Description

Cellular function is regulated by both intracellular and extracellular pH. Proton and bicarbonate transporters and exchangers, as well as ion channels permeable to protons and bicarbonate (HCO₃^–) participate to pH regulation (Chesler, 2003). Importantly, channels and transporters that regulate extracellular pH have the potential of influencing the function of nearby cells, making them particularly critical for the function of a tissue or organ as a whole. Extracellular pH regulation, and in particular buffering of extracellular acid loads, is especially important in the nervous system where high neuronal activity leads to the production and release in the extracellular space of a significant amount of acid.

We published that the ClC Cl^– channel CLH-1 is expressed in the Amphid sheath glial cells (AMsh glia) of the C. elegans amphid apparatus (Grant et al., 2015). These glial cells enwrap the sensory dendrites of 12 pairs of sensory neurons and are important for the structure and the function of these neurons (Bacaj et al., 2008, Singhvi et al., 2016, Raiders et al., 2021, Wang et al., 2008, Wang et al., 2012, Fernandez-Abascal et al., 2021, Wallace et al., 2016, Oikonomou et al., 2011, Razzauti and Laurent, 2021). Using the genetically encoded pH indicator Superecliptic pHlourin, we showed that CLH-1 is needed for influx of HCO₃^– into the AMsh glia at baseline pH, following removal of HCO₃^– from the extracellular space (Grant et al., 2015). However, by electrophysiological analysis of CLH-1 ionic currents in Xenopus oocytes and mammalian HEK cells, we also showed that this channel conducts ions preferably from the inside to the outside of the cell (Grant et al., 2015). This finding suggests that CLH-1 might mediate HCO₃^– efflux in vivo thereby resulting in regulation of the pH outside of AMsh glia, potentially even in the microenvironment between AMsh glia and sensory dendrites (Fig. 1A). Consistent with CLH-1-mediated efflux of ions, we found that CLH-1 mediates Cl^– efflux from AMsh glia needed for GABA regulation of ASH neurons’ excitability upon nose touch stimulation (Fernandez-Abascal et al., 2021).

To determine whether CLH-1 mediates HCO₃^– efflux in vivo, we compared pHlourin fluorescence in AMsh glia of wild type and clh-1 knockout mutants grown on control plates and on plates supplemented with 150 mM KHCO₃ (Johnson et al., 2020, Singhvi et al., 2016, Fernandez-Abascal et al., 2021) (Fig. 1B-C). pHlourin fluorescence intensity is proportional to the alkalinity of the cell, thus, higher fluorescence indicates that the intracellular environment is at a higher (more alkaline) pH. First, we found that AMsh glia of clh-1 knockout animals grown on control plates had on average a fluorescence intensity twice as high as in wild type (Fig. 1B-C). This result suggests that efflux of HCO₃^– from the AMsh glia is reduced in clh-1 knockout as compared to wild type. Second, AMsh glia pHlourin fluorescence of wild type animals grown on HCO₃^– enriched plates was like the fluorescence intensity seen in animals grown on control plates (Fig. 1B-C). This result indicates that in wild type AMsh glia an efficient HCO₃^– extrusion mechanism keeps the pH relatively stable despite the challenge with high concentration of HCO₃^–. On the contrary though, the AMsh glia of clh-1 knockout animals grown on HCO₃^– enriched plates had a fluorescence intensity that on average was one and half times higher than the fluorescence intensity of these cells in animals grown on control plates (Fig. 1B-C). This result confirms that HCO₃^– extrusion from AMsh glia is severely impaired in clh-1 knockout. These data also suggest that when challenged with 150 mM HCO₃^– AMsh transport HCO₃^– inside the cell in a CLH-1-independent manner. A CLH-1-independent HCO₃^– transport into AMsh glia was previously suggested by pH imaging of these cells under acid load challenge (Grant et al., 2015).

How do these results reconcile with our previous report (Grant et al., 2015)? The directionality of HCO₃^– flux through a channel depends on the driving force for the ion and the membrane potential. In our previous work that showed CLH-1-mediated HCO₃^– influx at baseline, pH imaging experiments were conducted on AMsh bathed in Hepes buffer and subsequently perfused with 20 mM HCO₃^–. Under those conditions, HCO₃^– was initially depleted from the intracellular milieu, resulting in an electrochemical gradient for HCO₃^– favoring inward current at negative resting membrane potential which was supported by our results. In the experiments reported here, growth in NGM control plates is expected to result in a physiological concentration of HCO₃^– of ~20 mM both inside and outside the AMsh cells (Harpur, 1974). Thus, at a negative resting membrane potential, HCO₃^– current is expected to be outward, which is supported by our results showing that clh-1 knockout leads to accumulation of HCO₃^– in the cell (Fig. 1C). When worms are grown in high HCO₃^– the reversal potential for this ion will tend to become more negative but it seems to remain more positive than the resting membrane potential, thus, continuing to favor HCO₃^– efflux through CLH-1. If clh-1 is knocked out, then efflux is impaired and accumulation of HCO₃^– is favored. Interestingly, we noticed that growth on high K⁺ (deriving from the dissociation of KHCO₃) does not seem to dramatically change the resting potential of the cell which appears to remain still more negative than the predicted reversal potential for HCO₃^– under these conditions (-47 mV). We note though that the exact concentration of K⁺ or HCO₃^– in worms grown on KHCO₃ plates is not known and may be different between the two ions.

To conclude, we propose that CLH-1 might regulate the pH outside AMsh glia, in particular in the microenvironment around the dendrites of the amphid sensory neurons, and that this might result in regulation of neuronal output. This CLH-1-dependent mechanism of regulation of neuronal function might be in addition to providing Cl^– ions for GABA signaling in nose touch avoidance (Fernandez-Abascal et al., 2021). Future imaging and behavioral experiments using an extracellular pH sensor and CLH-1 mutants with altered Cl^– and HCO₃^– permeability will test this hypothesis.