The amphid sheath glia of C. elegans are closely associated with 12 amphid sensory neurons. An important function of glia in general is the maintenance of the ionic composition and pH of the synaptic microenvironment. Previously, work from our lab showed that the pH sensitive DEG/ENaC channel ACD-1 is expressed in these glia, and that the activity of this channel is involved in modulation of chemosensory behavior in C. elegans. This highlights the likely importance of glial pH regulation in chemosensory signaling. However, to date no studies have been undertaken to determine the mechanisms by which the amphid sheath glia regulate pH. We expressed the GFP-based pH sensor Phlourin under the control of the glial-specific promoter PT02B11.3 to examine the mechanisms of intracellular pH regulation in amphid sheath glia, using in vivo fluorescent pH imaging. Incisions were made in the cuticle of the animals to allow for perfusion of solutions of different compositions over the glia. Initial experiments revealed that bicarbonate (HCO3-) transport is required for efficient buffering of pH in the glia. Interestingly however, HCO3- entry into the glia at baseline pH was not inhibited by removal of Na+ or Cl-, or by several pharmacological inhibitors of Na+/ HCO3- or Cl-/HCO3- transporters, which are commonly associated with HCO3- flux in many different cell types. RNA sequencing of AMsh cells revealed significant enrichment of clh-1 and clh-3 chloride channel transcripts in these cells. Knockout of clh-1, but not clh-3 blocked HCO3- entry into the glia. In addition, two- electrode voltage clamp experiments in Xenopus oocytes expressing CLH-1 showed that it is activated by decreases in extracellular pH and is permeable to HCO3-, revealing a role of CLH-1 in HCO3- flux and pH regulation. CLH-1 is also insensitive to the common chloride channel blockers DIDS (1 mM), NPPB (1mM), and niflumic acid (1 mM). This is in agreement with our earlier observation that these compounds do not inhibit HCO3- uptake in the amphid sheath glia. When examining behavioral responses of clh-1 animals, we found that they are are defective in the response to nose touch. We are now performing clh-1 rescue experiments in the amphid sheath glia to determine if the nose touch response does in fact require CLH-1 activity in glia. Our work identifies a novel molecular mechanism in glia for pH buffering mediated by an anion channel rather than a transporter and suggests that glial pH regulation plays a key role in the function of associated sensory neurons. .
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