Figure 1. Membrane anchored Gβγ binds to GIRK and activates the channel.

(A) Inhibitory neurotransmitters activate G_i/o G protein coupled receptors (GPCRs) in neuron membranes. The GPCRs facilitate the exchange of GDP to GTP on the G protein hetero-trimer, releasing the Gα_i/o subunit and Gβγ subunit. The membrane-anchored Gβγ subunit binds to and activates GIRK. (B) NTA lipid (head group modified with a Ni²⁺ chelator NTA, DOGS-NTA) is used to anchor non-lipid modified and His-tagged Gβγ (sGβγ-His10) onto the lipid membranes. 2 μM of sGβγ-His10 was used to fully saturate all NTA lipid on the membrane. The sGβγ-His10 density on the membrane can be controlled by the NTA lipid mole fraction. 32 μM C8-PIP₂ was included on the same side as sGβγ-His10. (C) Membrane-bound sGβγ-His10 activates GIRK to different levels depending on the NTA lipid mole fraction. Lipid modified Gβγ is used to fully activate GIRK at the end of each experiment. GIRK currents corresponding to different NTA lipid mole fractions are normalized to the fully activated value. A detailed description of the experiment is shown in Figure 1—figure supplement 1. Example current traces of activation by sGβγ-His10 and lipid modified Gβγ in liposomes are shown in Figure 1—figure supplement 2.

DOI: http://dx.doi.org/10.7554/eLife.15751.003

Figure 1—figure supplement 1. Details of the planar bilayer experiment.

To control Gβγ density in the membrane (A) GIRK2 channel proteoliposomes containing the corresponding mole fraction of DOGS-NTA-Ni²⁺ lipid were fused into planar lipid bilayers containing the same density of DOGS-NTA-Ni²⁺ lipid. (B) A high concentration KCl solution (1 M) was applied at the membrane to facilitate complete fusion of proteoliposomes attached to the membrane. (C) 1 mM NiSO₄ was applied at the membrane to ensure that all NTA groups were charged with Ni²⁺. (D) sGβγ-His10 and C8-PIP₂ were added to the top side of the membrane. GIRK2 channels with the cytoplasmic side facing the top side began to open. (E) A Na⁺ titration was then performed by stepwise addition of NaCl up to 32 mM final concentration. (F) After the Na⁺ titration, proteoliposomes of lipid modified Gβγ was fused to the membrane to maximize GIRK activation. (G) Maximum current of for each membrane is used for normalization of currents recorded in the same membrane.

DOI: http://dx.doi.org/10.7554/eLife.15751.004

Figure 1—figure supplement 2. Example traces of GIRK2 activation by sGβγ-His10 and lipid modified Gβγ in proteoliposomes.

Solution contains 10 mM potassium phosphate at pH 8.2 with 150 mM KCl on both sides of the membrane. 2 nM NiSO₄, 2 mM MgCl₂ and 32 μM C8-PIP₂ were also included on the cytosolic side of the channel. Membrane voltage was held at -50 mV. (A) 0.03 mol fraction of Ni-NTA lipid was included in the lipid bilayer. 2 μM of sGβγ-His10 was applied to the membrane at the time indicated by the arrow. The activation by sGβγ-His10 generally takes a few seconds to a few tens of seconds to reach equilibrium. After reaching equilibrium, the current is stable for many minutes. (B) Application of Gβγ in proteoliposome vesicles activates GIRK with a slower apparent kinetics. ~700 mM KCl was included in the vesicles to facilitate fusion with the membrane. The decrease in current immediately after application of the high salt vesicles is due to the change in local electro-chemical gradient of K⁺ near the membrane. Mixing restores the ionic conditions. To ensure saturation of Gβγ binding on the channel, Gβγ vesicles were applied several times until the current (after mixing) no long increases. With good membranes, after saturation is reached, the current is stable for minutes. Voltage families were recorded during the breaks indicated by '/ /' which take around 1 min each.

DOI: http://dx.doi.org/10.7554/eLife.15751.005