scholarly journals The human polycystin-2 protein represents an integral membrane protein with six membrane-spanning domains and intracellular N- and C-termini

2010 ◽  
Vol 433 (2) ◽  
pp. 285-294 ◽  
Author(s):  
Helen Hoffmeister ◽  
Anna-Rachel Gallagher ◽  
Anne Rascle ◽  
Ralph Witzgall
Keyword(s):  
Endoplasmic Reticulum ◽  
Experimental Evidence ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Integral Membrane Protein ◽  
Protein Product ◽  
C Terminus ◽  
Autosomal Dominant Polycystic Kidney ◽  
Transient Receptor ◽  
Membrane Spanning

PKD2 is one of the two genes mutated in ADPKD (autosomal-dominant polycystic kidney disease). The protein product of PKD2, polycystin-2, functions as a non-selective cation channel in the endoplasmic reticulum and possibly at the plasma membrane. Hydrophobicity plots and its assignment to the TRP (transient receptor potential) family of cation channels suggest that polycystin-2 contains six transmembrane domains and that both the N- and C-termini extend into the cytoplasm. However, no experimental evidence for this model has so far been provided. To determine the orientation of the different loops of polycystin-2, we truncated polycystin-2 within the predicted loops 1–5 and tagged the constructs at the C-terminus with an HA (haemagglutinin) epitope. After transient expression and selective membrane permeabilization, immunofluorescence staining for the HA epitope revealed that loops 1, 3 and 5 extend into the lumen of the endoplasmic reticulum or the extracellular space, whereas loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the N- and C-termini of polycystin-2. In accordance with the immunofluorescence data, protease protection assays from microsomal preparations yielded protected fragments when polycystin-2 was truncated in loops 1, 3 and 5, whereas no protected fragments could be detected when polycystin-2 was truncated in loops 2 and 4. The results of the present study therefore provide the first experimental evidence for the topological orientation of polycystin-2.

scholarly journals NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

2022 ◽  
Vol 23 (2) ◽  
pp. 892
Author(s):  
Mariia Belinskaia ◽  
Tomas Zurawski ◽  
Seshu Kumar Kaza ◽  
Caren Antoniazzi ◽  
J. Oliver Dolly ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Sensory Nerves ◽  
Neonatal Rat ◽  
Transient Receptor Potential Vanilloid ◽  
C Terminus ◽  
Low Concentrations ◽  
Transient Receptor ◽  
Neuronal Functions ◽  
Differential Ability

Nerve growth factor (NGF) is known to intensify pain in various ways, so perturbing pertinent effects without negating its essential influences on neuronal functions could help the search for much-needed analgesics. Towards this goal, cultured neurons from neonatal rat trigeminal ganglia—a locus for craniofacial sensory nerves—were used to examine how NGF affects the Ca2+-dependent release of a pain mediator, calcitonin gene-related peptide (CGRP), that is triggered by activating a key signal transducer, transient receptor potential vanilloid 1 (TRPV1) with capsaicin (CAP). Measurements utilised neurons fed with or deprived of NGF for 2 days. Acute re-introduction of NGF induced Ca2+-dependent CGRP exocytosis that was inhibited by botulinum neurotoxin type A (BoNT/A) or a chimera of/E and/A (/EA), which truncated SNAP-25 (synaptosomal-associated protein with Mr = 25 k) at distinct sites. NGF additionally caused a Ca2+-independent enhancement of the neuropeptide release evoked by low concentrations (<100 nM) of CAP, but only marginally increased the peak response to ≥100 nM. Notably, BoNT/A inhibited CGRP exocytosis evoked by low but not high CAP concentrations, whereas/EA effectively reduced responses up to 1 µM CAP and inhibited to a greater extent its enhancement by NGF. In addition to establishing that sensitisation of sensory neurons to CAP by NGF is dependent on SNARE-mediated membrane fusion, insights were gleaned into the differential ability of two regions in the C-terminus of SNAP-25 (181–197 and 198–206) to support CAP-evoked Ca2+-dependent exocytosis at different intensities of stimulation.

scholarly journals Pulsed infrared releases Ca2+ from the endoplasmic reticulum of cultured spiral ganglion neurons

2018 ◽  
Vol 120 (2) ◽  
pp. 509-524 ◽  
Author(s):  
John N. Barrett ◽  
Samantha Rincon ◽  
Jayanti Singh ◽  
Cristina Matthewman ◽  
Julio Pasos ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Infrared Radiation ◽  
Transient Receptor Potential ◽  
Trp Channels ◽  
Spiral Ganglion ◽  
Receptor Potential ◽  
Transient Temperature ◽  
Spiral Ganglion Neurons ◽  
Transient Receptor ◽  
Ganglion Neurons

Inner ear spiral ganglion neurons were cultured from day 4 postnatal mice and loaded with a fluorescent Ca2+ indicator (fluo-4, -5F, or -5N). Pulses of infrared radiation (IR; 1,863 nm, 200 µs, 200–250 Hz for 2–5 s, delivered via an optical fiber) produced a rapid, transient temperature increase of 6–12°C (above a baseline of 24–30°C). These IR pulse trains evoked transient increases in both nuclear and cytosolic Ca2+ concentration ([Ca2+]) of 0.20–1.4 µM, with a simultaneous reduction of [Ca2+] in regions containing endoplasmic reticulum (ER). IR-induced increases in cytosolic [Ca2+] continued in medium containing no added Ca2+ (±Ca2+ buffers) and low [Na+], indicating that the [Ca2+] increase was mediated by release from intracellular stores. Consistent with this hypothesis, the IR-induced [Ca2+] response was prolonged and eventually blocked by inhibition of ER Ca2+-ATPase with cyclopiazonic acid, and was also inhibited by a high concentration of ryanodine and by inhibitors of inositol (1,4,5)-trisphosphate (IP3)-mediated Ca2+ release (xestospongin C and 2-aminoethoxydiphenyl borate). The thermal sensitivity of the response suggested involvement of warmth-sensitive transient receptor potential (TRP) channels. The IR-induced [Ca2+] increase was inhibited by TRPV4 inhibitors (HC-067047 and GSK-2193874), and immunostaining of spiral ganglion cultures demonstrated the presence of TRPV4 and TRPM2 that colocalized with ER marker GRP78. These results suggest that the temperature sensitivity of IR-induced [Ca2+] elevations is conferred by TRP channels on ER membranes, which facilitate Ca2+ efflux into the cytosol and thereby contribute to Ca2+-induced Ca2+-release via IP3 and ryanodine receptors. NEW & NOTEWORTHY Infrared radiation-induced photothermal effects release Ca2+ from the endoplasmic reticulum of primary spiral ganglion neurons. This Ca2+ release is mediated by activation of transient receptor potential (TRPV4) channels and involves amplification by Ca2+-induced Ca2+-release. The neurons immunostained for warmth-sensitive channels, TRPV4 and TRPM2, which colocalize with endoplasmic reticulum. Pulsed infrared radiation provides a novel experimental tool for releasing intracellular Ca2+, studying Ca2+ regulatory mechanisms, and influencing neuronal excitability.

Structure of the human PKD1-PKD2 complex

Science ◽  
2018 ◽  
Vol 361 (6406) ◽  
pp. eaat9819 ◽  
Author(s):  
Qiang Su ◽  
Feizhuo Hu ◽  
Xiaofei Ge ◽  
Jianlin Lei ◽  
Shengqiang Yu ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Complex Structure ◽  
Transmembrane Helix ◽  
Receptor Potential ◽  
Trp Channel ◽  
Cryo Electron Microscopy ◽  
Disease Mechanisms ◽  
Autosomal Dominant Polycystic Kidney ◽  
Transient Receptor ◽  
Positively Charged

Mutations in two genes, PKD1 and PKD2, account for most cases of autosomal dominant polycystic kidney disease, one of the most common monogenetic disorders. Here we report the 3.6-angstrom cryo–electron microscopy structure of truncated human PKD1-PKD2 complex assembled in a 1:3 ratio. PKD1 contains a voltage-gated ion channel (VGIC) fold that interacts with PKD2 to form the domain-swapped, yet noncanonical, transient receptor potential (TRP) channel architecture. The S6 helix in PKD1 is broken in the middle, with the extracellular half, S6a, resembling pore helix 1 in a typical TRP channel. Three positively charged, cavity-facing residues on S6b may block cation permeation. In addition to the VGIC, a five–transmembrane helix domain and a cytosolic PLAT domain were resolved in PKD1. The PKD1-PKD2 complex structure establishes a framework for dissecting the function and disease mechanisms of the PKD proteins.

scholarly journals Molecular mapping of transmembrane mechanotransduction through the β1 integrin–CD98hc–TRPV4 axis

2020 ◽  
Vol 133 (20) ◽  
pp. jcs248823 ◽  
Author(s):  
Ratnakar Potla ◽  
Mariko Hirano-Kobayashi ◽  
Hao Wu ◽  
Hong Chen ◽  
Akiko Mammoto ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Molecular Mapping ◽  
Calcium Influx ◽  
Focal Adhesions ◽  
Receptor Potential ◽  
Transient Receptor Potential Vanilloid ◽  
Β1 Integrin ◽  
Mechanical Forces ◽  
C Terminus ◽  
Transient Receptor

ABSTRACTOne of the most rapid (less than 4 ms) transmembrane cellular mechanotransduction events involves activation of transient receptor potential vanilloid 4 (TRPV4) ion channels by mechanical forces transmitted across cell surface β1 integrin receptors on endothelial cells, and the transmembrane solute carrier family 3 member 2 (herein denoted CD98hc, also known as SLC3A2) protein has been implicated in this response. Here, we show that β1 integrin, CD98hc and TRPV4 all tightly associate and colocalize in focal adhesions where mechanochemical conversion takes place. CD98hc knockdown inhibits TRPV4-mediated calcium influx induced by mechanical forces, but not by chemical activators, thus confirming the mechanospecificity of this signaling response. Molecular analysis reveals that forces applied to β1 integrin must be transmitted from its cytoplasmic C terminus via the CD98hc cytoplasmic tail to the ankyrin repeat domain of TRPV4 in order to produce ultrarapid, force-induced channel activation within the focal adhesion.

Mediation of angiotensin II-induced Ca2+ signaling by polycystin 2 in glomerular mesangial cells

2008 ◽  
Vol 294 (4) ◽  
pp. F909-F918 ◽  
Author(s):  
Juan Du ◽  
Min Ding ◽  
Sherry Sours-Brothers ◽  
Sarabeth Graham ◽  
Rong Ma
Keyword(s):  
Transient Receptor Potential ◽  
Mesangial Cells ◽  
Rat Kidney ◽  
Receptor Potential ◽  
Cell Types ◽  
Protein Product ◽  
Dominant Negative ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Autosomal Dominant Polycystic Kidney

Ca+ influx across the plasma membrane is a major component of mesangial cell (MC) response to vasoconstrictors. Polycystin 2 (PC2), the protein product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, has been shown to function as a nonselective cation channel in a variety of cell types. The present study was performed to test the hypothesis that PC2 and its binding partners constitute a Ca2+-permeable channel and contribute to ANG II-induced Ca2+ signaling in MCs. Western blot and immunocytochemistry showed PC2 expression in cultured human MCs. The existence of PC2 in MCs was further confirmed by immunohistochemsitry in rat kidney sections. Coimmunoprecipitation displayed a selective interaction of PC2 with canonical transient receptor potential (TRPC) proteins TRPC1 and TRPC4. Cell-attached patch-clamp experiments revealed that ANG II-induced membrane currents were enhanced by overexpression of pkd2 but significantly inhibited by knock down of pkd2, 30 μM Gd3+ (a PC2 channel blocker), and dominant-negative pkd2 mutant (pkd2-D511V). Corresponding to the increase in channel currents, ANG II stimulation increased expression of PC2 on the cell surface of MCs and interaction with TRPC1 and TRPC4. Furthermore, ANG II-induced MC contraction was significantly reduced in pkd2-knocked down MCs. These data suggest that PC2 selectively assembles with TRPC1 and TRPC4 to form channel complexes mediating ANG II-induced Ca2+ responses in MCs.

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