National Academy of Sciences, Proceedings of the National Academy of Sciences, 23(110), p. 9553-9558, 2013
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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 121 KRWRK 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.