The volume-regulated anion channel (LRRC8) in nodose neurons is sensitive to acidic pH
Runping Wang, Yongjun Lu, Susheel Gunasekar, Yanhui Zhang, Christopher J. Benson, Mark W. Chapleau, Rajan Sah, François M. Abboud
Runping Wang, Yongjun Lu, Susheel Gunasekar, Yanhui Zhang, Christopher J. Benson, Mark W. Chapleau, Rajan Sah, François M. Abboud
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Research Article Cell biology

The volume-regulated anion channel (LRRC8) in nodose neurons is sensitive to acidic pH

Abstract

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.

Authors

Runping Wang, Yongjun Lu, Susheel Gunasekar, Yanhui Zhang, Christopher J. Benson, Mark W. Chapleau, Rajan Sah, François M. Abboud

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Figure 8

Protective effect of pHo-conditioned Cl– current during simulated ischemia and NMDA-induced apoptosis.

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Protective effect of pHo-conditioned Cl– current during simulated ischem...
(A) Lactate dehydrogenase (LDH) release from neurons in culture over a period of 1 and 3 hours exposure to “simulated ischemia” with acidic bathing solutions (pHo 5.0) that were oxygen deprived with glucose (open bars) or without glucose (striped bars). All responses are normalized to the mean value obtained after 3 hours of ischemia in each group (n = number of coverslips in each group). In each panel, control (Con) values were obtained over a period of 3 hours without ischemia and with or without glucose. The increase in LDH is negligible. The left panel shows progressive increases in LDH from 0.02 ± 0.01 to 0.22 ± 0.01 and 1.00 ± 0.19 at 1 and 3 hours of ischemia with glucose and from 0.08 ± 0.02 to 0.46 ± 0.03 and 1.00 ± 0.10 at 1 and 3 hours of ischemia without glucose. Activation of the low pHo–conditioned current (middle panel) reduces LDH release to corresponding values of 0.16 ± 0.03 and 0.48 ± 0.10 with glucose, and 0.29 ± 0.03 and 0.69 ± 0.06 without glucose, at 1 and 3 hours of ischemia, respectively; P < 0.05. When tamoxifen was added to prevent pHo conditioning (right panel), the protective effect is totally abrogated, and LDH release during “ischemia” was restored to 0.90 ± 0.13 at 3 hours with glucose and to 0.59 ± 0.08 and 0.89 ± 0.17 at 1 and 3 hour without glucose. (B) Neuronal apoptosis following NMDA exposure. The images show bright fields of all neurons on individual cover slips and the corresponding green fluorescent images of the apoptotic cells labeled with annexin V. In the absence of NMDA, there was essentially no detectable apoptosis over a period of 60 minutes (control). The percentile of apoptotic cells increased progressively to 15.9% ± 1.3% (n = 12), 32.6% ± 1.7% (n = 8), and 46.0% ± 4.3% (n = 8) after exposures to NMDA for 20, 40, and 60 minutes (left panel). Following activation of the Cl– current (middle panel), the % apoptotic cells is reduced dramatically to 6.2% ± 1.5% (n = 11), 10.3% ± 3.2% (n = 11), and 15.6% ± 3.1% (n = 12), respectively (P < 0.01). Tamoxifen (10 μM) partially reversed the protective effect of low pHo conditioning (right panel), and apoptosis increased to 11.9% ± 3.1% (n = 4), 21.6% ± 4.9% (n = 4), and 26.5% ± 1.5% (n = 4) for 20, 40, and 60 minutes NMDA exposure (P < 0.01). Bars include responses of individual coverslips with means ± SE; all statistical comparisons used 2-way ANOVA.