Anna Garcia-Elias

IP3 sensitizes TRPV4 channel to the mechano- and osmotransducing messenger 5′-6′-epoxyeicosatrienoic acid

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.

A loss-of-function nonsynonymous polymorphism in the osmoregulatory TRPV4 gene is associated with human hyponatremia

Disorders of water balance are among the most common and morbid of the electrolyte disturbances, and are reflected clinically as abnormalities in the serum sodium concentration. The transient receptor potential vanilloid 4 (TRPV4) channel is postulated to comprise an element of the central tonicity-sensing mechanism in the mammalian hypothalamus, and is activated by hypotonic stress in vitro. A nonsynonymous polymorphism in the TRPV4 gene gives rise to a Pro-to-Ser substitution at residue 19. We show that this polymorphism is significantly associated with serum sodium concentration and with hyponatremia (serum sodium concentration ≤135 mEq/L) in 2 non-Hispanic Caucasian male populations; in addition, mean serum sodium concentration is lower among subjects with the TRPV4P19S allele relative to the wild-type allele. Subjects with the minor allele were 2.4−6.4 times as likely to exhibit hyponatremia as subjects without the minor allele (after inclusion of key covariates). Consistent with these observations, a human TRPV4 channel mutated to incorporate the TRPV4P19S polymorphism showed diminished response to hypotonic stress (relative to the wild-type channel) and to the osmotransducing lipid epoxyeicosatrienoic acid in heterologous expression studies. These data suggest that this polymorphism affects TRPV4 function in vivo and likely influences systemic water balance on a population-wide basis.

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

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP2), although the structural rearrangements occurring on PIP2 binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP2 binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence 121KRWRK125, 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 PIP2 from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP2 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 PIP2-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP2 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–PIP2 interacting site and conditions that deplete PIP2 or restrict access of TRPV4 to PIP2—in the case of PACSIN3—change tail conformation and negatively affect channel activation by hypotonicity and heat.

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

Activation of the non-selective cation channel TRPV4 by mechanical and osmotic stimuli requires the involvement of phospholipase A2 and the subsequent production of the arachidonic acid metabolites, epoxieicosatrienoic acids (EET). Previous studies have shown that inositol trisphosphate (IP3) sensitizes TRPV4 to mechanical, osmotic, and direct EET stimulation. We now search for the IP3 receptor-binding site on TRPV4 and its relevance to IP3-mediated sensitization. Three putative sites involved in protein-protein interactions were evaluated: a proline-rich domain (PRD), a calmodulin (CaM)-binding site, and the last four amino acids (DAPL) that show a PDZ-binding motif-like. TRPV4-ΔCaM-(Δ812–831) channels preserved activation by hypotonicity, 4α-phorbol 12,13-didecanoate, and EET but lost their physical interaction with IP3 receptor 3 and IP3-mediated sensitization. Deletion of a PDZ-binding motif-like (TRPV4-ΔDAPL) did not affect channel activity or IP3-mediated sensitization, whereas TRPV4-ΔPRD-(Δ132–144) resulted in loss of channel function despite correct trafficking. We conclude that IP3-mediated sensitization requires IP3 receptor binding to a TRPV4 C-terminal domain that overlaps with a previously described calmodulin-binding site.

The TRPV4 Channel

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.

IP3 sensitizes TRPV4 channel to the mechano- and osmotransducing messenger 5'-6'-epoxyeicosatrienoic acid.

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.

Ion Channels in Asthma

Ion channels are specialized transmembrane proteins that permit the passive flow of ions following their electrochemical gradients. In the airways, ion channels participate in the production of epithelium-based hydroelectrolytic secretions and in the control of intracellular Ca2+ levels that will ultimately activate almost all lung cells, either resident or circulating. Thus, ion channels have been the center of many studies aiming to understand asthma pathophysiological mechanisms or to identify therapeutic targets for better control of the disease. In this minireview, we focus on molecular, genetic, and animal model studies associating ion channels with asthma.

Effect of Permanent Atrial Fibrillation on Cognitive Function in Patients With Chronic Heart Failure.

In patients with chronic heart failure (HF), cognitive impairment (CI) is associated with poorer treatment adherence and higher readmission and mortality rates. Previous studies suggest that atrial fibrillation (AF) could impair cognitive function. This study sought to assess the association between permanent AF (permAF) and CI in patients with HF. We evaluated cognitive function in 881 patients with stable HF (73 ± 11 years, 44% women, 48% with preserved ejection fraction) using the Mini-Mental State Examination test (n = 876) and the Pfeiffers Short Portable Mental Status Questionnaire (n = 848). CI was defined as a Mini-Mental State Examination score <24 or Short Portable Mental Status Questionnaire (errors) >2. The independent association between permAF and CI was assessed by binary logistic regression analysis. A total of 295 patients (33.5%) had CI, in 5.1% of cases moderate/severe. Patients with permAF had more frequently any degree of CI (43% vs 31%), and moderate/severe CI (8% vs 5%). In the multivariate analysis, CI was associated with permAF (odds ratio 1.54, 95% C.I. 1.05 to 2.28), an older age, female gender, diabetes mellitus, chronic kidney disease, previous stroke, New York Heart Association class III/IV, and lower systolic blood pressure. No interaction was found for AF and CI between patients with reduced and preserved ejection fraction. In conclusion, the presence of permAF is independently associated with CI in patients with HF, both with reduced and preserved ejection fraction. Given the clinical impact of CI in the HF population, active assessment of cognitive function is particularly warranted in patients with HF with permAF.

TRPV4 participates in the establishment of trailing adhesions and directional persistence of migrating cells.

Calcium signaling participates in different cellular processes leading to cell migration. TRPV4, a non-selective cation channel that responds to mechano-osmotic stimulation and heat, is also involved in cell migration. However, the mechanistic involvement of TRPV4 in cell migration is currently unknown. We now report that expression of the mutant channel TRPV4-121AAWAA (lacking the phosphoinositide-binding site 121KRWRK125 and the response to physiological stimuli) altered HEK293 cell migration. Altered migration patterns included periods of fast and persistent motion followed by periods of stalling and turning, and the extension of multiple long cellular protrusions. TRPV4-WT overexpressing cells showed almost complete loss of directionality with frequent turns, no progression, and absence of long protrusions. Traction microscopy revealed higher tractions forces in the tail of TRPV4-121AAWAA than in TRPV4-WT expressing cells. These results are consistent with a defective and augmented tail retraction in TRPV4-121AAWAA- and TRPV4-WT-expressing cells, respectively. The activity of calpain, a protease implicated in focal adhesion (FA) disassembly, was decreased in TRPV4-121AAWAA compared with TRPV4-WT-expressing cells. Consistently, larger focal adhesions were seen in TRPV4-121AAWAA compared with TRPV4-WT-expressing HEK293 cells, a result that was also reproduced in T47D and U87 cells. Similarly, overexpression of the pore-dead mutant TRPV4-M680D resumed the TRPV4-121AAWAA phenotype presenting larger FA. The migratory phenotype obtained in HEK293 cells overexpressing TRPV4-121AAWAA was mimicked by knocking-down TRPC1, a cationic channel that participates in cell migration. Together, our results point to the participation of TRPV4 in the dynamics of trailing adhesions, a function that may require the interplay of TRPV4 with other cation channels or proteins present at the FA sites.

Interaction between the Linker, Pre-S1, and TRP Domains Determines Folding, Assembly, and Trafficking of TRPV Channels

Functional transient receptor potential (TRP) channels result from the assembly of four subunits. Here, we show an interaction between the pre-S1, TRP, and the ankyrin repeat domain (ARD)-S1 linker domains of TRPV1 and TRPV4 that is essential for proper channel assembly. Neutralization of TRPV4 pre-S1 K462 resulted in protein retention in the ER, defective glycosylation and trafficking, and unresponsiveness to TRPV4-activating stimuli. Similar results were obtained with the equivalent mutation in TRPV1 pre-S1. Molecular dynamics simulations revealed that TRPV4-K462 generated an alternating hydrogen network with E745 (TRP box) and D425 (pre-S1 linker), and that K462Q mutation affected subunit folding. Consistently, single TRPV4-E745A or TRPV4-D425A mutations moderately affected TRPV4 biogenesis while double TRPV4-D425A/E745A mutation resumed the TRPV4-K462Q phenotype. Thus, the interaction between pre-S1, TRP, and linker domains is mandatory to generate a structural conformation that allows the contacts between adjacent subunits to promote correct assembly and trafficking to the plasma membrane.