Functional expression of TASK-1/TASK-3 heteromers in cerebellar granule cells

Abstract

TASK-1 and TASK-3 are functional members of the tandem-pore K+ (K2P) channel family, and mRNAs for both channels are expressed together in many brain regions. Although TASK-1 and TASK-3 subunits are able to form heteromers when their complementary RNAs are injected into oocytes, whether functional heteromers are present in the native tissue is not known. Using cultured cerebellar granule (CG) neurones that express mRNAs of both TASK-1 and TASK-3, we studied the presence of heteromers by comparing the sensitivities of cloned and native K+ channels to extracellular pH (pHo) and ruthenium red. The single-channel conductance of TASK-1, TASK-3 and a tandem construct (TASK-1/TASK-3) expressed in COS-7 cells were 14.2 +/- 0.4, 37.8 +/- 0.7 and 38.1 +/- 0.7 pS (-60 mV), respectively. TASK-3 and TASK-1/TASK-3 (and TASK-3/TASK-1) displayed nearly identical single-channel kinetics. TASK-3 and TASK-1/TASK-3 expressed in COS-7 cells were inhibited by 26 +/- 4 and 36 +/- 2 %, respectively, when pHo was changed from 8.3 to 7.3. In outside-out patches from CG neurones, the K+ channel with single channel properties similar to those of TASK-3 was inhibited by 31 +/- 7 % by the same reduction in pHo. TASK-3 and TASK-1/TASK-3 expressed in COS-7 cells were inhibited by 78 +/- 7 and 3 +/- 4 %, respectively, when 5 microm ruthenium red was applied to outside-out patches. In outside-out patches from CG neurones containing a 38 pS channel, two types of responses to ruthenium red were observed. Ruthenium red inhibited the channel activity by 77 +/- 5 % in 42 % of patches (range: 72-82 %) and by 5 +/- 4 % (range: 0-9 %) in 58 % of patches. When patches contained more than three 38 pS channels, the average response to ruthenium red was 47 +/- 6 % inhibition (n= 5). These electrophysiological studies show that native 38 pS K+ channels of the TASK family in cultured CG neurones consist of both homomeric TASK-3 and heteromeric TASK-1/TASK-3.

Figure 1. Sensitivity of whole-cell currents of TASK-1, TASK-3 and heteromer channels (TASK-1/TASK-3, TASK-3/TASK-1) expressed in COS-7 cells

A, whole-cell currents were recorded from COS-7 cells at three different pH_o values. The bath solution contained 5 mm KCl. B, current observed at pH 7.3 as a percent of that observed at pH 8.3. C, current observed at pH 6.3 as a percent of that observed at pH 8.3. Current levels in both B and C were determined at +60 mV. An asterisk indicates a significant difference between two values (P < 0.05). The numbers inside the bar represent the number of cells used. The data obtained from COS-7 cells expressing TASK-1/TASK-3 and TASK-3/TASK-1 were combined (Heteromer).

Figure 2. Single-channel kinetics of TASK-1, TASK-3 and heteromeric channels (TASK-1/TASK-3, TASK-3/TASK-1) expressed in COS-7 cells

A, cell-attached patches were formed and single channel openings recorded at various membrane potentials. Pipette and bath solutions contained 150 mm KCl. Channel openings typical of those obtained in five other experiments are shown. B, amplitude and open time duration histograms of TASK-1, TASK-3 and heteromeric channels were obtained from openings at –60 mV, and fitted by a single exponential function and Gaussian function, respectively. C, single channel amplitudes were determined from amplitude histogram at each membrane potential to draw the current-voltage relationship. Each point is the mean ± S.D. of five determinations. Current–voltage relationships of TASK-3 and both heteromers could be superimposed.

Figure 3. pH_o Sensitivity of single channel currents of TASK-1, TASK-3 and heteromeric channels (TASK-1/TASK-3, TASK-3/TASK-1) expressed in COS-7 cells

A, single channel currents from outside-out patches are shown as inward currents recorded at –60 mV in symmetrical 150 mm KCl. Channel openings typical of those obtained in five other experiments are shown. B, current observed at pH 7.3 as a percent of that observed at pH 8.3. Channel current (channel activity × amplitude) was determined at –60 mV for TASK-1, TASK-3 and heteromers. The results obtained from both heteromers were combined. C, current observed at pH 6.3 as a percent of that observed at pH 8.3. An asterisk indicates a significant difference between two values (P < 0.05). The numbers inside the bar represent the number of cells (n) used.

Figure 4. Single-channel properties of K⁺ channels similar to TASK-3 cerebellar granule cells

A, channel openings of TASK-3-like channels in neurones are shown at five different membrane potentials in cell-attached patches in symmetrical 150 mm KCl. B, amplitude and duration histograms of the K⁺ channel were determined from openings at –60 mV. Current amplitudes were determined at each membrane potential and used to draw the current–voltage relationship. Current–voltage relationships for TASK-3 and TASK-1/TASK-3 channels expressed in COS-7 cells are also shown for comparison (n= 5). C, single channel currents from an outside-out patch of a cerebellar granule cell are shown at three different pH_o values. Inward currents observed at –60 mV in symmetrical 150 mm KCl are shown. Corresponding amplitude histograms are also shown to indicate the decreases in amplitude and conductance by changes in pH_o. D, current observed at pH 7.3 as a percent of that observed at pH 8.3. The results from both heteromers (TASK-1/3 and TASK-3/1) were combined. E, the same as D except that the pH_o was changed from 8.3 to 6.3. An asterisk indicates a significant difference between two values (P < 0.05). The numbers inside the bar represent the number of cells (n) used.

Figure 5. Sensitivity to ruthenium red of whole-cell currents of TASK-1, TASK-3 and heteromeric channels (TASK-1/TASK-3 and TASK-3/TASK-1) expressed in COS-7 cells

A, whole-cell currents were recorded during a 1 second ramp pulse (–120 mV to +60 mV) in a bath solution containing 5 mm KCl. B, current observed in the presence of ruthenium red as a percent of control current measured at +60 mV. An asterisk indicates a significant difference between two values (P < 0.05). The numbers inside the bar represent the number of cells (n) used.

Figure 6. Sensitivity to ruthenium red of single-channel currents of TASK-1, TASK-3 and heteromeric channels (TASK-1/TASK-3 and TASK-3/TASK-1) expressed in COS-7 cells

A-D, using outside-out patches, 5 μm ruthenium red was applied to the bath solution for 1–2 minutes and then washed off. Expanded current tracings are shown at indicated time points (lower case letters). E, effect of ruthenium red on the relative current of TASK-1, TASK-3, and the heteromers. The asterisk indicates a significant difference from the control observed before treatment of ruthenium red (P < 0.05). F, single channel openings from an outside-out patch showing only one TASK-3 channel are shown before and after application of ruthenium red. Amplitude and duration histograms obtained from these openings show the large inhibitory effect of ruthenium red on single channel TASK-3 conductance. TASK-3 control: 1.1 ± 0.2 ms and 2.3 ± 0.1 pA (–60 mV); after ruthenium red: 0.7 ± 0.1 ms and 0.9 ± 0.2 pA (– 60 mV).

Figure 7. K⁺ channels similar to TASK-1 and TASK-3 in cerebellar granule neurones

A, single channel currents from outside-out patches containing only K⁺ channels similar to TASK-3 are shown before and after treatment with ruthenium red. In patches containing one open K⁺ channel, two types of responses were observed. In Type 1 response, ruthenium red had a very small inhibitory effect (∼5 %). In Type 2 response, ruthenium red produced ∼ 80 % inhibition. In patches that contained several channels (Type 3 response), ruthenium red inhibited the channel activity by approximately half (48 %). The cell membrane potential was held at –60 mV. Pipette and bath solutions contained 150 mm KCl. B, single channel currents from outside-out patches containing only K⁺ channels similar to TASK-1, are shown before and after treatment with ruthenium red. C, summary of the effect of ruthenium red on K⁺ channel currents in CG neurones, showing the three types of responses. The asterisk indicates a significant difference from the Type 1 response (P < 0.05).