Cholesterol up-regulates neuronal G protein-gated inwardly rectifying potassium (GIRK) channel activity in the hippocampus

Abstract

Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease.

Keywords: G protein-gated inwardly rectifying potassium channel; cholesterol; cholesterol-induced channel activation; electrophysiology; hippocampal CA1 pyramidal neurons; lipid bilayer; lipid-protein interaction; molecular docking; neuron; structure-function.

Figure 1.

Cholesterol levels in pyramidal neurons are increased in hypercholesterolemic rats. A and B, neuron-specific, enolase-associated immunofluorescence signal was detected from (A) pyramidal neurons from the CA1 hippocampal region, but not from (B) cerebral artery myocytes. C–E, filipin-associated fluorescence signal of hippocampal CA1 pyramidal neurons from rats on control (C) versus (D) a high-cholesterol diet. E, summary data of filipin-associated fluorescence signals of hippocampal CA1 pyramidal neurons from rats on control and on a high-cholesterol diet (n = 12–13). F, summary data of filipin-associated fluorescence signals obtained from control, cholesterol-depleted, and cholesterol-enriched isolated hippocampal CA1 pyramidal neurons (n = 12–14). Significant difference is indicated by an asterisk (*, p ≤ 0.05).

Figure 2.

Cholesterol up-regulates neuronal GIRK channels in neurons from the CA1 hippocampal region. A, representative traces of pre-baclofen (basal), baclofen-induced, and baclofen-induced tertiapin-Q-blocked GIRK currents. B, representative traces of baclofen-induced tertiapin-blocked control, cholesterol-enriched and cholesterol-depleted GIRK currents. In A and B, V_holding = −80 mV. Summary data: (C) inward current densities at −100 mV and (D) outward current densities at +60 mV. E, I-V curves. Significant difference is indicated by an asterisk (*, p ≤ 0.05).

Figure 3.

Cholesterol leads to an increase in the open probability of GIRK2^∧. Representative traces of GIRK2^∧ reconstituted in lipid bilayers with 12.5 μm diC₈-PI(4,5)P₂ (A) at −100 mV without and with 100 nm of the channel blocker tertiapin-Q; B at −100 and 100 mV without and with 33 mol % cholesterol; and C, at −100 mV with 33 mol % cholesterol and without and with 100 nm of the channel blocker tertiapin-Q. D, summary data showing the effect of 12.5 μm diC₈-PI(4,5)P₂, 100 nm tertiapin-Q, and 33 mol % cholesterol on the open probability of GIRK2^∧ (n = 3–6). E, summary data showing the effect of 33 mol % cholesterol on the open probability of GIRK2^∧ in the presence of 12.5 μm diC₈-PI(4,5)P₂. F, current-voltage curves obtained from single channel recordings of GIRK2^∧ reconstituted in lipid bilayers with and without cholesterol. The conductance was calculated from the slope of the linear fit of the currents recorded between −100 and −70 mV. G, summary data showing that cholesterol does not affect the conductance of GIRK2^∧ in the presence of 12.5 μm diC₈-PI(4,5)P₂.

Figure 4.

Putative cholesterol binding sites in GIRK2^∧. A, location of the cholesterol molecule (yellow and magenta) at two putative cholesterol-binding sites of GIRK2^∧ between two adjacent subunits of the channel (cyan and violet) as obtained from molecular docking. B, enlargement of the channel region that includes the two putative cholesterol-binding sites. Stick (C) and surface (D) presentations depicting the residues that surround the cholesterol molecule (yellow) in site 1 including Val⁹⁹ and Leu¹⁷⁴. Stick (E) and surface (F) presentations depicting the residues that surround the cholesterol molecule (magenta) in site 2 including Leu⁸⁶, Val¹⁰¹, and Val¹⁸³. G, surface presentation of the cholesterol molecule of site 2 showing the direct interaction between the cholesterol molecule and Val¹⁰¹ and Val¹⁸³ as well as its indirect interaction with Leu⁸⁶ via Phe¹⁸⁶.

Figure 5.

The effect of cholesterol on GIRK2^∧ depends on residues in the transmembrane domain of the channel. A and B, cholesterol up-regulates GIRK2^∧ when expressed in Xenopus oocytes. A, representative traces at −80 and +80 mV. B, summary data at −80 mV (n = 6–9). C, a ribbon presentation of GIRK2 channel showing the representative transmembrane residues (in ball presentation) whose effect on cholesterol sensitivity of the channel was tested in mutagenesis studies displayed in D. The location of the residues is depicted in ball presentation in two adjacent subunits of the channel. D, whole cell basal currents recorded in Xenopus oocytes at −80 mV showing the effect of cholesterol enrichment on GIRK2^∧ and L86V, V99I, M100L, V101A, L174V, V183I, and I195M (n = 11–23). Significant difference is indicated by an asterisk (*, p ≤ 0.05).