Fig 1.
Gi/o- and Gq-coupled receptors increased the amplitudes of the THIK-1 channel current.
(A) GABABR activates THIK-1. Ramp pulses from -120 mV to +40 mV (400 ms) were repetitively applied every 5 sec. Shown in left panel is the current trace recorded from a cell co-transfected with THIK-1 and GABABR. The black bar indicates the application timing of 100 μM GABA. The current-voltage relationships before, during and after application of 100 μM GABA (black, red and blue lines, respectively) are shown in the right panel. (B) Effects of Gi/o-Rs. Amplitude at 0 mV of 5th trace after the agonist application was set as Iagonist and that before the application was as I0. Circles indicate the normalized agonist-induced current amplitude to I0 (Iagonist/I0) in each cell without (open circles) or with PTX treatment (filled circles). Mean and S.D. are shown as bars (n = 5–9). Glutamate (200 μM) and oxo-M (10 μM) were applied to activate mGlu2 and M2R, respectively. (C) mGlu1 activates THIK-1 channel. Shown are the current traces recorded from cells co-transfected with THIK-1 and mGlu1 treated without (left) or with (right) U731222 (2 μM, 10 min). Application timing of glutamate (1 mM) is indicated by black bars. (D) Effect of Gq-Rs (Iagonist/I0) in each cell without (open circles) or with treatment of U73122 (filled circles) or U73343 (gray circles). Iagosnist was the amplitude of the 7th trace after the agonist application. Oxo-M (10 μM) was applied to activate M1R and M3R. Mean and S.D. are shown as bars (n = 7–12). *: 0.01<p≤ 0.05, **: 0.001<p≤ 0.01, ***: p≤ 0.001. The current densities before application of agonists (I0) were not significantly changed by treatments of PTX or PLC inhibitor (see Table 2).
Table 1.
Efficacy of agonists.
Table 2.
Basal current (I0) density of THIK-1 channel.
Fig 2.
Effects of Gs-coupled A2aR on the THIK-1 channel current.
(A) THIK-1 channel current before and after activation of Gs-coupled A2aR. The current trace obtained from the cell co-transfected with THIK-1 and A2aR is shown (left). Applications of 10 μM NECA is indicated by a black bar on the current trace. Shown in the right panel are the current-voltage relationships before and during the NECA application (black and red lines, respectively). (B) Effects of Gs-coupled A2aR on the THIK-1 channel current. Circles indicate the current density before and 50 sec after the application of NECA in each cell (n = 6). (C) Effect of A2aR on the current increase induced by GABABR. The current traces obtained from cells co-transfected with THIK-1, A2aR and GABABR are shown. For control experiment, only GABA (100 μM) was applied (black bars on the traces). For the analysis of the effect of A2aR, NECA (10 μM) was additionally applied (open bar). (D) Effects of A2aR on the Gi/o-R-induced increases in the THIK-1 channel current. Increments of the 15th (blue in C) and 5th current (red in C) amplitudes from I0 were measured and ΔI15th/ΔI5th was calculated in each cell to evaluate the effect of A2aR. Similar experiments were performed in cells transfected with M2R and A2aR. Oxo-M (10 μM) was applied to activate M2R. Circles indicate the ratios (ΔI15th/ΔI5th) in each cell without or with additional application of NECA (open or filled circles, respectively). Mean and S.D. are shown as bars (n = 4–7). n.s.: p> 0.05.
Fig 3.
Effects of mutations at the consensus Gβγ binding motif of THIK-1 channel on the current.
(A) Effects of Gβγ on the THIK-1 channel. The basal current densities (I0) in each cell transfected with wild type THIK-1 channel or the triple mutants (N-Ala; E15A/D16A/N17A, C-Ala; S365A/E366A/M367A) are indicated as open circles. The I0 from cells additionally co-transfected with Gβγ, are shown as filled circles. Mean and S.D. are shown as bars (n = 5–11). Co-expression of Gβγ increased the I0 of wild type but not of N-Ala and C-Ala mutants. (B) Effects of Gi/o-Rs on wild type and C-Ala THIK-1 channels. Circles show the ratios of the current amplitude after the agonist application to the basal one (Iagonist/I0) in each cell transfected with indicated receptors and wild type THIK-1 channel or C-Ala mutant (open and filled circles, respectively). Mean and S.D. are shown as bars (n = 4–7). (C) Effects of Gq-Rs on wild type and C-Ala THIK-1 channels. Circles show Iagonist/I0 in each cell transfected with indicated receptors and wild type THIK-1 channel or C-Ala mutant (open and filled circles, respectively). Mean and S.D. are shown as bars (n = 6). The receptors were activated by the application of agonists as written in the legend of Fig 1. *: 0.01<p≤ 0.05, **: 0.001<p≤ 0.01, n.s.: p> 0.05.
Fig 4.
Effect of the PIP2 hydrolysis on THIK-1 channel current.
(A) Current traces of KCNQ2/3 channels with or without VSP. Cells expressing KCNQ2/3 without (left) or with (right) VSP were depolarized to +40 mV for 8 sec from the holding potential of -80 mV. Traces show the macroscopic currents normalized by the maximal current amplitudes. (B) Effect of the PIP2 hydrolysis on KCNQ2/3 channels. Circles show the ratio of the current amplitude at the end of the depolarization to the maximal one (Iend/Imax) in each cell without or with VSP (open and filled circles, respectively). Mean and S.D. are shown as bars (n = 9). (C) Current traces of THIK-1 channel with or without VSP. Cells expressing THIK-1 alone (left) or with VSP (right) were depolarized to +40 mV for 8 sec from the holding potential of -80 mV. Traces normalized by the maximal amplitudes are shown. (D) Effect of the PIP2 hydrolysis on the THIK-1 channel. Circles show the ratio (Iend/Imax) in each cell without or with VSP (open and filled circles, respectively). Mean and S.D. are shown as bars (n = 7–8). **: 0.001<p≤ 0.01, n.s.: p> 0.05.
Fig 5.
Effects of OAG on THIK-1 channel current.
(A) Current and membrane potential relationships before and after application of OAG. Ramp pulses were applied ever 5 sec before and after application of OAG. The current and membrane potential relationships before (black) and 50 sec after application of the indicated concentration of OAG (red) are shown. (B) Effects of OAG on THIK-1 channel. Circles indicate the ratio of the current amplitudes after application of OAG or DMSO to the amplitudes before application of them (I10th/I0). Mean and S.D. are shown as bars (n = 8–12). n.s.: p> 0.05.
Table 3.
Effects of PKC inhibitor on M1R-induced potentiation of THIK-1 channel.
Fig 6.
mGlu2 and M1R increased the amplitudes of wild type and ΔN-THIK-2 channel currents.
(A) Current-voltage relationships before and after stimulation of mGlu2. Ramp pulses were repetitively applied as written before. Shown are the current-voltage relationships of the THIK-2 channels, before and after application of 200 μM glutamate (black and red lines, respectively). (B) Effects of mGlu2 on the THIK-2 channels. Symbols indicate the basal current densities (I0 in left panel) and the effect of the mGlu2 activation (Iglu/I0 in right panel) in cells transfected with mGlu2-YFP alone (open circles), mGlu2-YFP and THIK-2 (filled circle) and mGlu2-YFP and ΔN-THIK-2 (open squares). Mean and S.D. are indicated as bars (n = 7). (C) Effects of PTX treatment on the mGlu2-induced current increase. Open and filled circles indicate Iglu/I0 from control cells and PTX treated cells, respectively. Mean and S.D. are shown as bars (n = 6). (D) Current-voltage relationships before and after stimulation of M1R. Shown are the current-voltage relationships before and after application of 10 μM oxo-M (black and red lines, respectively). (E) Effects of M1R on THIK-2 channels. Symbols indicate I0 (left panel) and the effect of the M1R activation (Ioxo/I0 in right panel) in cells transfected with M1R-YFP alone (open circles), M1R-YFP and THIK-2 (filled circle) and M1R-YFP and ΔN-THIK-2 (open squares). Mean and S.D. are shown as bars (n = 8–9). (F) Effect of the PLC inhibitor. The effect of M1R was inhibited by the treatment of cells with U73122 but not with U73343 (filled circles and open squares, respectively). Mean and S.D. are shown as bars (n = 6). *: 0.01<p≤ 0.05, **: 0.001<p≤ 0.01, ***: p≤ 0.001, n.s.: p> 0.05.
Fig 7.
Effect of C-Ala mutation or OAG on ΔN-THIK-2 channel.
(A) Current-voltage relationships before and after stimulation of mGlu2. The current-voltage relationships of ΔN-THIK-2 channels are shown before and after application of 200 μM glutamate (black and red lines, respectively). (B) Effects of the mGlu2 activation on the ΔN-THIK-2 and C-Ala mutants. Symbols indicate the potentiating effect of glutamate (Iglu/I0) in each cell transfected with mGlu2 and the ΔN-THIK-2 channel or the C-Ala mutant (open and filled circles, respectively). Mean and S.D. are shown as bars (n = 11–12). (C) Current-voltage relationships before and after application of OAG. The current-voltage relationships before and 50 sec after application of OAG are shown (black and red lines, respectively). The tested concentration of OAG was 10 μM (left) and 30 μM (right). (D) Effects of OAG on the ΔN-THIK-2 channel. The ratio (I10th/I0) are not different between OAG applied group (filled circles) and vehicle applied group (open circles). Mean and S.D. are shown as bars (n = 7–11). **: 0.001<p≤ 0.01, n.s.: p> 0.05.
Fig 8.
Gi/o- and Gq-R dependent regulation of the heterodimeric THIK channels.
(A) Basal current densities of the tandem THIK channels. Symbols show the basal current density (I0) of each cell transfected with the indicated dimeric channels. Mean and S.D. are shown as bars (n = 9–10). (B) Effect of mGlu2 on the tandem THIK channels. Symbols show the effects of the mGlu2 activation (Iglu/I0) in each cell transfected with the indicated THIK channel dimers. 200 μM glutamate were applied to activate mGlu2. Mean and S.D. are shown as bars (n = 9–10). (C) Basal current densities of the tandem THIK channels. Symbols show the basal current density (I0) of each cell transfected with the indicated dimeric channels. Mean and S.D. are shown as bars (n = 9–10). (D) Effects of M1R on the tandem THIK channels. 10 μM oxo-M were applied to activate M1R. Symbols show effects of the M1R activation (Ioxo/I0) in each cell transfected with the indicated THIK channel dimers. Mean and S.D. are shown as bars (n = 9–10). ***: p≤ 0.001, *: 0.01<p≤ 0.05.