Intrinsic Molecular Proton Sensitivity Underlies GPR4 Effects on Retrotrapezoid Nucleus Neuronal Activation and...

An interoceptive homeostatic reflex monitors levels of CO₂/H⁺ to maintain blood gas homeostasis and rapidly regulate tissue acid-base balance by driving lung ventilation and CO₂ excretion-this CO₂-evoked increase in respiration is the hypercapnic ventilatory reflex (HCVR). Retrotrapezoid nucleus (RTN) neurons provide crucial excitatory drive to downstream respiratory rhythm/pattern-generating circuits, and their activity is directly modulated by changes in CO₂/H⁺ RTN neurons express GPR4 and TASK-2, global deletion of which abrogates CO₂/H⁺ activation of RTN neurons and the HCVR. It has not been determined if the intrinsic pH sensitivity of these proton detectors is required for these effects. We used CRISPR/Cas9 genome editing to generate mice with mutations in either of two pH-sensing histidine residues in GPR4 to determine effects on RTN neuronal CO₂/H⁺ sensitivity and the HCVR. In global GPR4(H81F) and GPR4(H167F) mice, CO₂-stimulated breathing and CO₂-induced RTN neuronal activation were strongly blunted, with no effect on hypoxia-stimulated breathing. In brainstem slices from GPR4(H81F) mice, peak firing of RTN neurons during bath acidification was significantly reduced compared with GPR4 wild-type mice, and a subpopulation of RTN neurons was rendered pH-insensitive, phenocopying previous results from GPR4-deleted mice. These effects were independent of changes in RTN number/distribution, neuronal excitability or transcript levels for GPR4 and TASK-2. CO₂-stimulated breathing was reduced to a similar extent in GPR4(H81F) and TASK-2-deleted mice, with combined mutation yielding no additional deficit in the HCVR. Together, these data demonstrate that the intrinsic pH sensitivity of GPR4 is necessary for full elaboration of the HCVR.