Blood gas and tissue pH regulation depend on the ability of the brain to sense CO2 and/or H+ and alter breathing appropriately, a homeostatic process called central respiratory chemosensitivity. We show that selective expression of the proton-activated receptor GPR4 in chemosensory neurons of the mouse retrotrapezoid nucleus (RTN) is required for CO2-stimulated breathing. Genetic deletion of GPR4 disrupted acidosis-dependent activation of RTN neurons, increased apnea frequency and blunted ventilatory responses to CO2. Reintroduction of GPR4 into RTN neurons restored CO2-dependent RTN neuronal activation and rescued the ventilatory phenotype. Additional elimination of TASK-2, a pH-sensitive K+ channel expressed in RTN neurons, essentially abolished the ventilatory response to CO2. The data identify GPR4 and TASK-2 as distinct, parallel and essential central mediators of respiratory chemosensitivity.

Phox2b-expressing glutamatergic neurons of the retrotrapezoid nucleus (RTN) display properties expected of central respiratory chemoreceptors; they are directly activated by CO 2 /Hϩ via an unidentified pH-sensitive background K ϩ channel and, in turn, facilitate brainstem networks that control breathing. Here, we used a knock-out mouse model to examine whether TASK-2 (K2P5), an alkaline-activated background K ϩ channel, contributes to RTN neuronal pH sensitivity. We made patch-clamp recordings in brainstem slices from RTN neurons that were identified by expression of GFP (directed by the Phox2b promoter) or ␤-galactosidase (from the gene trap used for TASK-2 knock-out). Whereas nearly all RTN cells from control mice were pH sensitive (95%, n ϭ 58 of 61), only 56% of GFP-expressing RTN neurons from TASK-2 Ϫ/Ϫ mice (n ϭ 49 of 88) could be classified as pH sensitive (Ͼ30% reduction in firing rate from pH 7.0 to pH 7.8); the remaining cells were pH insensitive (44%). Moreover, none of the recorded RTN neurons from TASK-2 Ϫ/Ϫ mice selected based on ␤-galactosidase activity (a subpopulation of GFP-expressing neurons) were pH sensitive. The alkaline-activated background K ϩ currents were reduced in amplitude in RTN neurons from TASK-2 Ϫ/Ϫ mice that retained some pH sensitivity but were absent from pH-insensitive cells. Finally, using a working heart-brainstem preparation, we found diminished inhibition of phrenic burst amplitude by alkalization in TASK-2 Ϫ/Ϫ mice, with apneic threshold shifted to higher pH levels. In conclusion, alkaline-activated TASK-2 channels contribute to pH sensitivity in RTN neurons, with effects on respiration in situ that are particularly prominent near apneic threshold.

The activity of background potassium and sodium channels determines neuronal excitability, but physiological roles for "leak" Na ϩ channels in specific mammalian neurons have not been established. Here, we show that a leak Na ϩ channel, Nalcn, is expressed in the CO 2 /H ϩ -sensitive neurons of the mouse retrotrapezoid nucleus (RTN) that regulate breathing. In RTN neurons, Nalcn expression correlated with higher action potential discharge over a more alkalized range of activity; shRNA-mediated depletion of Nalcn hyperpolarized RTN neurons, and reduced leak Na ϩ current and firing rate. Nalcn depletion also decreased RTN neuron activation by the neuropeptide, substance P, without affecting pH-sensitive background K ϩ currents or activation by a cotransmitter, serotonin. In vivo, RTN-specific knockdown of Nalcn reduced CO 2 -evoked neuronal activation and breathing; hypoxic hyperventilation was unchanged. Thus, Nalcn regulates RTN neuronal excitability and stimulation by CO 2 , independent of direct pH sensing, potentially contributing to respiratory effects of Nalcn mutations; transmitter modulation of Nalcn may underlie state-dependent changes in breathing and respiratory chemosensitivity.

We discuss recent evidence which suggests that the principal central respiratory chemoreceptors are located within the retrotrapezoid nucleus (RTN) and that RTN neurons are directly sensitive to [H(+) ]. RTN neurons are glutamatergic. In vitro, their activation by [H(+) ] requires expression of a proton-activated G protein-coupled receptor (GPR4) and a proton-modulated potassium channel (TASK-2) whose transcripts are undetectable in astrocytes and the rest of the lower brainstem respiratory network. The pH response of RTN neurons is modulated by surrounding astrocytes but genetic deletion of RTN neurons or deletion of both GPR4 and TASK-2 virtually eliminates the central respiratory chemoreflex. Thus, although this reflex is regulated by innumerable brain pathways, it seems to operate predominantly by modulating the discharge rate of RTN neurons, and the activation of RTN neurons by hypercapnia may ultimately derive from their intrinsic pH sensitivity. RTN neurons increase lung ventilation by stimulating multiple aspects of breathing simultaneously. They stimulate breathing about equally during quiet wake and non-rapid eye movement (REM) sleep, and to a lesser degree during REM sleep. The activity of RTN neurons is regulated by inhibitory feedback and by excitatory inputs, notably from the carotid bodies. The latter input operates during normo- or hypercapnia but fails to activate RTN neurons under hypocapnic conditions. RTN inhibition probably limits the degree of hyperventilation produced by hypocapnic hypoxia. RTN neurons are also activated by inputs from serotonergic neurons and hypothalamic neurons. The absence of RTN neurons probably underlies the sleep apnoea and lack of chemoreflex that characterize congenital central hypoventilation syndrome.

Genetic variation in GRK4gamma was associated with HT in the subjects studied.