Details

Autor(en) / Beteiligte

• 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.

Titel

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

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 2013-06, Vol.110 (23), p.9553-9558

Ort / Verlag

United States: National Academy of Sciences

Erscheinungsjahr

2013

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.