Receptor in the brain controls breathing: Control of breathing in mammals depends primarily not on sensing oxygen, but rather on detecting concentrations of carbon dioxide in the blood. Failure of this system can cause potentially deadly sleep apnias. Taking a hint from insects, which use a heterotrimeric guanine nucleotide–binding protein-coupled receptor (GPCR) to sense carbon dioxide, Kumar et al. demonstrate that the GPCR GPR4 is essential to control breathing in mice. GPR4 senses protons generated by the formation of carbonic acid in the blood and works with a pH-sensitive potassium channel called TASK-2 in a set of brain cells that control breathing. Science , this issue p. 1255

Phox2b-expressing glutamatergic neurons of the retrotrapezoid nucleus (RTN) display properties expected of central respiratory chemoreceptors; they are directly activated by CO₂/H⁺via an unidentified pH-sensitive background K⁺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⁺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 β-galactosidase (from the gene trap used for TASK-2 knock-out). Whereas nearly all RTN cells from control mice were pH sensitive (95%,n= 58 of 61), only 56% of GFP-expressing RTN neurons fromTASK-2^−/−mice (n= 49 of 88) could be classified as pH sensitive (>30% 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 fromTASK-2^−/−mice selected based on β-galactosidase activity (a subpopulation of GFP-expressing neurons) were pH sensitive. The alkaline-activated background K⁺currents were reduced in amplitude in RTN neurons fromTASK-2^−/−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 inTASK-2^−/−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 respirationin situthat are particularly prominent near apneic threshold.

The activity of background potassium and sodium channels determines neuronal excitability, but physiological roles for “leak” Na⁺ channels in specific mammalian neurons have not been established. Here, we show that a leak Na⁺ channel, Nalcn, is expressed in the CO₂/H⁺-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⁺ current and firing rate. Nalcn depletion also decreased RTN neuron activation by the neuropeptide, substance P, without affecting pH-sensitive background K⁺ currents or activation by a cotransmitter, serotonin. In vivo, RTN-specific knockdown of Nalcn reduced CO₂-evoked neuronal activation and breathing; hypoxic hyperventilation was unchanged. Thus, Nalcn regulates RTN neuronal excitability and stimulation by CO₂, 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. SIGNIFICANCE STATEMENT Breathing is an essential, enduring rhythmic motor activity orchestrated by dedicated brainstem circuits that require tonic excitatory drive for their persistent function. A major source of drive is from a group of CO₂/H⁺-sensitive neurons in the retrotrapezoid nucleus (RTN), whose ongoing activity is critical for breathing. The ionic mechanisms that support spontaneous activity of RTN neurons are unknown. We show here that Nalcn, a unique channel that generates “leak” sodium currents, regulates excitability and neuromodulation of RTN neurons and CO₂-stimulated breathing. Thus, this work defines a specific function for this enigmatic channel in an important physiological context.

Objective: To perform association studies of polymorphisms of the potential candidate essential hypertension (HT) genes GRK4, PTP1B and HSD3B1. Methods: Subjects consisted of 168 unrelated, Caucasian essential hypertensive (HT) patients and 312 normotensive (NT) controls. Biological power was increased by ensuring subjects in each group had parents with the same blood pressure (BP) status as theirs. Three GRK4γ variants (R65L, A142V and A486V), one HSD3B1 variant (T←C Leu338) and one PTP1B variant (1484insG) were genotyped by polymerase chain reaction and restriction enzyme digestion or by homogenous MassEXTENDTM Assay. Results: The V allele of the A486V variant of GRK4γ, but not the R65L or A142V variants, showed an association with HT (P = 0.02). The V allele was also associated with an elevation in systolic blood pressure (SBP) (P = 0.002). Although the L65 and the V142 alleles tracked with elevation in diastolic (DBP), this was seen only in male HTs (P = 0.009; P = 0.002, respectively). Haplotype frequencies differed between the HT and NT groups, particularly for the R, V, V haplotype combination of R65L, A142V and A486V, respectively. Neither of the HSD3B1 or PTP1B variants were associated with HT. Conclusion: Genetic variation in GRK4γ was associated with HT in the subjects studied.

Background: Ketamine is a commonly used anesthetic, but the mechanistic basis for its clinically relevant actions remains to be determined. The authors previously showed that HCN1 channels are inhibited by ketamine and demonstrated that global HCN1 knockout mice are twofold less sensitive to hypnotic actions of ketamine. Although that work identified HCN1 channels as a viable molecular target for ketamine, it did not determine the relevant neural substrate. Methods: To localize the brain region responsible for HCN1-mediated hypnotic actions of ketamine, the authors used a conditional knockout strategy to delete HCN1 channels selectively in excitatory cells of the mouse forebrain. A combination of molecular, immunohistochemical, and cellular electrophysiologic approaches was used to verify conditional HCN1 deletion; a loss-of-righting reflex assay served to ascertain effects of forebrain HCN1 channel ablation on hypnotic actions of ketamine. Results: In conditional knockout mice, HCN1 channels were selectively deleted in cortex and hippocampus, with expression retained in cerebellum. In cortical pyramidal neurons from forebrain-selective HCN1 knockout mice, effects of ketamine on HCN1-dependent membrane properties were absent; notably, ketamine was unable to evoke membrane hyperpolarization or enhance synaptic inputs. Finally, the EC₅₀ for ketamine-induced loss-of-righting reflex was shifted to significantly higher concentrations (by approximately 31%). Conclusions: These data indicate that forebrain principal cells represent a relevant neural substrate for HCN1-mediated hypnotic actions of ketamine. The authors suggest that ketamine inhibition of HCN1 shifts cortical neuron electroresponsive properties to contribute to ketamine-induced hypnosis.