Structural Basis of TRPV4 N Terminus Interaction with Syndapin/PACSIN1-3 and PIP2

Goretzki, Benedikt; Glogowski, Nina A.; Diehl, Erika; Duchardt‐Ferner, Elke; Hacker, Carolin; Gaudet, Rachelle; Hellmich, Ute A.

doi:10.1016/j.str.2018.08.002

Cited by 33 publications

(54 citation statements)

References 68 publications

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“…Molecular weights and secondary structure of recombinant proteins as determined by circular dichroism (CD) spectroscopy were as expected (Supplementary Fig. 3c, d ) 17 , 40 . Additionally, purified RhoA protein was functional in a luminescence-based GTPase assay (Supplementary Fig.…”

Section: Resultssupporting

confidence: 72%

Neuropathy-causing TRPV4 mutations disrupt TRPV4-RhoA interactions and impair neurite extension

et al. 2021

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TRPV4 is a cell surface-expressed calcium-permeable cation channel that mediates cell-specific effects on cellular morphology and function. Dominant missense mutations of TRPV4 cause distinct, tissue-specific diseases, but the pathogenic mechanisms are unknown. Mutations causing peripheral neuropathy localize to the intracellular N-terminal domain whereas skeletal dysplasia mutations are in multiple domains. Using an unbiased screen, we identified the cytoskeletal remodeling GTPase RhoA as a TRPV4 interactor. TRPV4-RhoA binding occurs via the TRPV4 N-terminal domain, resulting in suppression of TRPV4 channel activity, inhibition of RhoA activation, and extension of neurites in vitro. Neuropathy but not skeletal dysplasia mutations disrupt TRPV4-RhoA binding and cytoskeletal outgrowth. However, inhibition of RhoA restores neurite length in vitro and in a fly model of TRPV4 neuropathy. Together these results identify RhoA as a critical mediator of TRPV4-induced cell structure changes and suggest that disruption of TRPV4-RhoA binding may contribute to tissue-specific toxicity of TRPV4 neuropathy mutations.

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Section: Resultssupporting

confidence: 72%

Neuropathy-causing TRPV4 mutations disrupt TRPV4-RhoA interactions and impair neurite extension

et al. 2021

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“…A recent cryoEM structure of Xenopus tropicalis TRPV4 was used to model the position of p.S94L and other previously described neuropathy-causing TRPV4 mutations (figure 2). 7 Consistent with previous proteolysis protection 10 and Nuclear Magnetic Resonance studies, 11 the N-terminal region—up to residue 147 (human numbering), thus including p.S94—was disordered and not modeled in the cryoEM structure. However, p.S94L is located N-terminal to 2 important regulatory regions: a phosphoinositide-binding domain; residues 121–125 in humans) that interacts with phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 10 and a proline-rich region (PRR; residues 135–144 in humans) that interacts with the SH3 domain of PACSIN3.…”

Section: Resultssupporting

confidence: 78%

Homozygous TRPV4 mutation causes congenital distal spinal muscular atrophy and arthrogryposis

et al. 2019

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ObjectiveTo identify the genetic cause of disease in a form of congenital spinal muscular atrophy and arthrogryposis (CSMAA).MethodsA 2-year-old boy was diagnosed with arthrogryposis multiplex congenita, severe skeletal abnormalities, torticollis, vocal cord paralysis, and diminished lower limb movement. Whole-exome sequencing (WES) was performed on the proband and family members. In silico modeling of protein structure and heterologous protein expression and cytotoxicity assays were performed to validate pathogenicity of the identified variant.ResultsWES revealed a homozygous mutation in the TRPV4 gene (c.281C>T; p.S94L). The identification of a recessive mutation in TRPV4 extends the spectrum of mutations in recessive forms of the TRPV4-associated disease. p.S94L and other previously identified TRPV4 variants in different protein domains were compared in structural modeling and functional studies. In silico structural modeling suggests that the p.S94L mutation is in the disordered N-terminal region proximal to important regulatory binding sites for phosphoinositides and for PACSIN3, which could lead to alterations in trafficking and/or channel sensitivity. Functional studies by Western blot and immunohistochemical analysis show that p.S94L increased TRPV4 activity-based cytotoxicity and resultant decreased TRPV4 expression levels, therefore involves a gain-of-function mechanism.ConclusionsThis study identifies a novel homozygous mutation in TRPV4 as a cause of the recessive form of CSMAA.

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“…29 Further studies are required to delineate the molecular determinants that control how the CEC TRPV4 channels respond to extrinsic and intrinsic stressors, and whether lack of activation by strain involves masking of the N-terminal region by PACSIN SH3 domains. 90 The present study identifies the TRPV4-hemichannel-ATP signaling axis as a potential modulator of paracrine, mechanically, and hypotonically induced corneal excitation. TRPV4induced ATP release was attenuated by HC-06 and BAPTA-AM, demonstrating an obligatory link to [Ca 2+ ] i homeostasis and channel activation.…”

Section: Discussionmentioning

confidence: 71%

Section: Discussionmentioning

confidence: 99%

Polymodal Sensory Transduction in Mouse Corneal Epithelial Cells

Lapajne

Lakk

Yarishkin

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. Contact lenses, osmotic stressors, and chemical burns may trigger severe discomfort and vision loss by damaging the cornea, but the signaling mechanisms used by corneal epithelial cells (CECs) to sense extrinsic stressors are not well understood. We therefore investigated the mechanisms of swelling, temperature, strain, and chemical transduction in mouse CECs. METHODS. Intracellular calcium imaging in conjunction with electrophysiology, pharmacology, transcript analysis, immunohistochemistry, and bioluminescence assays of adenosine triphosphate (ATP) release were used to track mechanotransduction in dissociated CECs and epithelial sheets isolated from the mouse cornea. RESULTS. The transient receptor potential vanilloid (TRPV) transcriptome in the mouse corneal epithelium is dominated by Trpv4, followed by Trpv2, Trpv3, and low levels of Trpv1 mRNAs. TRPV4 protein was localized to basal and intermediate epithelial strata, keratocytes, and the endothelium in contrast to the cognate TRPV1, which was confined to intraepithelial afferents and a sparse subset of CECs. The TRPV4 agonist GSK1016790A induced cation influx and calcium elevations, which were abolished by the selective blocker HC067047. Hypotonic solutions, membrane strain, and moderate heat elevated [Ca 2+ ] CEC with swelling-and temperature-, but not strain-evoked signals, sensitive to HC067047. GSK1016790A and swelling evoked calcium-dependent ATP release, which was suppressed by HC067027 and the hemichannel blocker probenecid. CONCLUSIONS. These results demonstrate that cation influx via TRPV4 transduces osmotic and thermal but not strain inputs to CECs and promotes hemichannel-dependent ATP release. The TRPV4-hemichannel-ATP signaling axis might modulate corneal pain induced by excessive mechanical, osmotic, and chemical stimulation.

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Structural Basis of TRPV4 N Terminus Interaction with Syndapin/PACSIN1-3 and PIP2

Cited by 33 publications

References 68 publications

Neuropathy-causing TRPV4 mutations disrupt TRPV4-RhoA interactions and impair neurite extension

Neuropathy-causing TRPV4 mutations disrupt TRPV4-RhoA interactions and impair neurite extension

Homozygous TRPV4 mutation causes congenital distal spinal muscular atrophy and arthrogryposis

Polymodal Sensory Transduction in Mouse Corneal Epithelial Cells

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