A Novel Mechanism of pH Buffering inC. elegansGlia: Bicarbonate Transport via the Voltage-Gated ClC Cl⁻Channel CLH-1

Abstract

An important function of glia is the maintenance of the ionic composition and pH of the synaptic microenvironment. In terms of pH regulation, HCO 3 Ϫ buffering has been shown to be important in both glia and neurons. Here, we used in vivo fluorescent pH imaging and RNA sequencing of the amphid sheath glia of Caenorhabditis elegans to reveal a novel mechanism of cellular HCO 3 Ϫ uptake. While the classical mechanism of HCO 3 Ϫ uptake involves Na ϩ /HCO 3 Ϫ cotransporters, here we demonstrate that the C. elegans ClC Cl Ϫ channel CLH-1 is highly permeable to HCO 3 Ϫ and mediates HCO 3 Ϫ uptake into amphid sheath glia. CLH-1 has homology and electrophysiological properties similar to the mammalian ClC-2 Cl Ϫ channel. Our data suggest that, in addition to maintaining synaptic Cl Ϫ concentration, these channels may also be involved in maintenance of synaptic pH via HCO 3 Ϫ flux. These findings provide an exciting new facet of study regarding how pH is regulated in the brain.

Roles of the ClC Chloride Channel CLH-1 in Food-associated Salt Chemotaxis Behavior ofC. elegans

“…It has previously been reported that clh-1 is expressed in hypodermal cells, seam cells, D-cells of the vulva, and neuronal and glial cells of the head (Grant et al, 2015; Nehrke et al, 2000). These expression patterns were confirmed with a transcriptional reporter ( clh-1p::nls4::mTFP , see methods).…”

“…The mutant phenotype was rescued when cDNA was expressed either pan-neuronally or specifically in ASER, suggesting that clh-1 acts in the nervous system including ASER (Figure 3 b , similar results for pe577 , Figure 3—figure supplement 1 a ). On the other hand, clh-1(wt) cDNA failed to rescue clh-1(pe572) when expressed either in amphid sheath (AmSh) cells, where CLH-1 function as a pH mediator (Grant et al, 2015), or in the left-sided ASE neuron (ASEL) (Figure 3 b ). Unexpectedly, combined expression of the transgene in ASER and AmSh cells or expression in all ciliated neurons also failed to rescue the phenotype (Figure 3 b ), albeit it recovered chemotaxis of very limited population of animals.…”

“…A recent study showed that AmSh glia regulate the function and shape of AFD thermosensory neuron’s microvilli by modulating efflux of Cl - to the extracellular space of the receptive endings (Singhvi et al, 2016). In addition, it has been elucidated that CLH-1 transports anions to maintain intracellular pH of AmSh cells (Grant et al, 2015). These results implied the possibility that the receptive ending of ASER may be impaired in clh-1(pe) mutants.…”

“…Previous studies showed that CLH-1 mediates flux of univalent anions including chloride and bicarbonate. Furthermore, electrophysiological characterization of CLH-1 using Xenopus oocytes indicated that CLH-1 is an inwardly rectifying channel that conduct Cl - (Grant et al, 2015; Nehrke et al, 2000). Our genetic analyses suggested the clh-1(pe) mutations might be neomorphic.…”

“…CLH-1 shares the highest (37%) identity with mammalian ClC-2. Functional studies using heterologous expression in Xenopus oocytes and mammalian cells demonstrated that both CLH-1 and ClC-2 are inwardly rectifying chloride channels (Grant et al, 2015; Nehrke et al, 2000; Staley et al, 1996; Thiemann et al, 1992). On the other hand, two recent studies showed that ClC-2 contributes to Cl − influx in neurons depending on the electrochemical potential of Cl − (Ratté and Prescott, 2011; Rinke et al, 2010).…”