The leucine rich repeat containing protein 8A (LRRC8A), or SWELL1, is an essential component of the volume-regulated anion channel (VRAC) that is activated by cell swelling and ionic strength. We report here for the first time to our knowledge its expression in a primary cell culture of nodose ganglia neurons and its localization in the soma, neurites, and neuronal membrane. We show that this neuronal VRAC/SWELL1 senses low external pH (pHo) in addition to hypoosmolarity. A robust sustained chloride current is seen in 77% of isolated nodose neurons following brief exposures to extracellular acid pH. Its activation involves proton efflux, intracellular alkalinity, and an increase in NOX-derived H2O2. The molecular identity of both the hypoosmolarity-induced and acid pHo–conditioned VRAC as LRRC8A (SWELL1) was confirmed by Cre-flox–mediated KO, shRNA-mediated knockdown, and CRISPR/Cas9-mediated LRRC8A deletion in HEK cells and in primary nodose neuronal cultures. Activation of VRAC by low pHo reduces neuronal injury during simulated ischemia and N-methyl-D-aspartate–induced (NMDA-induced) apoptosis. These results identify the VRAC (LRRC8A) as a dual sensor of hypoosmolarity and low pHo in vagal afferent neurons and define the mechanisms of its activation and its neuroprotective potential.
Runping Wang, Yongjun Lu, Susheel Gunasekar, Yanhui Zhang, Christopher J. Benson, Mark W. Chapleau, Rajan Sah, François M. Abboud
Schematic summary of activation mechanisms of volume-regulated anion channel (VRAC/LRRC8) by pHo conditioning and hypoosmolarity.
In nodose neuron, extracellular low pH activates transient acid-sensing ion channels (ASICs), inhibits a K+ current, and activates a large and prolonged pH-conditioned Cl– conductance similar to the VRAC/LRRC8, causing depolarization. Extracellular low pH induces the pH-conditioned VRAC current by causing intracellular alkalinity. Proton leak through the plasma membrane during acidic pH exposures, causing a transient drop in pHi, which in turn activates Na+/H+ exchangers (NHE) and proton channels to increase pHi. Alkalinity sustains NOX activation. The released H2O2 opens a pH-conditioned VRAC/LRRC8 channel (efflux of Cl– ions produces large inward currents that cause depolarization). The same VRAC is induced by hypoosmolarity believed to cause a swelling-mediated mechanical stimulation of angiotensin II AT1 receptors (AT1R), which also activate NOX to produce H2O2. The dual NOX activation by intracellular alkalinity or by stimulation of AT1R mediates a prolonged and pronounced Cl– efflux and membrane depolarization of nodose neurons. Activation of this pHo-conditioned current reduces neuronal ishemic damage and NMDA-induced apoptosis. Thus, VRAC/LRRC8 provides dual sensory neuroprotective effect for vagal afferents against low pH and cell swelling. CaCC, Ca2+-dependent Cl– channels.