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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 110; no. 23; pp. 9553 - 9558
• Main Authors: Garcia-Elias, Anna, Mrkonjic, Sanela, Pardo-Pastor, Carlos, Inada, Hitoshi, Hellmich, Ute A., Rubio-Moscardó, Fanny, Plata, Cristina, Gaudet, Rachelle, Vicente, Rubén, Valverde, Miguel A.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 04-06-2013; National Acad Sciences
• Subjects: Analysis of Variance; Ankyrins; Binding sites; Biological Sciences; Calcium; Calcium - metabolism; Cell membranes; Cells; Cells, Cultured; Cloning, Molecular; Cytoplasm - metabolism; Epithelial cells; Fluorescence; Fluorescence Resonance Energy Transfer; Gene expression; HEK293 Cells; HeLa Cells; Humans; Hypotonicity; Intracellular Signaling Peptides and Proteins - metabolism; Patch-Clamp Techniques; Phorbols - metabolism; Phosphatidylinositol 4,5-Diphosphate - metabolism; Phosphatidylinositols; Physiological regulation; Physiological stimulation; Protein Structure, Tertiary; Proteins; T cell receptors; TRPV Cation Channels - metabolism; regulation; thermosensitivity; structure
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1220231110

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP ₂), although the structural rearrangements occurring on PIP ₂ binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP ₂ binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence ¹²¹KRWRK ¹²⁵, which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP ₂ from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP ₂ facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5′-6’-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP ₂-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP ₂ mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4–PIP ₂ interacting site and conditions that deplete PIP ₂ or restrict access of TRPV4 to PIP ₂—in the case of PACSIN3—change tail conformation and negatively affect channel activation by hypotonicity and heat.