TASK channels determine pH sensitivity in select respiratory neurons but do not contribute to central respiratory chemosensitivity

Abstract

Central respiratory chemoreception is the mechanism by which the CNS maintains physiologically appropriate pH and PCO2 via control of breathing. A prominent hypothesis holds that neural substrates for this process are distributed widely in the respiratory network, especially because many neurons that make up this network are chemosensitive in vitro. We and others have proposed that TASK channels (TASK-1, K(2P)3.1 and/or TASK-3, K(2P)9.1) may serve as molecular sensors for central chemoreception because they are highly expressed in multiple neuronal populations in the respiratory pathway and contribute to their pH sensitivity in vitro. To test this hypothesis, we examined the chemosensitivity of two prime candidate chemoreceptor neurons in vitro and tested ventilatory responses to CO2 using TASK channel knock-out mice. The pH sensitivity of serotonergic raphe neurons was abolished in TASK channel knock-outs. In contrast, pH sensitivity of neurons in the mouse retrotrapezoid nucleus (RTN) was fully maintained in a TASK null background, and pharmacological evidence indicated that a K+ channel with properties distinct from TASK channels contributes to the pH sensitivity of rat RTN neurons. Furthermore, the ventilatory response to CO2 was completely retained in single or double TASK knock-out mice. These data rule out a strict requirement for TASK channels or raphe neurons in central respiratory chemosensation. Furthermore, they indicate that a non-TASK K+ current contributes to chemosensitivity of RTN neurons, which are profoundly pH-sensitive and capable of driving respiratory output in response to local pH changes in vivo.

Figure 1.

Generation of TASK-1 and TASK-3 knock-out mice. Targeting constructs for TASK-1 (A) and TASK-3 (D) were transfected into ES cells to generate “floxed” alleles containing loxP sites flanking exon II of the respective genes. Initial crosses between germ-line chimeric animals and C57BL/6 mice yielded progeny with heterozygous floxed (f/+) TASK alleles. These animals were crossed with a deleter Hs-Cre1 mouse strain to generate heterozygous (+/−) offspring, and those animals were intercrossed to produce mice homozygous for the deleted alleles (−/−). Tail DNA was assayed by Southern blot analysis (B, E) using the indicated probes and by multiplex PCR (C, F) across the downstream loxP site using the indicated primers. The probed restriction fragments and PCR products matched the predicted sizes indicated in the panels. WT, Wild type.

Figure 2.

Characterization of TASK channel knock-out mice. A, In situ hybridization using [³³P]-labeled cRNA probes specific for exon 2 of either TASK-1 (left) or TASK-3 (right) in sagittal sections of brains from TASK-1^−/− (top) and TASK-3^−/− (bottom) mice; note the absence of hybridization for the cognate gene in each knock-out, despite using twice the probe concentration (x2). B, TASK-1 and TASK-3 mRNA levels were assayed by qRT-PCR in brainstem samples from control (n = 22) and TASK knock-out (n = 11 each) mice (*p < 0.0001 vs control by ANOVA). C, D, Control and TASK knock-out mice were examined for their ability to run on an accelerating rotating rod (C; n ≥ 8 per group) and to remove their tails from a radiant heat source, at either low or high intensity (D; n ≥ 7 per group). None of the TASK knock-out mice lines showed any obvious gross sensorimotor deficits.

Figure 3.

TASK channels confer pH sensitivity to serotonergic raphe neurons. A, Left, Composite map shows location of dorsal raphe neurons from each genotype recovered after recording: ■, control; ♦, TASK-1^−/−; •, TASK-3^−/−; ▴, TASK^−/− (double knock-outs). The area encompassed by dashed lines is an enlargement of the corresponding square defining the dorsal raphe region. Right, pH-sensitive raphe neuron filled with biocytin and immunostained with an antibody for tryptophan hydroxylase (TrpH-ir); the overlay image confirms the serotonergic phenotype of the recorded raphe neuron (white arrow), which was located in a cluster of other serotonergic neurons (red arrows). B, Non-isotopic in situ hybridization for TASK-1 and TASK-3 in dorsal raphe of control and TASK^−/− double knock-out mice. C, Averaged firing rate at the indicated extracellular pH of raphe neurons from control (n = 11), TASK-1^−/− (n = 7), TASK-3^−/− (n = 6), and TASK^−/− (n = 7) mice; deletion of TASK subunits, singly or in combination, eliminated pH sensitivity. D, Averaged I–V relationships reveal a prominent weakly rectifying pH-sensitive K⁺ current in raphe neurons from control mice (■; n = 14); this current was absent in raphe neurons from TASK-1^−/− (♦; n = 7), TASK-3^−/− (•; n = 5), or TASK^−/− (▴; n = 4) mice (*p < 0.05, two-way RM-ANOVA). Scale bars, 50 μm.

Figure 4.

RTN neurons from TASK channel knock-out mice retain pH sensitivity and express a pH-sensitive K⁺ current like their wild-type counterparts. A, Effects on firing rate of changing bath pH in representative RTN neurons from control (left) and TASK^−/− (right) mice. B, Averaged firing rate at the indicated extracellular pH of RTN neurons from control (n = 8), TASK-1^−/− (n = 8), TASK-3^−/− (n = 9), and TASK^−/− (n = 5) mice; there was no difference in pH sensitivity of RTN neurons from control and TASK knock-out mice. C, Averaged I–V relationships of pH-sensitive currents expressed by RTN neurons in control and TASK knock-out mice (n = 7, 7, 9, and 5; p = 0.3, by two-way RM-ANOVA).

Figure 5.

The pH-sensitive current expressed by rat RTN chemoreceptors is not sensitive to halothane. A, Traces of holding current and conductance in a rat RTN neuron (V_h of −60 mV) during changes in bath pH under control conditions and during exposure to halothane (3%). Note that halothane decreased current and conductance, an effect opposite to that expected for TASK channels, and that effects of pH were similar in magnitude under control conditions and in the presence of halothane. (*, trace blanked and corrected for artifact in recording). B, The I–V relationship of the halothane-sensitive current shows a weakly rectifying profile with a reversal near E_K, suggesting inhibition of a background K⁺ current. C, I–V relationships of pH-sensitive currents under control conditions and in halothane were not different, indicating that TASK channels do not contribute to the pH-sensitive current in rat RTN neurons.

Figure 6.

TASK channels are not the molecular substrate of central chemosensitivity. Whole animal ventilatory response to CO₂ was measured in control animals and TASK knock-out mice by plethysmography. A, Exemplar records of respiratory frequency, tidal volume (normalized to body weight), and minute ventilation (product of tidal volume and respiratory frequency) from control and TASK^−/− mice during exposure to incrementing inspired CO₂. B, Averaged values of minute ventilation with each of the indicated inspired gas concentrations; there were no significant differences in CO₂ sensitivity between control and TASK knock-out animals (p = 0.21, by two-way RM-ANOVA, n ≥ 5 per group).