Intrinsic molecular proton sensitivity underlies GPR4 effects on retrotrapezoid nucleus (RTN) neuronal activation and CO2-stimulated breathing.

Abstract

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 to 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.Significance Statement Among the critical mechanisms for whole-body homeostasis, the hypercapnic ventilatory reflex (HCVR) regulates lung ventilation in order to maintain physiological levels of arterial PCO2 and acid-base balance. GPR4 is a proton-activated receptor and putative molecular proton sensor in retrotrapezoid nucleus (RTN) neurons, which are a crucial neural component of this respiratory reflex. In this work, we developed multiple lines of mice in which the intrinsic pH sensitivity of GPR4 was globally disabled by mutations in key histidine residues; in those mice, CO2/H+-sensitivity of RTN neurons and the HCVR were strongly blunted. These data support a role for GPR4 as a direct molecular sensor for CO2/H+ in support of this important homeostatic reflex.