TASK-2 Channels Contribute to pH Sensitivity of Retrotrapezoid Nucleus Chemoreceptor Neurons

TASK-2 deletion leads to blunted or absent pH sensitivity in RTN neurons. A–C, Representative firing rate responses to changes in bath pH obtained by cell-attached recordings in GFP-expressing RTN neurons from TASK-2^+/+ (A) and TASK-2^−/− (B, C) mice. Plots of firing rate at different bath pH levels are provided for each individual neuron (right column). D, Frequency distribution of the percentage decrease in firing from pH 7.0 to pH 7.8 in RTN neurons from TASK-2^+/+ (n = 61; blue) and TASK-2^−/− (n = 88; red) mice; a cutoff of >30% decrease in firing was used to classify cells as either pH sensitive or pH insensitive. E, The percentage of cells that were pH sensitive and pH insensitive were significantly different between TASK-2^+/+ and TASK-2^−/− mice (*p < 0.0001 by χ² analysis). F, The frequency (and cumulative probability; inset) distributions of pH₅₀ values for pH-sensitive RTN neurons from TASK-2^+/+ and TASK-2^−/− mice; data were fitted with Gaussian distributions that yielded mean pH₅₀ values that were significantly different between genotypes (7.45 ± 0.1 vs 7.73 ± 0.2 for TASK-2^+/+ and TASK-2^−/−, p < 0.0001). G, The relationship between firing rate and pH for RTN neurons from TASK-2^+/+ mice and for the subpopulations of RTN neurons from TASK-2^−/− mice that were classified as pH sensitive and pH insensitive. *p < 0.05 and **p < 0.01 versus TASK-2^+/+ by two-way ANOVA.

Alkaline-activated background K⁺ current is diminished or absent in TASK-2^−/− RTN neurons. Voltage-clamp recordings were obtained from GFP-expressing RTN neurons during bath acidification and alkalization; before gaining whole-cell access, each cell was first characterized as pH sensitive or pH insensitive based on firing responses to pH changes in the cell-attached configuration (Fig. 2). A, In a pH-sensitive TASK-2^+/+ RTN neuron, acidification decreased holding current (top trace) and conductance (bottom trace) and alkalization caused a reversible outward shift in current with an increase in conductance. B, In a pH-insensitive TASK-2^−/− RTN neuron, changes in bath pH had little effect on holding current or conductance. C, Averaged I–V relationship of the pH-sensitive current density (pH 8.0 minus pH 7.0) for RTN neurons from TASK-2^+/+ mice (n = 13) and for RTN neurons from TASK-2^−/− mice that were classified as pH sensitive (n = 12) and pH insensitive (n = 7). Note that the weakly rectifying alkaline-activated K⁺ current seen in TASK-2^+/+ neurons was reduced in pH-sensitive RTN neurons from TASK-2^−/− mice and absent in pH-insensitive TASK-2^−/− RTN neurons. **p < 0.01 versus TASK-2^+/+ by two-way ANOVA.

TASK-2 is strongly expressed in a subset of Phox2b-expressing RTN neurons. A, Photomicrographs of histochemical staining for X-gal (left) and GFP and TH (right) in the RTN region of the rostroventrolateral medulla. The boxed region depicted in the lower-magnification image (top) is shown at greater magnification (bottom). Note that, among the double-positive neurons (GFP⁺/X-gal⁺; white arrowheads), there were also some GFP⁺ neurons in the RTN that were not stained with X-gal (yellow arrowheads). B, Top, Schematic of the brainstem region containing the RTN, delimiting the landmarks used to align sections for cell counts. Bottom, Quantification of the total number of Phox2b-expressing chemoreceptor neurons in each section (defined as GFP⁺/TH⁻) and of the percentage of those RTN neurons that also showed detectable X-gal staining. Overall, β-galactosidase expression was observed in ∼63% (321 ± 40 of 504 ± 25 RTN neurons) of these RTN neurons, with an even fractional distribution throughout the rostrocaudal extent of the nucleus. 7n, Facial nerve; VII, facial nucleus; VRG, ventral respiratory group; NA, nucleus ambiguus. C, scPCR from a representative sample of GFP-expressing dissociated RTN neurons (lanes 1–6) revealed expression of Phox2b and VGlut2 in all cells tested, with no evidence for GAD1 and TH, as expected for chemosensitive RTN neurons. Notably, we detected TASK-2 in all but one of those RTN neurons (lane 5). The positive control GAPDH transcript was universally detectable, and no-template negative controls were included in all reactions (nt, lane 7). D, Verification by scPCR of TASK-2 deletion from individual Phox2b- and VGlut2-expressing RTN neurons obtained from TASK-2^−/− mice (lanes 1–6); TASK-2 expression was evident in two of three wild-type RTN neurons processed concurrently. E, Quantification of scPCR data revealed TASK-2 expression in 85% of RTN neurons (n = 52/61).

TASK-2 deletion ablates pH sensitivity in all RTN neurons selected for β-galactosidase activity. A, Representative whole-cell current-clamp recordings of membrane potential (top plots) and the associated firing rate (bottom traces) from RTN neurons stained with FDG, a fluorogenic β-galactosidase substrate, in slices from heterozygous TASK-2^+/− and homozygous TASK-2^−/− mice during exposure to bath solutions equilibrated with different levels of CO₂ (3, 5, and 10%, corresponding to pH 7.6, pH 7.4, and pH 7.1). The TASK-2^+/− cell showed typical pH-dependent changes in membrane potential and firing rate, whereas the TASK-2^−/− RTN neuron was insensitive to both acidification and alkalization. All FDG-labeled TASK-2^−/− neurons failed to respond to changes in pH. B, The relationship between firing rate and CO₂ (pH) for FDG-fluorescent RTN neurons from TASK-2^+/− (n = 14) and TASK-2^−/− (n = 11) mice. *p < 0.05, **p < 0.0001 versus TASK-2^+/−. C, Using whole-cell voltage clamp under conditions that block most voltage-dependent K⁺ channels (tetraethylammonium, 10 mm; 4-AP, 3 mm; and barium, 10 μm), currents were evoked with a series of voltage steps in an acidified (left, 10% CO₂, pH 7.1) and alkalized bath (right, 3% CO₂, pH 7.6) in FDG-labeled RTN neurons from TASK-2^+/− (top) and TASK-2^−/− (bottom) mice. D, Averaged I–V relationship of pH-sensitive current density (10% CO₂ minus 3% CO₂) for RTN neurons from TASK-2^+/− (n = 10) and TASK-2^−/− (n = 5) mice. Note that the alkaline-activated K⁺ current seen in TASK-2^+/− neurons was totally absent in FDG-fluorescent TASK-2^−/− RTN neurons.