IP3 Receptor Binds to and Sensitizes TRPV4 Channel to Osmotic Stimuli via a Calmodulin-binding Site

Abstract

The TRPV4 2 cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues where it participates in the generation of a Ca 2ϩ signal and/or depolarization of the membrane potential (1). TRPV4 participation in osmo-and mechanotransduction (1, 2) contributes to important functions including cellular (3, 4) and systemic volume homeostasis (5, 6), arterial dilation (7,8), nociception (9), epithelial hydroelectrolyte transport (10, 11), bladder voiding (12), and ciliary beat frequency regulation (13)(14)(15). TRPV4 also responds to temperature (16,17), endogenous arachidonic acid (AA) metabolites (18), and phorbol esters including the inactive 4␣-phorbol 12,13-didecanoate (4␣PDD) (19,20) and participates in receptor-operated Ca 2ϩ entry (15), thus showing multiple modes of activation.TRPV4 channel activity can be sensitized by co-application of different stimuli (9,21,22) or participation of different cell signaling pathways (14), suggesting the presence of different regulatory sites. In this sense, several proteins have been proposed to modulate TRPV4 subcellular localization and/or function: microtubuleassociated protein 7 (23), calmodulin (CaM, (24)), with no lysine protein kinases (25), and PACSIN3 (26). We have also recently reported a close functional and physical interaction between the inositol trisphosphate receptor 3 (IP 3 R3) and TRPV4 that sensitizes the latter to the mechano-and osmotransducing messenger 5Ј-6Ј-epoxieicosatrienoic acid (EET) (14).In this study, we reveal that IP 3 -mediated sensitization of TRPV4 to EET requires IP 3 R3 binding to TRPV4 and identify the IP 3 R3-binding site in a C-terminal region of the TRPV4 that coincides with a previously described CaM-binding site (24). Besides, our study has provided further evidence of the importance of the TRPV4 N terminus in channel gating as deletion of a proline-rich domain prevents channel activation by different stimuli despite correct localization and oligomerization. EXPERIMENTAL PROCEDURESGeneration of TRPV4 Mutants-TRPV4 mutations were generated by site-directed mutagenesis (QuikChange II XL site-directed mutagenesis kit, Stratagene) deleting: 13 amino acids (positions 132-144) to generate the mutation lacking the N-terminal PRD domain (TRPV4-⌬PRD); 20 amino acids (positions 812-831) for the mutation lacking the calmodulinbinding domain (TRPV4-⌬CaM); and the last 4 amino acids of the channel (positions 868 -871) for the mutation lacking the C-terminal PDZ domain (TRPV4-⌬DAPL). All TRPV4 constructs were tagged with YFP (or cyan fluorescent protein) at the C terminus, and sequences were verified by PCR.Cell Transfection-HEK293 and HeLa cells were transiently transfected with ExGen500 (Fermentas MBI) using 8 eq of polyethyl enimine together with 3 g of cDNA of the following plasmids: 1.5 g of rat pcDNA3-IP 3 R3 plasmid and 1.5 g of human TRPV4-YPF plasmids, WT, or mutants. Cells were cultured from 24 to 48 h before use.Confocal Imaging-HeLa cells transiently transfected with WT and TRPV4 mutants were fixed w...

TRPV4 channel contributes to serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension

“…A recent study suggests that acetylcholine stimulates TRPV4-dependent Ca 2ϩ influx in endothelial cells through activation of PKC␣ (1). Besides PKC, InsP 3 can sensitize TRPV4 to the stimulation of 5=,6=-EET in ciliated epithelial cells, presumably through binding of the InsP 3 receptor to a TRPV4 COOH-terminal domain (19,21). Moreover, TRPV4 forms heteromeric channels with TRPC1 in endothelial cells and contributes to store-operated Ca 2ϩ entry (42-44), which is a major Ca 2ϩ influx pathway activated by InsP 3 -induced Ca 2ϩ release.…”

A TRPV4 Channel C-terminal Folding Recognition Domain Critical for Trafficking and Function

“…The cytosolic C terminus of TRPV4 harbors many binding sites for various interacting proteins and factors, such as F-actin, tubulin, MAP7, inositol trisphosphate receptor, and calmodulin, that are relevant to regulation of subunit assembly, trafficking to the plasma membrane, and channel activity (33,34,36,37). It is of interest to note that the proposed C-terminal folding recognition domain turns out to overlap with the calmodulin-binding site previously shown to induce conformational changes and Ca 2ϩ -dependent potentiation of TRPV4 (36,53).…”

“…Dysfunctional TRP channels that fail to fold or assemble properly in the ER are usually characterized by a lack of complex glycosylation (29 -32). The intracellular C-terminal region of TRPV4 has been shown to interact with various structural or modulatory proteins that regulate channel activity and surface expression, including MAP7, actin/tubulin, inositol trisphosphate receptor, TRPP2, and calmodulin (33)(34)(35)(36)(37). Fluorescence resonance energy transfer (FRET) and confocal imaging studies have attributed to the TRPV4 C terminus an important role in channel subunit oligomerization and trafficking (38,39).…”

Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli

“…EETindependent TRPV4 activation by membrane stretch in excised patches from oocytes also has been reported (22), in apparent contradiction to early reports claiming lack of activation by membrane stretch (1). Several studies have characterized TRPV4 domains implicated in channel regulation by calmodulin (23, 24), protein kinase C and casein kinase substrate in neurons 3 (PACSIN3) (25), intracellular ATP (24) and inositol-trisphosphate receptor (16,26). However, little is known about the domains relevant for TRPV4 activation by different stimuli, apart from the interaction between the TRPV4 activator 4α-phorbol 12,13-didecanoate (4α-PDD) and transmembrane domains 3 and 4 (27).…”