Dual activation of neuronal G protein-gated inwardly rectifying potassium (GIRK) channels by cholesterol and alcohol.
- Authors
- Glaaser, Ian W; Slesinger, Paul A
- Year
- 2017
- Journal
- Scientific reports
- PMID
- 28676630
- DOI
- 10.1038/s41598-017-04681-x
- PMCID
- PMC5496853
Activation of G protein-gated inwardly rectifying potassium (GIRK) channels leads to a hyperpolarization of the neuron's membrane potential, providing an important component of inhibition in the brain. In addition to the canonical G protein-activation pathway, GIRK channels are activated by small molecules but less is known about the underlying gating mechanisms. One drawback to previous studies has been the inability to control intrinsic and extrinsic factors. Here we used a reconstitution strategy with highly purified mammalian GIRK2 channels incorporated into liposomes and demonstrate that cholesterol or intoxicating concentrations of ethanol, i.e., >20 mM, each activate GIRK2 channels directly, in the absence of G proteins. Notably, both activators require the membrane phospholipid PIP but appear to interact independently with different regions of the channel. Elucidating the mechanisms underlying G protein-independent pathways of activating GIRK channels provides a unique strategy for developing new types of neuronal excitability modulators.
Brain PIP2 and soluble PIP2 activate reconstituted brain GIRK2 channels in the absence of other proteins. (a) Graph shows purification of mouse GIRK2 from Pichia pastoris. The absorbance is plotted as a function of elution volume, with molecular weight (MW) standards; Blue - 440 kDa, Black - 158 kDa, Red - 75 kDa (arrows). The major peak elutes at a volume consistent with the size of a GIRK2 tetramer (T). Inset, Coomassie blue staining of a gradient SDS-PAGE protein gel shows two bands, one for tetramer (T) and one for monomer (M). Inset, wild-type GIRK2 was truncated at the amino- and carboxy-termini (see methods). (b) Example of K+ flux assay with GIRK2 reconstituted into PE/PG liposomes in the absence or presence of brain PIP2 (1%) (see methods). The fluorescence is normalized and plotted as a function of time (‘Relative K+ flux’). Arrows indicate addition of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the GIRK2 channel blocker MTS-HE (HE, 100 μM) and valinomycin (VM). Note the quenching of fluorescence for GIRK2 with PIP2 (green) upon addition of CCCP, which is attenuated with addition of MTS-HE. Dashed line represents no flux. (c) Representative fluorescent traces of GIRK2 alone (grey), GIRK2 and MTS-HE (red), and GIRK2 with acute application of 30 μM diC8-PIP2 (green). The decay was fit with a single exponential to determine that rate constant (1/τ, s-1). (d) Bar graph shows the average ( ± SEM) fractional activation with diC8-PIP2 based on the change in steady-state fluorescence (green bar vs. grey bar). MTS-HE (red bar) significantly inhibits diC8-PIP2-activated GIRK2 channels (n = 7, P < 0.0001).
LLM interpretation
This figure consists of four panels characterizing the purification and activation of reconstituted GIRK2 channels. Panel (a) shows a size-exclusion chromatography trace and SDS-PAGE gel confirming the purification of GIRK2 as a tetramer. Panels (b) and (c) display fluorescence-based $K^+$ flux assays over time, showing that PIP2 (or diC8-PIP2) induces a rapid decrease in relative fluorescence (increased flux) that is attenuated by the blocker MTS-HE. Panel (d) is a bar graph quantifying fractional activation, showing a significant increase with diC8-PIP2 (green bar) compared to GIRK2 alone (grey bar), which is significantly inhibited by HE (red bar, $P < 0.0001$).
Potency of PIP2 activation of GIRK2 channels. (a) Normalized fluorescent traces (mean ± SEM) show K+ flux for GIRK2 following addition of the indicated concentration of diC8-PIP2 or MTS-HE (100 μM) (n = 9). (b) Plot of the rate of K+ flux as a function of diC8-PIP2 concentration. Line shows best fit using the Hill equation with apparent EC50 of 25.1 ± 3.3 μM and Hill coefficient of 1.7 ± 0.1 (n = 10). (c) Normalized fluorescent traces (mean ± SEM) show K+ flux for GIRK2-containing liposomes with 1% brain PIP2 upon addition of the indicated concentration of neomycin, a competitive inhibitor of PIP2 (n = 4). (d) Plot of the fractional inhibition of K+ flux as a function of neomycin concentration. Fraction inhibition was calculated using the apparent steady-state K+ flux measurement. Line shows best fit using the Hill equation with IC50 for neomycin inhibition 11.1 ± 2.4 μM and with a Hill coefficient of 1.3 ± 0.3 (n = 4).
LLM interpretation
This figure consists of four panels analyzing the potency of PIP2 activation and inhibition of GIRK2 channels. Panels (a) and (c) show normalized fluorescent traces of relative $\text{K}^+$ flux over time, demonstrating that increasing concentrations of $\text{diC8-PIP}_2$ increase flux (a) and increasing concentrations of neomycin inhibit flux in the presence of brain $\text{PIP}_2$ (c). Panels (b) and (d) are dose-response plots using the Hill equation, showing the rate of $\text{K}^+$ flux as a function of $[\text{diC8-PIP}_2]$ and fractional inhibition as a function of $[\text{Neo}]$, respectively.
Alcohol directly activates GIRK2 channels in absence of G proteins. (a,b) Normalized fluorescent traces (mean ± SEM) show K+ flux for GIRK2-containing liposomes with 1% brain PIP2 and increasing concentrations of ethanol (EtOH) (a) (n = 12) (green traces) or propanol (PrOH) (b) (n = 4–6) (blue traces). (c) The normalized rate of K+ flux is plotted as a function of ethanol (green) and propanol (blue) concentration. Inset, zoom of the dose-response curves over the physiological alcohol concentrations. * p < 0.05 vs. 10 mM using one-way ANOVA and Dunnett’s post hoc test (n = 5–6 for PrOH and 11–12 for EtOH). (d) Normalized fluorescent traces (mean ± SEM) show K+ flux for GIRK2 in the presence of 100 mM PrOH in the absence (light blue, n = 3) or presence of 50 μM diC8-PIP2 (dark blue, n = 4). GIRK2 in the absence of both PrOH and diC8-PIP2 is shown for comparison (black, n = 4). (e) Normalized fluorescent traces (mean ± SEM) show K+ flux for GIRK2-containing liposomes with 1% brain PIP2 and 1 mM β-OG (left) (n = 4, magenta) or 30 μM capsaicin (right) (n = 4, blue). MTS-HE inhibition is shown for comparison (red). (f) The normalized rate of K+ flux is plotted as a function of different β-OG (pink) or capsaicin (blue) concentrations, and is compared to 100 mM EtOH (green). #not significant vs. 3 μM Capsaicin, $not significant vs. 0.3 mM β-OG, ** P < 0.05 vs. 3 μM Capsaicin or 0.3 mM β-OG using one-way ANOVA and Dunnett’s post hoc test.
LLM interpretation
This figure consists of fluorescent traces (a, b, d, e) and dose-response plots (c, f) measuring $K^+$ flux in GIRK2-containing liposomes. Panels (a-c) show that increasing concentrations of ethanol (green) and propanol (blue) increase the normalized rate of $K^+$ flux, with propanol exhibiting a stronger effect. Panel (d) demonstrates that the addition of $diC8-PIP_2$ enhances $K^+$ efflux in the presence of propanol compared to propanol alone. Panels (e-f) compare the effects of $\beta$-OG (pink) and capsaicin (blue) to ethanol (green), showing that $\beta$-OG and capsaicin produce significantly lower normalized rates of $K^+$ flux.
Cholesterol directly activates GIRK2 channels. (a) Normalized fluorescent traces (mean ± SEM) show K+ flux for GIRK2-containing liposomes with 1% brain PIP2 in the absence (black) or presence of 5% cholesterol (orange) (n = 6). Inset, chemical structure of cholesterol is shown. (b) Bar graph shows the increase in the rate of K+ flux with different cholesterol concentrations (1%, 5%, and 10%). Statistical significance * p < 0.05, ** p < 0.01 (n = 6).
LLM interpretation
Figure (a) shows normalized fluorescent traces of relative $\text{K}^+$ flux over time for GIRK2-containing liposomes, where the addition of 5% cholesterol (orange) results in a faster decay compared to the control (black). Figure (b) is a bar graph quantifying the rate of $\text{K}^+$ flux across different cholesterol concentrations (0%, 1%, 5%, and 10%). The rate increases with higher cholesterol concentrations, with statistically significant increases observed at 5% ($p < 0.05$) and 10% ($p < 0.01$) compared to the control.
Cholesterol enhances sensitivity of GIRK2 channels to PIP2. (a) Normalized fluorescent traces (mean ± SEM) show K+ flux for GIRK2-containing liposomes with 5% cholesterol in response to increasing concentrations of diC8-PIP2 (green) or MTS-HE (100 μM, red). Note little change in K+ flux in the absence of PIP2 (black) similar to that of liposomes without cholesterol (n = 7). (b) The rate of K+ flux is plotted as a function of different diC8-PIP2 concentrations for GIRK2 in the presence of 5% cholesterol (mustard circles). Line shows best fit using the Hill equation with EC50 of 12.2 ± 2.5 μM and a Hill coefficient of 1.5 ± 0.2 (n = 6). For reference, dose-response without cholesterol from Fig. 2B is shown (green circles). (c) Normalized fluorescent traces (mean ± SEM) show K+ flux for GIRK2-containing liposomes with 1% brain PIP2 / 5% cholesterol with different concentrations of neomycin (n = 5). (d) The rate of K+ flux for GIRK2/brain PIP2/5% cholesterol is plotted as a function of different neomycin concentrations. Line shows best fit using the Hill equation with an IC50 15.0 ± 4.6 μM and Hill coefficient of 1.2 ± 0.3 (n = 5).
LLM interpretation
This figure consists of four panels analyzing the effect of cholesterol on GIRK2 channel sensitivity to $\text{PIP}_2$. Panels (a) and (c) show normalized fluorescent traces of relative $\text{K}^+$ flux over time, demonstrating that increasing concentrations of $\text{diC}_8\text{-PIP}_2$ (green) and increasing concentrations of neomycin (blue) modulate flux in the presence of 5% cholesterol. Panel (b) is a dose-response curve showing a higher rate of $\text{K}^+$ flux for GIRK2 with 5% cholesterol (mustard) compared to without cholesterol (green) across $\text{PIP}_2$ concentrations. Panel (d) is a dose-response plot showing the fractional inhibition of $\text{K}^+$ flux as a function of neomycin concentration for GIRK2 with 5% cholesterol and brain $\text{PIP}_2$.
Cholesterol does not affect the ethanol sensitivity of GIRK2 channels. (a) Normalized fluorescent traces (mean ± SEM) show the K+ flux for GIRK2-containing liposomes with 1% brain PIP2 (black trace), and either 100 mM EtOH (green), 5% cholesterol (orange), or both 100 mM EtOH and 5% cholesterol (blue trace) (n = 4–12) (b) The rate of K+ flux for GIRK2 with PIP2 and 5% cholesterol (blue) or PIP2 alone (green) is plotted as a function of different EtOH concentrations. Inset, the normalized rate of K+ flux is plotted as a function of EtOH concentration.
LLM interpretation
Figure (a) shows normalized fluorescent traces of relative $\text{K}^+$ flux over time for GIRK2-containing liposomes under various conditions: basal (black), EtOH (green), cholesterol (orange), and EtOH + cholesterol (blue), with HE as a control (red). Figure (b) is a line graph plotting the rate of $\text{K}^+$ flux ($1/\tau, \text{s}^{-1}$) against ethanol concentration ([EtOH], mM) for conditions with 5% cholesterol (blue) and no cholesterol (green). The data indicate that the addition of cholesterol increases the rate of $\text{K}^+$ flux and enhances the sensitivity of the channels to increasing concentrations of ethanol, as further shown in the normalized rate inset.
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In this knowledge base
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|---|---|---|
| Genetics of Alcohol Use Disorder: A Role for Induced Pluripotent Stem Cells? | 2018 | 29897633 |
External
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|---|---|---|---|---|
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