NMDAR in cultured astrocytes: Flux‐independent pH sensor and flux‐dependent regulator of mitochondria and plasma membrane‐mitochondria bridging

Abstract

Glutamate N-methyl-D-aspartate (NMDA) receptor (NMDAR) is critical for neu-rotransmission as a Ca2+ channel. Nonetheless, flux-independent signaling has also been demonstrated. Astrocytes express NMDAR distinct from its neuronal counter-part, but cultured astrocytes have no electrophysiological response to NMDA. We recently demonstrated that in cultured astrocytes, NMDA at pH6 (NMDA/pH6) act-ing through the NMDAR elicits flux-independent Ca2+ release from the Endoplasmic Reticulum (ER) and depletes mitochondrial membrane potential (mΔΨ). Here we show that Ca2+ release is due to pH6 sensing by NMDAR, whereas mΔΨ deple-tion requires both: pH6 and flux-dependent NMDAR signaling. Plasma membrane (PM) NMDAR guard a non-random distribution relative to the ER and mitochon-dria. Also, NMDA/pH6 induces ER stress, endocytosis, PM electrical capacitance reduction, mitochondria-ER, and -nuclear contacts. Strikingly, it also produces the formation of PM invaginations near mitochondria along with structures referred to here as PM-mitochondrial bridges (PM-m-br). These and earlier data strongly sug-gest PM-mitochondria communication. As proof of the concept of mass transfer, we found that NMDA/pH6 provoked mitochondria labeling by the PM dye FM-4-64FX. NMDA/pH6 caused PM depolarization, cell acidification, and Ca2+ release from most mitochondria. Finally, the MCU and microtubules were not involved in mΔΨ depletion, while actin cytoskeleton was partially involved. These findings demon-strate that NMDAR has concomitant flux-independent and flux-dependent actions in cultured astrocytes. This work has been recently accepted for publication in The FASEB J.