A, left, representative trace of TASK1 currents in an inside-out macropatch, measured at +100 mV. Applications of 25 μm AASt PIP₂ and 3 μg ml⁻¹ poly Lys are indicated by the horizontal lines. Right, statistical summary of the data. The average current level in the cell-attached configuration was 65 ± 21 pA (n= 3). B, left, a representative trace for TASK3 currents is shown, measured at +40 mV. Applications of 5 μm AASt PIP₂ and 3 μg ml⁻¹ poly Lys are indicated by the horizontal lines. Right, statistical summary of the data, normalized to the cell-attached current level. The average current in the cell-attached level was 580 ± 160 pA (n= 8). C, left, representative trace of TREK1 currents measured in macropatches at +100 mV. Applications of 2.5 μm AASt PIP₂ and 3 μg ml⁻¹ poly Lys are indicated by the horizontal lines. Right, statistical summary of the data (n= 6–10), normalized to the cell-attached level. The average current in the cell-attached level was 143 ± 35 pA (n= 10). D, left, representative trace for TRAAK currents is shown, measured in macropatches at +100 mV. Applications of 100 μm diC₈ PIP₂, 5 μm AASt PIP₂ and 3 μg ml⁻¹ poly Lys are indicated by the horizontal lines. Right, statistical summary of the data (n= 4), normalized to the cell-attached current level. The average current in the cell-attached level was 80 ± 9 pA (n= 8).

A, effect of wortmannin on whole-cell current recovery from carbachol inhibition for TREK1 channels. Left, a typical normalized recovery from inhibition for TREK1 channels. Right, summary data for recovery of inhibition after 200 s of removal of ACh in the presence or absence of wortmannin (n= 16). Oocytes were treated with 10 μm wortmannin during the recovery and for at least 10 min prior to ACh application. B, effect of the PKC inhibitor calphostin-C (10 μm) on ACh-induced inhibition. Summary data of whole-cell current inhibition by either 10 nm PMA or 10 μm ACh in either presence or absence of calphostin-C (n= 5–7). C, effect of 10 μm SAG and 10 μm SLG on TREK1 currents on inside-out macropatches. Left, a typical inside-out macropatch current at +90 mV; SAG was applied as indicated. Establishment of the inside-out configuration is indicated by the arrow. Right, the summary data of effects of SAG and SLG on basal current. D, effects of 10 μm SAG and 10 μm SLG on PIP₂-activated TREK1 currents on inside-out macropatches. Left, a typical inside-out macropatch current at +90 mV, 25 μm diC₈ PIP₂ and SAG were applied as indicated. Right, summary data of effects of SAG and SLG on inside-out macropatch currents activated by diC₈ PI(4,5)P₂.

A, typical response of TASK3 channels measured in oocyte macropatches to a depolarizing voltage step to +50 mV from a −150 mV holding voltage before and after 60 s application of poly Lys. B, typical response of TASK3 channels measured in oocyte macropatches to a voltage ramp (0.25 mV ms⁻¹) before and after 300 s of PIP₂Ab application. C, TREK1, TASK1 and TASK3 currents were measured in oocyte macropatches. Left, typical inhibition of either TREK1, TASK1 or TASK3 currents by poly Lys; right, summary data of kinetics of inhibition by poly Lys (30 μg ml⁻¹, see Methods) for TREK1 (n= 5) TASK1 (n= 7) and TASK3 (n= 5) channels. D, typical effect of poly Lys application after PIP₂Ab application. After PIP₂Ab is bound to PIP₂, poly Lys application had no effect on TASK3 channels (n= 6). E, summary data for inhibition of the whole-cell current by 60–90 min treatment with 10 μm wortmannin (n= 10–18). F, average whole-cell current recovery from carbachol inhibition for TREK1, TASK1 and TASK3 channels.

TASK1 and TASK3 currents were assessed by two-electrode voltage clamp. A, normalized effect of 1 μm carbachol application on whole-cell currents measured at −100 mV in 80 mm K⁺ extracellular solution, average of n= 5–6 cells. B, summary data for current inhibition by various agonists: angiotensin II (10 nm); lysophosphatidic acid (0.5 μm) and carbachol (1 μm) (for experiments done as in A). C, left, representative trace of the effect of 10 μm ACh on whole-cell TREK1 currents measured at +100 and −100 mV using a ramp protocol. Centre, representative trace of the effect of ACh on whole-cell TRAAK currents measured at +100 and −100 mV using a ramp protocol. Right, summary of the ACh-induced inhibition measured at −100 mV (n= 6 and n= 8 for TREK1 and TRAAK, respectively). Current level was estimated using the difference between current measured in high K⁺ (mm: 96 KCl, 1 NaCl, 1 MgCl₂, 5 Hepes, pH 7.4) and current measured in low K⁺ (mm: 2 KCl, 95 NaCl, 1 MgCl₂, 5 Hepes, pH 7.4) solutions at −100 mV. Note activation and fast inactivation of native Cl⁻ current by ACh indicating expression of M₁ receptors despite the lack of TRAAK inhibition.

A, left, time course of whole-cell currents for oocytes expressing TREK1 channels at +80 and −100 mV during ACh application in high K⁺ (96 mm) solution. Right, whole-cell currents in response to a slow voltage ramp (0.1 mV ms⁻¹) before and after 1 min (red) and steady-state (green) ACh inhibition. B, left, plot of the chord conductance (I/V) measured using the current–voltage relationship shown in A. Continuous lines represent a Boltzmann fit of the data. Right, summary data of the effects of ACh on V_¹/1. V_¹/1 was estimated by the conductance half-way between the conductance at −100 and +100 mV (n= 12). C, left, activation of TREK1 currents in inside-out macropatches by 100 μm DiC₈ PIP₂ at +90 and −90 mV in symmetrical high K⁺ (96 mm) solutions. Right, currents in response to a slow voltage ramp (0.16 mV ms⁻¹) during (black) and after (green) application of PIP₂. D, left, plot of the chord conductance (I/V) measured using the current–voltage relationship shown in C. Continuous lines represent a Boltzmann fit of the data. Right, summary data of effects of the application of DiC₈ PIP₂ on V_¹/1 estimated by the voltage where the conductance was half-way between of the conductance measured at +100 and −150 mV (n= 6). E, left, representative current traces of KCNQ1/KCNE1 channels, activated by a voltage step from the holding potential of −80 mV to +60 mV and then −40 mV before and after the application of ACh. Right, current–voltage relationship measured from the tail currents at −40 mV after steps to different voltages. F, left, normalized current–voltage relationship before and after the application of ACh. Right, summary data (n= 15). All conductance measurements were fit by a Boltzmann equation (continuous line).