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 7

The pHo-conditioned Cl– current is dependent on intracellular alkalinity.

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The pHo-conditioned Cl– current is dependent on intracellular alkalinity...
(A) The pHo-conditioned current is significantly reduced when changes in pHi are buffered effectively with 40 mM HEPES (3.0 ± 0.6 pA/pF, n = 4 mice) compared with 10 mM HEPES (11.8 ± 2.0 pA/pF, n = 4 mice, **P < 0.01). However, the effect of hypoosmolarity is still preserved (23.7 ± 5.1, n = 2 mice in 40mM HEPES, vs. 31.0 ± 4.4 pA/pF, n = 4 mice in 10 mM HEPES, P > 0.05). (B) The pH-sensitive fluorescence of BCECF is changed from “blue” at pHo 7.4 (first image) to purple during exposures to pHo 6.0 (middle image) and to green, yellow, and red after the exposure (right image), reflecting variable increases in pHi. Horizontal bar in the right panel represent 20 μm. (C) Tracings represent changes in fluorescence recorded from 5 different neurons simultaneously first during their transient exposures to pHo 6.0 and then following the increases in pH to a variable degree in each neuron. Those increases are rapidly reduced when Zn2+ and amiloride are added (see arrow). (D) Normal pHi of 7.2 ± 0.04 (n = 40 neurons from 3 mice at pHo =7.4) drops to 7.11 ± 0.01 (n = 40 neurons from 3 mice, **P < 0.01) during the transient exposures to pHo 6.0 and then increases to pHi 7.54 ± 0.05 during the peak conditioned current (n = 40 neurons from 3 mice, **P < 0.001). (E) pHi drops (ΔpHi) by 0.13 ± 0.01 units (n = 40 neurons from 3mice, **P < 0.01) during exposures to pHo 6.0, increases by 0.29 ± 0.05 units (n = 40 from 3 mice, 2 data points not shown, **P < 0.01) after exposure and then rapidly declines by 0.13 ± 0.02 units (n = 39 from 3 mice, one point not shown, **P < 0.01) with the addition of Zn2+ and amiloride. (F) The pHo-conditioned current (black arrow) is inhibited from 23.4 ± 6.5 (n = 5 mice) to 3.6 ± 1.1 pA/pF (n = 4 mice, *P < 0.05) with amiloride and Zn2+, but the hypoosmolar-induced current remains intact (25.9 ± 6.3, n = 4 mice, vs. 28.6 ± 4.5 pA/pF, n = 4 mice, P > 0.05). (G) A drop in pHi from 7.25 down to 7.0 and 6.0 does not induce a significant pH-conditioned current, with values of 1.4 ± 0.6 (n = 2 mice), 0.4 ± 0.1 (n = 2 mice), and 1.8 ± 0.8 (n = 2 mice) pA/pF, respectively. In contrast, increases in pHi to 7.5, 7.6, and 7.8 cause progressively larger and prolonged inward currents, averaging 9.2 ± 1.8 (n = 3 mice), 16.3 ± 4.1 (n = 4 mice), and 27.5 ± 8.5 pA/pF (n = 3 mice), respectively (P = 0.021 by ANOVA). All panels include responses of individual neurons with means ± SE. Statistical comparisons are unpaired 2-tailed Student’s t test, except G.