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An interoceptive homeostatic reflex monitors levels of CO 2 /H + to maintain blood gas homeostasis and rapidly regulate tissue acid-base balance by driving lung ventilation and CO 2 excretion-this CO 2 -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 CO 2 /H + RTN neurons express GPR4 and TASK-2, global deletion of which abrogates CO 2 /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 CO 2 /H + sensitivity and the HCVR. In global GPR4(H81F) and GPR4(H167F) mice, CO 2 -stimulated breathing and CO 2 -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. CO 2 -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., Competing Interests: The authors declare no competing financial interest., (Copyright © 2024 Gonye et al.)

GPR4 is a proton-sensing G protein-coupled receptor implicated in many peripheral and central physiological processes. GPR4 expression has previously been assessed only via detection of the cognate transcript or indirectly, by use of fluorescent reporters. In this work, CRISPR/Cas9 knock-in technology was used to encode a hemagglutinin (HA) epitope tag within the endogenous locus of Gpr4 and visualize GPR4-HA in the mouse central nervous system using a specific, well characterized HA antibody; GPR4 expression was further verified by complementary Gpr4 mRNA detection. HA immunoreactivity was found in a limited set of brain regions, including in the retrotrapezoid nucleus (RTN), serotonergic raphe nuclei, medial habenula, lateral septum, and several thalamic nuclei. GPR4 expression was not restricted to cells of a specific neurochemical identity as it was observed in excitatory, inhibitory, and aminergic neuronal cell groups. HA immunoreactivity was not detected in brain vascular endothelium, despite clear expression of Gpr4 mRNA in endothelial cells. In the RTN, GPR4 expression was detected at the soma and in proximal dendrites along blood vessels and the ventral surface of the brainstem; HA immunoreactivity was not detected in RTN projections to two known target regions. This localization of GPR4 protein in mouse brain neurons corroborates putative sites of expression where its function has been previously implicated (e.g., CO 2 -regulated breathing by RTN), and provides a guide for where GPR4 could contribute to other CO 2 /H + modulated brain functions. Finally, GPR4-HA animals provide a useful reagent for further study of GPR4 in other physiological processes outside of the brain. Significance Statement GPR4 is a proton-sensing G-protein coupled receptor whose expression is necessary for a number of diverse physiological processes including acid-base sensing in the kidney, immune function, and cancer progression. In the brain, GPR4 has been implicated in the hypercapnic ventilatory response mediated by brainstem neurons. While knockout studies in animals have clearly demonstrated its necessity for normal physiology, descriptions of GPR4 expression have been limited due to a lack of specific antibodies for use in mouse models. In this paper, we implemented a CRISPR/Cas9 knock-in approach to incorporate the coding sequence for a small epitope tag into the locus of GPR4. Using these mice, we were able to describe GPR4 protein expression directly for the first time., (Copyright © 2024 Gonye et al.)

An interoceptive homeostatic system monitors levels of CO 2 /H + and provides a proportionate drive to respiratory control networks that adjust lung ventilation to maintain physiologically appropriate levels of CO 2 and rapidly regulate tissue acid-base balance. It has long been suspected that the sensory cells responsible for the major CNS contribution to this so-called respiratory CO 2 /H + chemoreception are located in the brainstem-but there is still substantial debate in the field as to which specific cells subserve the sensory function. Indeed, at the present time, several cell types have been championed as potential respiratory chemoreceptors, including neurons and astrocytes. In this review, we advance a set of criteria that are necessary and sufficient for definitive acceptance of any cell type as a respiratory chemoreceptor. We examine the extant evidence supporting consideration of the different putative chemoreceptor candidate cell types in the context of these criteria and also note for each where the criteria have not yet been fulfilled. By enumerating these specific criteria we hope to provide a useful heuristic that can be employed both to evaluate the various existing respiratory chemoreceptor candidates, and also to focus effort on specific experimental tests that can satisfy the remaining requirements for definitive acceptance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gonye and Bayliss.)

The interoceptive homeostatic mechanism that controls breathing, blood gases and acid-base balance in response to changes in CO 2 /H + is exquisitely sensitive, with convergent roles proposed for chemosensory brainstem neurons in the retrotrapezoid nucleus (RTN) and their supporting glial cells. For astrocytes, a central role for NBCe1, a Na + -HCO 3 - cotransporter encoded by Slc4a4, has been envisaged in multiple mechanistic models (i.e. underlying enhanced CO 2 -induced local extracellular acidification or purinergic signalling). We tested these NBCe1-centric models by using conditional knockout mice in which Slc4a4 was deleted from astrocytes. In GFAP-Cre;Slc4a4 fl/fl mice we found diminished expression of Slc4a4 in RTN astrocytes by comparison to control littermates, and a concomitant reduction in NBCe1-mediated current. Despite disrupted NBCe1 function in RTN-adjacent astrocytes from these conditional knockout mice, CO 2 -induced activation of RTN neurons or astrocytes in vitro and in vivo, and CO 2 -stimulated breathing, were indistinguishable from NBCe1-intact littermates; hypoxia-stimulated breathing and sighs were likewise unaffected. We obtained a more widespread deletion of NBCe1 in brainstem astrocytes by using tamoxifen-treated Aldh1l1-Cre/ERT2;Slc4a4 fl/fl mice. Again, there was no difference in effects of CO 2 or hypoxia on breathing or on neuron/astrocyte activation in NBCe1-deleted mice. These data indicate that astrocytic NBCe1 is not required for the respiratory responses to these chemoreceptor stimuli in mice, and that any physiologically relevant astrocytic contributions must involve NBCe1-independent mechanisms. KEY POINTS: The electrogenic NBCe1 transporter is proposed to mediate local astrocytic CO 2 /H+ sensing that enables excitatory modulation of nearby retrotrapezoid nucleus (RTN) neurons to support chemosensory control of breathing. We used two different Cre mouse lines for cell-specific and/or temporally regulated deletion of the NBCe1 gene (Slc4a4) in astrocytes to test this hypothesis. In both mouse lines, Slc4a4 was depleted from RTN-associated astrocytes but CO 2 -induced Fos expression (i.e. cell activation) in RTN neurons and local astrocytes was intact. Likewise, respiratory chemoreflexes evoked by changes in CO 2 or O 2 were unaffected by loss of astrocytic Slc4a4. These data do not support the previously proposed role for NBCe1 in respiratory chemosensitivity mediated by astrocytes., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Select neuronal populations display steady rhythmic neuronal firing that provides tonic excitation to drive downstream networks and behaviors. In noradrenergic neurons of the locus coeruleus (LC), circadian neurons of the suprachiasmatic nucleus (SCN), and CO 2 /H + -activated neurons of the brainstem retrotrapezoid nucleus (RTN), large subthreshold membrane potential oscillations contribute to the pacemaker-like action potential discharge. The oscillations and firing in LC and SCN involve contributions from leak sodium (NALCN) and L-type calcium channels while recent work from RTN suggested an additional pivotal role for a secondary calcium-activated and voltage-gated cationic current sensitive to TRPM4 channel blockers. Here, we tested whether TRPM4 contributes to subthreshold oscillations in mouse LC and SCN. By RNAscope in situ hybridization, Trpm4 transcripts were detected in both cell groups. In whole-cell recordings from acute slice preparations, prominent voltage-dependent membrane potential oscillations were revealed in LC and SCN after blocking action potentials. These oscillations were inhibited by two chemically-distinct blockers of TRPM4, 9-phenanthrol (9-pt) and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). Under whole-cell voltage clamp, inward currents evoked by oscillation voltage waveforms were inhibited in LC by blocking L-type calcium channels and TRPM4. These data implicate TRPM4 in the large subthreshold membrane potential oscillations that underlie tonic action potential discharge in LC and SCN, providing a voltage-dependent and calcium-dependent cationic current to augment the depolarizing inward Na + and Ca 2+ currents previously associated with this distinctive electroresponsive property., (Copyright © 2021 Li et al.)

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism., (© 2021. The Author(s).)

Brainstem networks that control regular tidal breathing depend on excitatory drive, including from tonically active, CO 2 /H + -sensitive neurons of the retrotrapezoid nucleus (RTN). Here, we examine intrinsic ionic mechanisms underlying the metronomic firing activity characteristic of RTN neurons. In mouse brainstem slices, large-amplitude membrane potential oscillations are evident in synaptically isolated RTN neurons after blocking action potentials. The voltage-dependent oscillations are abolished by sodium replacement; blocking calcium channels (primarily L-type); chelating intracellular Ca 2+ ; and inhibiting TRPM4, a Ca 2+ -dependent cationic channel. Likewise, oscillation voltage waveform currents are sensitive to calcium and TRPM4 channel blockers. Extracellular acidification and serotonin (5-HT) evoke membrane depolarization that augments TRPM4-dependent oscillatory activity and action potential discharge. Finally, inhibition of TRPM4 channels in the RTN of anesthetized mice reduces central respiratory output. These data implicate TRPM4 in a subthreshold oscillation that supports the pacemaker-like firing of RTN neurons required for basal, CO 2 -stimulated, and state-dependent breathing., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Signaling bias is the propensity for some agonists to preferentially stimulate G protein-coupled receptor (GPCR) signaling through one intracellular pathway versus another. We previously identified a G protein-biased agonist of the D 2 dopamine receptor (D2R) that results in impaired β-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with β-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired β-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the β 2 -adrenergic receptor (β2R) to build β2R-WT and β2R-Y199 5.38 A models in complex with the full β2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in β2R-Y199 5.38 A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in β2R-Y199 5.38 A, which is predicted to affect its interactions with β-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)