponentially increase at temperatures above 35°C (8 -10). It has also been shown in current measurements on oocytes and by cytoplasmic Ca 2ϩ ([Ca 2ϩ ] i) measurements in HEK cells that TRPV4 is activated in both expression systems at temperature above 30°C (11). TRPM8 is activated by temperatures below 22°C and is therefore a candidate for cold reception (12, 13).So far, the molecular mechanism of channel activation by heat for any of these channels is not known, and functional measurements of ...

Section: Discussionmentioning

confidence: 99%

Heat-evoked Activation of TRPV4 Channels in a HEK293 Cell Expression System and in Native Mouse Aorta Endothelial Cells

Watanabe

Vriens

Suh

et al. 2002

613

457

“…Alternative splicing is a major contributor to protein diversity (50). Within the TRP family of ion channels several splice variants have been identified, some of them resulting in lack of responses to typical stimuli, others modifying the pore properties, and those exerting dominant negative effects (42,(51)(52)(53)(54). Group II TRPV4 splice variants have been identified in two unrelated, human airway epithelial cell lines (CFT1-LCFSN and HBE).…”

mentioning

confidence: 99%

Human TRPV4 Channel Splice Variants Revealed a Key Role of Ankyrin Domains in Multimerization and Trafficking

Arniges

Fernández-Fernández

Albrecht

et al. 2006

167

150

The TRPV4 cation channel exhibits a topology consisting of six predicted transmembrane domains (TM) with a putative pore loop between TM5 and TM6 and intracellular N-and C-tails, the former containing at least three ankyrin domains. Functional transient receptor potential (TRP) channels are supposed to result following the assembly of four subunits. However, the rules governing subunit assembly and protein domains implied in this process are only starting to emerge. The ankyrin, TM, and the C-tail domains have been identified as important determinants of the oligomerization process. We now describe the maturation and oligomerization of five splice variants of the TRPV4 channel. The already known TRPV4-A and TRPV4-B (⌬384 -444) variants and the new TRPV4-C (⌬237-284), TRPV4-D (⌬27-61), and TRPV4-E (⌬237-284 and ⌬384 -444) variants. All alternative spliced variants involved deletions in the cytoplasmic N-terminal region, affecting (except for TRPV4-D) the ankyrin domains. Subcellular localization, fluorescence resonance energy transfer, co-immunoprecipitation, glycosylation profile, and functional analysis of these variants permitted us to group them into two classes: group I (TRPV4-A and TRPV4-D) and group II (TRPV4-B, TRPV4-C, and TRPV4-E). Group I, unlike group II variants, were correctly processed, homo-and heteromultimerized in the endoplasmic reticulum, and were targeted to the plasma membrane where they responded to typical TRPV4 stimuli. Our results suggest that: 1) TRPV4 biogenesis involves core glycosylation and oligomerization in the endoplasmic reticulum followed by transfer to the Golgi apparatus for subsequent maturation; 2) ankyrin domains are necessary for oligomerization of TRPV4; and 3) lack of TRPV4 oligomerization determines its accumulation in the endoplasmic reticulum.The non-selective cation channel TRPV4 is a member of the transient receptor potential (TRP) 3 family of channels (1). TRPV4 shows multiple modes of activation and regulatory sites, enabling it to respond to various stimuli, including osmotic cell swelling (2-5), mechanical stress (6 -8), heat (9), acidic pH (7), endogenous ligands (10), high viscous solutions (11), and synthetic agonists such as 4␣-phorbol 12,13-didecanoate (4␣-PDD) (12). TRPV4 mRNA is expressed in a broad range of tissues (13), although functional tests have only been carried out in a few: endothelial (14), epithelial (5,11,15), smooth muscle (16), keratinocytes (17), and DRG neurons (18). An alternative splice variant lacking the seventh exon has also been cloned from human aortic endothelial cells (19), although this variant was not further investigated because of its unresponsiveness to hypotonic stimuli.The general topology of a TRP subunit consists of six predicted transmembrane domains (TM) with a putative pore loop between TM5 and TM6 and intracellular N-and C-terminal regions of variable length, the former containing multiple ankyrin (ANK) repeats in the TRPC, TRPA, TRPN, and TRPV subfamilies (1). ANK repeats are modular protein interaction domains, ...

“…The cloning of VR1 resulted in the identification of many genes with sequence homology, for example, heat-sensitive vanilloid receptor-like (VRL1) channel and osmotically activated channel (VR-OAC) (19,20). In addition, splicing variants of VR1, such as stretchinhibitable channel (SIC) and VR.5Јsv, were also identified (21,22). Interestingly, none of these homologues respond to capsaicin when expressed heterologously.…”

mentioning

confidence: 99%

Agonist Recognition Sites in the Cytosolic Tails of Vanilloid Receptor 1

Jung

Lee

Hwang

et al. 2002

149

110

Vanilloid receptor 1 (VR1), a ligand-gated ion channel activated by vanilloids, acid, and heat, is a molecular detector that integrates multiple modes of pain. Although the function and the biophysical properties of the channel are now known, the regions of VR1 that recognize ligands are largely unknown. By the stepwise deletion of VR1 and by chimera construction using its capsaicin-insensitive homologue, VRL1, we localized key amino acids, Arg-114 and Glu-761, in the N-and C-cytosolic tails, respectively, that determine ligand binding. Point mutations of the two key residues resulted in a loss of sensitivity to capsaicin and a concomitant loss of specific binding to [ 3 H]resiniferatoxin, a potent vanilloid. Furthermore, changes in the charges of the two amino acids blocked capsaicin-sensitive currents and ligand binding without affecting current responses to heat. Thus, these two regions in the cytoplasmic tails of VR1 provide structural elements for its hydrophilic interaction with vanilloids and might constitute a long-suspected binding pocket.Capsaicin, the principal pungent ingredient of hot peppers, excites sensory neurons by opening an ion channel, the vanilloid receptor 1 (VR1), 1 thereby causing pain. VR1 is a ligand-gated, cationic channel that is present mainly in small nociceptive sensory neurons (1-3). The presence of VR1 in sensory neurons leads to questions concerning the existence of endogenous capsaicin-like substances, and various lipid metabolic products of lipoxygenases or anandamide have been suggested as candidates, because they activate VR1 and are structurally similar to capsaicin (4, 5). Accordingly, a role for lipoxygenase products in the activation of VR1 during inflammation was suggested (5), and in fact, bradykinin, a potent pain-causing inflammatory mediator, is now known to activate VR1 via the lipoxygenase/VR1 pathway (6). In addition, bradykinin also has a potential to sensitize VR1 via a phospholipase C or protein kinase C pathway (7-9).VR1 is also activated by acid or heat at over 43°C, a threshold temperature for pain (3, 10 -12). Moreover, because ischemic or inflamed tissues become acidic, the acid activation of VR1 is a pathologically relevant event (13). More direct evidence for the pathophysiological role of VR1 in the production of inflammatory pain came from knock-out experiments. In mice lacking VR1, thermal hyperalgesia evoked by inflammation is reduced (14, 15). Furthermore, hyperalgesia induced by the key inflammatory mediators, bradykinin and nerve growth factor, is reduced in mice lacking VR1 (8). Thus, VR1 is now considered a primary molecular transducer that mediates inflammatory hyperalgesia (13).The putative topology of VR1 indicates that it belongs to a class of transient receptor potential channels possessing six transmembrane domains and two cytosolic domains at each Nand C terminus (3, 16). VR1 appears to form a homotetramer when expressed heterologously (17). However, VR1 may form a heteromultimer with another temperature-sensitive channel, transient rec...