Motoneurons express heteromeric TWIK-related acid-sensitive K+ (TASK) channels containing TASK-1 (KCNK3) and TASK-3 (KCNK9) subunits

Abstract

Background potassium currents carried by the KCNK family of two-pore-domain K+ channels are important determinants of resting membrane potential and cellular excitability. TWIK-related acid-sensitive K+ 1 (TASK-1, KCNK3) and TASK-3 (KCNK9) are pH-sensitive subunits of the KCNK family that are closely related and coexpressed in many brain regions. There is accumulating evidence that these two subunits can form heterodimeric channels, but this evidence remains controversial. In addition, a substantial contribution of heterodimeric TASK channels to native currents has not been unequivocally established. In a heterologous expression system, we verified formation of heterodimeric TASK channels and characterized their properties; TASK-1 and TASK-3 were coimmunoprecipitated from membranes of mammalian cells transfected with the channel subunits, and a dominant negative TASK-1(Y191F) construct strongly diminished TASK-3 currents. Tandem-linked heterodimeric TASK channel constructs displayed a pH sensitivity (pK approximately 7.3) in the physiological range closer to that of TASK-1 (pK approximately 7.5) than TASK-3 (pK approximately 6.8). On the other hand, heteromeric TASK channels were like TASK-3 insofar as they were activated by high concentrations of isoflurane (0.8 mm), whereas TASK-1 channels were inhibited. The pH and isoflurane sensitivities of native TASK-like currents in hypoglossal motoneurons, which strongly express TASK-1 and TASK-3 mRNA, were best represented by TASK heterodimeric channels. Moreover, after blocking homomeric TASK-3 channels with ruthenium red, we found a major component of motoneuronal isoflurane-sensitive TASK-like current that could be attributed to heteromeric TASK channels. Together, these data indicate that TASK-1 and TASK-3 subunits coassociate in functional channels, and heteromeric TASK channels provide a substantial component of background K(+) current in motoneurons with distinct modulatory properties.

Figure 1.

TASK-1 coimmunoprecipitates with TASK-3 in HEK 293 cells. A, Characterization of TASK-3 antibody. Top, Lanes were loaded with 5 μg of membrane preparations from HEK 293 cells transfected with the indicated constructs, and proteins were separated by 10% SDS-PAGE. Epitope-tagged HA-TASK-3 (T3) was identified with the anti-TASK-3 antibody; no immunoreactivity was seen for the corresponding HA-TASK-1 (T1) channel construct, or for HA-TASK-3 when the antibody was preadsorbed with excess antigenic peptide (pep). Essentially identical bands were recognized with an antibody to the epitope (α-HA), which was also used to confirm expression of HA-TASK-1. Bottom, Immunohistochemistry was used to detect TASK-3 distribution in sections of rat brain and adrenal gland. TASK-3-like immunoreactivity was detected in a distribution pattern that was strikingly similar to previous TASK-3 in situ hybridization reports (Karschin et al., 2001; Talley et al., 2001; Vega-Saenz de Miera et al., 2001; Bayliss et al., 2003). Especially strong labeling was seen in cell bodies from the following regions (clockwise from top left): hypoglossal motor nucleus (HMs), locus ceruleus (LC), adrenal gland, anterodorsal thalamic nucleus (ThAD), dorsal raphe nucleus (RDo), and striatum. Scale bars: striatum, adrenal inset, 50 μm; others, 100 μm. B, HEK 293 cells were cotransfected with TASK-3 and HA-TASK-1 constructs. The TASK-3 antibody coimmunoprecipitated (IP) TASK-3 (left) and HA-TASK-1 (middle) but did not immunoprecipitate transferrin receptor (TfrR), an endogenously expressed membrane protein (right); a control nonspecific rabbit IgG did not immunoprecipitate either TASK subunit. Molecular weight markers (kilodaltons) are indicated on the left; arrowheads indicate locations of precipitated proteins.

Figure 2.

A TASK-1 construct bearing a point mutation in the second pore domain (Y191F) acts as a dominant negative of TASK-1 (T1) and TASK-3 (T3) channels. HEK 293 cells were transfected with the indicated constructs and recorded under whole-cell voltage clamp by using a ramp voltage protocol (-130 to 20 mV; 0.2 V/sec). Whole-cell conductance was calculated as the slope of the current evoked between -130 and - 60 mV, and the pH-sensitive conductance was determined as the difference in conductance at pH 8.4 and 5.9. A, Expression of TASK-1(Y191F) by itself generated no pH-sensitive conductance, and coexpression of TASK-1(Y191F) abolished the pH-sensitive conductance generated by wild-type TASK-1 (left; from 1.3 ± 0.2 to 0.1 ± 0.02 nS; ^*p < 0.05, ANOVA). A total of 6 μg of DNA was transfected in all cases, with TASK channel constructs transfected at 3 μg each and the balance provided by empty pcDNA3 vector. B, Importantly, TASK-1(Y191F) also eliminated pH-sensitive conductance from coexpressed TASK-3 channels (right; from 14.2 ± 3.8 to 3.0 ± 0.8 nS; ^*p < 0.05, t test). A total of 9 μg of DNA was transfected in each experiment, with GFP-TASK-3 at 2 μg and either TASK-1(Y191F) or pcDNA3 at 7 μg.

Figure 3.

The pH sensitivity of TASK-like conductance in motoneurons resembles that of TASK-1/TASK-3 tandem heterodimeric channels. HEK 293 cells were transfected with cloned rat TASK-1, TASK-3, or tandem heterodimeric TASK-1/TASK-3 and TASK-3/TASK-1 constructs. A, Time series illustrating effects of changes in extracellular pH on whole-cell conductance (measured between -130 and -60 mV) in representative cells expressing TASK-1 (top), TASK-3 (middle), and TASK-1/TASK-3 (bottom). B, Left, pH sensitivity curves for TASK-1 (squares), TASK-3 (diamonds), and tandem heterodimers (pooled data from TASK-1/TASK-3 and TASK-3/TASK-1; triangles). Slope conductance at each bath pH was normalized to the total pH-sensitive conductance (pH 8.4-5.9) in individual cells and averaged; data points (mean ± SEM) were fitted with logistic equations (solid lines). The pK of the tandem heterodimer TASK channel constructs (pK ∼7.3; n = 11) was closer to that of TASK-1 (pK ∼7.5; n = 6) than to that of TASK-3 (pK ∼6.8; n = 5). Right, The pH sensitivity of motoneuronal TASK-like current was determined in hypoglossal motoneurons recorded in brainstem slices from neonatal rats (Talley et al., 2000). The pH sensitivity of the native motoneuronal TASK-like currents (circles) overlays the fitted curve of the pH sensitivity of the tandem heterodimer TASK channels, with a pK of ∼7.4 (n = 9).

Figure 4.

The isoflurane sensitivity of the TASK-like current in motoneurons resembles that of TASK-1/TASK-3 tandem heterodimeric channels. HEK 293 cells were transfected with rat TASK channel constructs and exposed to isoflurane at concentrations of 0.13, 0.4, and 0.8 mm. A, Whole-cell current was measured from cells expressing TASK-1 (top), TASK-3 (middle), and TASK-1/TASK-3 (bottom) during depolarizing ramps (-130 to 20mV; 0.2 V/sec) and plotted against membrane potential (Em) under control conditions, pH 7.3, and during bath acidification, pH 5.9, bath alkalization, pH 8.4, and exposure to isoflurane in an alkalized bath (0.8 mm isoflurane). Insets, Time series illustrating effects on whole-cell conductance of changing bath pH and of 0.8 mm isoflurane for each cell represented in the corresponding I-V plots. B, Left, Dose-response curves illustrating effects of isoflurane on rat TASK-1 (squares), TASK-3 (diamonds), or the tandem heterodimeric TASK-1/TASK-3 construct (triangles). Slope conductance at each isoflurane concentration was normalized to the total pH-sensitive conductance in individual cells and averaged; data points (mean ± SEM) were fitted with logistic equations (solid lines). Differences in effects of isoflurane on the TASK constructs were highly significant (F_(2,37) = 90; p < 0.0001, ANOVA); at the highest concentration, isoflurane increased TASK-3 and TASK-1/TASK-3 channel currents (both p < 0.05; n = 5; n = 8), whereas it inhibited TASK-1 current (p < 0.05; n = 5). Right, Effects of isoflurane on the native TASK-like conductance were determined in rat hypoglossal motoneurons, normalized to total pH-sensitive conductance, and averaged (±SEM; circles); the isoflurane sensitivity of the motoneuronal conductance overlays most closely that of the TASK channel heterodimer.

Figure 5.

The pH sensitivity of native motoneuronal currents, together with the isoflurane sensitivity of ruthenium red-resistant currents, indicates a contribution from TASK channel heterodimers. Whole-cell voltage-clamp recordings were obtained at -60 mV from hypoglossal motoneurons in brainstem slices from rat pups to determine effects of bath pH and a discriminating concentration of isoflurane (0.8 mm) on membrane current. A, Relative to control conditions, pH 7.3, bath acidification to pH 5.9 decreased motoneuronal outward current, whereas bath alkalization to pH 8.4 increased outward current, with the difference representing the total pH-sensitive current. Isoflurane caused a robust increase in outward current typical for effects on TASK-3 or TASK channel heterodimers. B, After defining the pH-sensitive current, ruthenium red (10 μm) was applied to block TASK-3 channels; application of ruthenium red invariably increased outward current, suggesting effects on multiple conductances. In the continuous presence of ruthenium red, 0.8 mm isoflurane caused a large increase in membrane current. C, The averaged isoflurane-sensitive current (±SEM) in the absence (control, filled circles; n = 5) and presence (shaded circles; n = 8) of ruthenium red was plotted against membrane potential and was well fitted by using the Goldman-Hodgkin-Katz current equation indicating open-rectifier K⁺ conductance. Inset, The isoflurane-sensitive conductance was normalized to the total pH-sensitive conductance in each cell and averaged (±SEM) for control conditions and in the presence of 10 μm ruthenium red. ^*p < 0.05, t test.

Figure 6.

The measured pH and isoflurane sensitivity of motoneurons are well described by curves derived from estimates of the relative contributions of TASK homodimers and heterodimers to native currents. Data from experiments with combined application of ruthenium red and isoflurane were used to estimate the fractional contribution of TASK-1 homodimers, TASK-3 homodimers, and TASK-1/TASK-3 heterodimers to the whole-cell pH-sensitive current in motoneurons (TASK-1, ∼14%; TASK-3, ∼34%; TASK heterodimers, ∼52%; see Results). These fractional contributions were applied as weighting factors to the pH and isoflurane sensitivity curves determined for cloned TASK channels, which were then summed and fitted to determine a predicted pH (A) and isoflurane (B) sensitivity of motoneuronal TASK-like currents. In both cases, these predicted sensitivity curves (dashed lines) closely matched data obtained from motoneurons (circles).