The temporal dynamics of morphogen presentation impacts transcriptional responses and tissue patterning (1). However, the mechanisms controlling morphogen release are far from clear. We found that inwardly rectifying potassium (Irk) channels regulate endogenous transient increases in intracellular calcium and Bone Morphogenetic Protein (BMP/Dpp) release for Drosophila wing development (2). Inhibition of Irk channels reduces BMP/Dpp signaling, and ultimately disrupts wing morphology (2, 3). Ion channels impact development of several tissues and organisms in which BMP signaling is essential (2-15). In neurons and pancreatic beta cells, Irk channels modulate membrane potential to affect intracellular Ca++ to control secretion of neurotransmitters and insulin (15-21). Based on Irk activity in neurons, we hypothesized that electrical activity controls endoplasmic reticulum Ca++ release into the cytoplasm to regulate the release of BMP. To test this hypothesis, we reduced expression of proteins that control endoplasmic reticulum calcium (Stim, Orai, SERCA, SK, and Best2) and documented wing phenotypes. We found that reduced Stim and SERCA function decreases amplitude and frequency of endogenous calcium transients in the wing disc and reduced Dpp/BMP release in the wing disc. Together, our results suggest control of endoplasmic reticulum is required for Dpp/BMP release.
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Photopharmacological modulation of native CRAC channels using azoboronate photoswitches
Store-operated calcium entry through calcium release–activated calcium (CRAC) channels replenishes intracellular calcium stores and plays a critical role in cellular calcium signaling. CRAC channels are activated by tightly regulated interaction between the endoplasmic reticulum (ER) calcium sensor STIM proteins and plasma membrane (PM) Orai channels. Our current understanding of the role of STIM–Orai-dependent calcium signals under physiologically relevant conditions remains limited in part due to a lack of spatiotemporally precise methods for direct manipulation of endogenous CRAC channels. Here, we report the synthesis and characterization of azoboronate light-operated CRAC channel inhibitors (LOCIs) that allow for a dynamic and fully reversible remote modulation of the function of native CRAC channels using ultraviolet (UV) and visible light. We demonstrate the use of LOCI-1 to modulate gene expression in T lymphocytes, cancer cell seeding at metastatic sites, and pain-related behavior.
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- Award ID(s):
- 2116412
- NSF-PAR ID:
- 10320054
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 119
- Issue:
- 13
- ISSN:
- 0027-8424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Key points Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage.
Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level.
We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure.
Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell‐extracellular matrix contacts and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse.
This study helps elucidate basic mechanotransduction properties that may contribute to intraocular pressure regulation in the vertebrate eye.
Abstract Chronic elevations in intraocular pressure (IOP) can cause blindness by compromising the function of trabecular meshwork (TM) cells in the anterior eye, but how these cells sense and transduce pressure stimuli is poorly understood. Here, we demonstrate functional expression of two mechanically activated channels in human TM cells. Pressure‐induced cell stretch evoked a rapid increase in transmembrane current that was inhibited by antagonists of the mechanogated channel Piezo1, Ruthenium Red and GsMTx4, and attenuated in Piezo1‐deficient cells. The majority of TM cells exhibited a delayed stretch‐activated current that was mediated independently of Piezo1 by TRPV4 (transient receptor potential cation channel, subfamily V, member 4) channels. Piezo1 functions as the principal TM transducer of physiological levels of shear stress, with both shear and the Piezo1 agonist Yoda1 increasing the number of focal cell‐matrix contacts. Analysis of TM‐dependent fluid drainage from the anterior eye showed significant inhibition by GsMTx4. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force‐sensing contexts. Piezo1‐dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure‐dependent outflow suggests potential for a novel therapeutic target in treating glaucoma.
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Abstract Small-conductance calcium-activated potassium (SK) channels show a ubiquitous distribution on neurons, in both somatodendritic and axonal regions. SK channels are associated with neuronal activity regulating action potential frequency, dendritic excitability, and synaptic plasticity. Although the physiology of SK channels and the mechanisms that control their surface expression levels have been investigated extensively, little is known about what controls SK channel diffusion in the neuronal plasma membrane. This aspect is important, as the diffusion of SK channels at the surface may control their localization and proximity to calcium channels, hence increasing the likelihood of SK channel activation by calcium. In this study, we successfully investigated the diffusion of SK channels labeled with quantum dots on human embryonic kidney cells and dissociated hippocampal neurons by combining a single-particle tracking method with total internal reflection fluorescence microscopy. We observed that actin filaments interfere with SK mobility, decreasing their diffusion coefficient. We also found that during neuronal maturation, SK channel diffusion was gradually inhibited in somatodendritic compartments. Importantly, we observed that axon barriers formed at approximately days in vitro 6 and restricted the diffusion of SK channels on the axon initial segment (AIS). However, after neuron maturation, SK channels on the AIS were strongly immobilized, even after disruption of the actin network, suggesting that crowding may cause this effect. Altogether, our work provides insight into how SK channels diffuse on the neuronal plasma membrane and how actin and membrane crowding impacts SK channel diffusion.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1073/pnas.2118160119
