Store-Operated Calcium Entry in Müller Glia Is Controlled by Synergistic Activation of TRPC and Orai Channels

Abstract

The endoplasmic reticulum (ER) is at the epicenter of astrocyte Ca 2ϩ signaling. We sought to identify the molecular mechanism underlying store-operated calcium entry that replenishes ER stores in mouse Müller cells. Store depletion, induced through blockade of sequestration transporters in Ca 2ϩ -free saline, induced synergistic activation of canonical transient receptor potential 1 (TRPC1) and Orai channels. Store-operated TRPC1 channels were identified by their electrophysiological properties, pharmacological blockers, and ablation of the Trpc1 gene. Ca 2ϩ release-activated currents (I CRAC ) were identified by ion permeability, voltage dependence, and sensitivity to selective Orai antagonists Synta66 and GSK7975A. Depletion-evoked calcium influx was initiated at the Müller end-foot and apical process, triggering centrifugal propagation of Ca 2ϩ waves into the cell body. EM analysis of the end-foot compartment showed high-density ER cisternae that shadow retinal ganglion cell (RGC) somata and axons, protoplasmic astrocytes, vascular endothelial cells, and ER-mitochondrial contacts at the vitreal surface of the end-foot. The mouse retina expresses transcripts encoding both Stim and all known Orai genes; Müller glia predominantly express stromal interacting molecule 1 (STIM1), whereas STIM2 is mainly confined to the outer plexiform and RGC layers. Elimination of TRPC1 facilitated Müller gliosis induced by the elevation of intraocular pressure, suggesting that TRPC channels might play a neuroprotective role during mechanical stress. By characterizing the properties of storeoperated signaling pathways in Müller cells, these studies expand the current knowledge about the functional roles these cells play in retinal physiology and pathology while also providing further evidence for the complexity of calcium signaling mechanisms in CNS astroglia.

Expression of peptidylarginine deiminase 4 in an alkali injury model of retinal gliosis

“…The finding that PADs are present in the uninjured retina indicates that they are expressed at basal levels, allowing potentially rapid response to changes in calcium concentrations [33],[34]. Calcium levels increase in Müller glial cells after stretching [35], which cause alterations in cytoskeletal mechanical function [36].…”

“…PAD2 is known to citrullinate vimentin [37]. PADs 1–4 are more enzymatically active in the presence of calcium, creating an ideal system to respond to changes that are induced by enhanced mechanosensitivity from Müller glia on increases in intraocular pressure [38],[33]. The propagation of calcium waves between Müller glia and astrocytes also provides a mechanism to synchronize the retinal response across the retina [39],[40],[41].…”

Subcellular propagation of calcium waves in Müller glia does not require autocrine/paracrine purinergic signaling

“…Our findings therefore argue against purinergic autofeedback as a contributory factor to SOCE-induced [Ca 2C ] i elevations in M€ uller glia. To address whether P 2 signaling contributes to subcellular propagation of SOCE-initiated Ca 2C waves, 12 we assessed the kinetics of initiation and vertical propagation of the waves in cells with depleted Ca 2C stores. In our previous report 12 we demonstrated that the 10%-90% rise time of [Ca 2C ] SOCE in the end-foot process is decreased compared to the apical process and somata, however, the probability of wave origination was about equal for the two processes ([Ca 2C ] SOCE was initiated in the endfoot in »58% of cells and in the apical process in »42% of cells).…”

“…Our previous study showed that depletion synergistically activates Orai and TRPC1 channels, most likely in combination with the STIM1 depletion sensor 12 ; however the mechanism of wave propagation remained unclear. We know that Ca 2C waves in M€ uller cells can be evoked by numerous stimuli that include light, mechanical stress and hypotonicity 14,17,18 and that wave propagation can involve intrinsic P 2 receptors, ryanodine receptors, glutamate receptors and TRPV4 channels 12,16,18 possibly in association with AQP4. 20 As in cortical astrocytes, the fundamental step leading to regenerative Ca 2C events is likely to involve regenerative activation of the ER Ca 2C pool via the activation of GPCRs, phospholipase C and production of IP 3 ; whereas it remains to be seen whether mammalian M€ uller cells use ryanodine receptor signaling in wave propagation.…”

“…10,11 We reported that acutely dissociated mouse M€ uller glial cells express prominent I CRAC that was associated with elevations in [Ca 2C ] i . 12 Interestingly, depletion-evoked transmembrane currents and [Ca 2C ] i elevations in M€ uller cells were characterized by » equal activations of Orai and TRPC1 channels, suggesting that these radial glia are capable of finetuning their signaling through synergistic activation of dissimilar SOC channels. Another intriguing feature of store depletion was the transcellular propagation of Ca 2C reminiscent of wave-like phenomena evoked by light, glutamate spillover, mechanical activation, osmotic swelling, activation of TRPV4 channels and ryanodine release channels, and spontaneous waves observed in M€ uller glia in the absence of stimuli.…”