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.structure | regulation | thermosensitivity T he transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel that responds to osmotic (1-4), mechanical (5-7), and temperature stimulation (8), thereby contributing to many different physiological functions, including cellular (4, 9) and systemic volume homeostasis (10), vasodilation (11, 12), nociception (13), epithelial hydroelectrolyte transport (14), bladder voiding (15), ciliary beat frequency regulation (7,16,17), chondroprotection (18), and skeletal regulation (19). The osmotic (20) and mechanical (7, 16) sensitivity of TRPV4 depends on the activation of phospholipase A 2 and subsequent production of the arachidonic acid metabolite 5′-6′-epoxyeicosatrienoic acid (EET), whereas the mechanism leading to temperature-mediated activation (observed only in intact cells) is not known at present (21). 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), prot...

The widely distributed TRPV4 cationic channel participates in the transduction of both physical (osmotic, mechanical, and heat) and chemical (endogenous, plant-derived, and synthetic ligands) stimuli. In this chapter we will review TRPV4 expression, biophysics, structure, regulation, and interacting partners as well as physiological and pathological insights obtained in TRPV4 animal models and human genetic studies.

Mechanical and osmotic sensitivity of the transient receptor potential vanilloid 4 (TRPV4) channel depends on phospholipase A2 (PLA2) activation and the subsequent production of the arachidonic acid metabolites, epoxyeicosatrienoic acid (EET). We show that both high viscous loading and hypotonicity stimuli in native ciliated epithelial cells use PLA2–EET as the primary pathway to activate TRPV4. Under conditions of low PLA2 activation, both also use extracellular ATP-mediated activation of phospholipase C (PLC)–inositol trisphosphate (IP3) signaling to support TRPV4 gating. IP3, without being an agonist itself, sensitizes TRPV4 to EET in epithelial ciliated cells and cells heterologously expressing TRPV4, an effect inhibited by the IP3 receptor antagonist xestospongin C. Coimmunoprecipitation assays indicated a physical interaction between TRPV4 and IP3 receptor 3. Collectively, our study suggests a functional coupling between plasma membrane TRPV4 channels and intracellular store Ca2+ channels required to initiate and maintain the oscillatory Ca2+ signal triggered by high viscosity and hypotonic stimuli that do not reach a threshold level of PLA2 activation.

Long QT syndrome, short QT syndrome, Brugada syndrome and catecholaminergic polymorphic ventricular tachycardia are inherited primary electrical disorders that predispose to sudden cardiac death in the absence of structural heart disease. Also known as cardiac channelopathies, primary electrical disorders respond to mutations in genes encoding cardiac ion channels and/or their regulatory proteins, which result in modifications in the cardiac action potential or in the intracellular calcium handling that lead to electrical instability and life-threatening ventricular arrhythmias. These disorders may have low penetrance and expressivity, making clinical diagnosis often challenging. However, because sudden cardiac death might be the first presenting symptom of the disease, early diagnosis becomes essential. Genetic testing might be helpful in this regard, providing a definite diagnosis in some patients. Yet important limitations still exist, with a significant proportion of patients remaining with no causative mutation identifiable after genetic testing. This review aims to provide the latest knowledge on the genetic basis of cardiac channelopathies and discuss the role of the affected proteins in the pathophysiology of each one of these diseases.