Regulation of neuropathic pain by microglial Orai1 channels.

Microglia are important mediators of neuroinflammation, which underlies neuropathic pain. However, the molecular checkpoints controlling microglial reactivity are not well-understood. Here, we investigated the role of Orai1 channels for microglia-mediated neuroinflammation following nerve injury and find that deletion of Orai1 in microglia attenuates Ca2+ signaling and the production of inflammatory cytokines by proalgesic agonists. Conditional deletion of Orai1 attenuated microglial proliferation in the dorsal horn, spinal cytokine levels, and potentiation of excitatory neurotransmission following peripheral nerve injury. These cellular effects were accompanied by mitigation of pain hyperalgesia in microglial Orai1 knockout mice. A small-molecule Orai1 inhibitor, CM4620, similarly mitigated allodynia in male mice. Unexpectedly, these protective effects were not seen in female mice, revealing sexual dimorphism in Orai1 regulation of microglial reactivity and hyperalgesia. Together, these findings indicate that Orai1 channels are key regulators of the sexually dimorphic role of microglia for the neuroinflammation that underlies neuropathic pain.

The basic residues in the Orai1 channel inner pore promote opening of the outer hydrophobic gate.

Store-operated Orai1 channels regulate a wide range of cellular functions from gene expression to cell proliferation. Previous studies have shown that gating of Orai1 channels is regulated by the outer pore residues V102 and F99, which together function as a hydrophobic gate to block ion conduction in resting channels. Opening of this gate occurs through a conformational change that moves F99 away from the permeation pathway, leading to pore hydration and ion conduction. In addition to this outer hydrophobic gate, several studies have postulated the presence of an inner gate formed by the basic residues R91, K87, and R83 in the inner pore. These positively charged residues were suggested to block ion conduction in closed channels via mechanisms involving either electrostatic repulsion or steric occlusion by a bound anion plug. However, in contrast to this model, here we find that neutralization of the basic residues dose-dependently abolishes both STIM1-mediated and STIM1-independent activation of Orai1 channels. Molecular dynamics simulations show that loss of the basic residues dehydrates the pore around the hydrophobic gate and stabilizes the pore in a closed configuration. Likewise, the severe combined immunodeficiency mutation, Orai1 R91W, closes the channel by dewetting the hydrophobic stretch of the pore and stabilizing F99 in a pore-facing configuration. Loss of STIM1-gating in R91W and in the other basic residue mutants is rescued by a V102A mutation, which restores pore hydration at the hydrophobic gate to repermit ion conduction. These results indicate that the inner pore basic residues facilitate opening of the principal outer hydrophobic gate through a long-range effect involving hydration of the outer pore.

Enantiomeric Aß peptides inhibit the fluid shear stress response of PIEZO1.

Traumatic brain injury (TBI) elevates Abeta (Aß) peptides in the brain and cerebral spinal fluid. Aß peptides are amphipathic molecules that can modulate membrane mechanics. Because the mechanosensitive cation channel PIEZO1 is gated by membrane tension and curvature, it prompted us to test the effects of Aß on PIEZO1. Using precision fluid shear stress as a stimulus, we found that Aß monomers inhibit PIEZO1 at femtomolar to picomolar concentrations. The Aß oligomers proved much less potent. The effect of Aßs on Piezo gating did not involve peptide-protein interactions since the D and L enantiomers had similar effects. Incubating a fluorescent derivative of Aß and a fluorescently tagged PIEZO1, we showed that Aß can colocalize with PIEZO1, suggesting that they both had an affinity for particular regions of the bilayer. To better understand the PIEZO1 inhibitory effects of Aß, we examined their effect on wound healing. We observed that over-expression of PIEZO1 in HEK293 cells increased cell migration velocity ~10-fold, and both enantiomeric Aß peptides and GsMTx4 independently inhibited migration, demonstrating involvement of PIEZO1 in cell motility. As part of the motility study we examined the correlation of PIEZO1 function with tension in the cytoskeleton using a genetically encoded fluorescent stress probe. Aß peptides increased resting stress in F-actin, and is correlated with Aß block of PIEZO1-mediated Ca2+ influx. Aß inhibition of PIEZO1 in the absence of stereospecific peptide-protein interactions shows that Aß peptides modulate both cell membrane and cytoskeletal mechanics to control PIEZO1-triggered Ca2+ influx.

Heterogeneous Cytoskeletal Force Distribution Delineates the Onset Ca2+ Influx Under Fluid Shear Stress in Astrocytes.

Mechanical perturbations increase intracellular Ca2+ in cells, but the coupling of mechanical forces to the Ca2+ influx is not well understood. We used a microfluidic chamber driven with a high-speed pressure servo to generate defined fluid shear stress to cultured astrocytes, and simultaneously measured cytoskeletal forces using a force sensitive actinin optical sensor and intracellular Ca2+. Fluid shear generated non-uniform forces in actinin that critically depended on the stimulus rise time emphasizing the presence of viscoelasticity in the activating sequence. A short (ms) shear pulse with fast rise time (2 ms) produced an immediate increase in actinin tension at the upstream end of the cell with minimal changes at the downstream end. The onset of Ca2+ rise began at highly strained areas. In contrast to stimulus steps, slow ramp stimuli produced uniform forces throughout the cells and only a small Ca2+ response. The heterogeneity of force distribution is exaggerated in cells having fewer stress fibers and lower pre-tension in actinin. Disruption of cytoskeleton with cytochalasin-D (Cyt-D) eliminated force gradients, and in those cells Ca2+ elevation started from the soma. Thus, Ca2+ influx with a mechanical stimulus depends on local stress within the cell and that is time dependent due to viscoelastic mechanics.

Flow induced adherens junction remodeling driven by cytoskeletal forces.

Adherens junctions (AJs) are a key structural component for tissue organization and function. Under fluid shear stress, AJs exhibit dynamic assembly/disassembly, but how shear stress couples to AJs is unclear. In MDCK cells we measured simultaneously the forces in cytoskeletal α-actinin and the density and length of AJs using a genetically coded optical force sensor, actinin-sstFRET, and fluorescently labeled E-cadherin (E-cad). We found that shear stress of 0.74dyn/cm2 for 3h significantly enhanced E-cad expression at cell-cell contacts and this phenomenon has two phases. The initial formation of segregated AJ plaques coincided with a decrease in cytoskeletal tension, but an increase in tension was necessary for expansion of the plaques and the formation of continuous AJs in the later phase. The changes in cytoskeletal tension and reorganization appear to be an upstream process in response to flow since it occurred in both wild type and dominant negative E-cad cells. Disruption of F-actin with a Rho-ROCK inhibitor eliminated AJ growth under flow. These results delineate the shear stress transduction paths in cultured cells, which helps to understand pathology of a range of diseases that involve dysfunction of E-cadherin.