Abstract
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 PIP2 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.
Highlights
Many modulatory neurotransmitters in the brain, such as dopamine, acetylcholine, serotonin and GABA, inhibit neuronal activity by stimulating G protein-coupled receptors (GPCRs) that couple to G protein-gated inwardly rectifying (GIRK, referred to as Kir3) channels[1]
In GIRK2-containing liposomes loaded with K+ and incubated with the membrane permeable pH-sensitive ACMA dye, the addition of the proton ionophore CCCP results in quenching of the ACMA dye if GIRK channels are open, i.e., H+ enter via CCCP if K+ can exit the proteoliposome (Fig. 1b)
The canonical pathway for GIRK channel activation is mediated by G protein Gβγ subunits, which occurs following stimulation of a Gi/o-coupled GPCRs, such as GABAB, dopamine D2 or mu opioid receptors[1, 2]
Summary
Many modulatory neurotransmitters in the brain, such as dopamine, acetylcholine, serotonin and GABA, inhibit neuronal activity by stimulating G protein-coupled receptors (GPCRs) that couple to G protein-gated inwardly rectifying (GIRK, referred to as Kir3) channels[1]. Following stimulation of GPCRs that couple to pertussis toxin-sensitive Gi/o G proteins, the G protein Gβγ subunits bind directly to the channel, and induce a conformational change that opens the channel in a manner that depends on the membrane phospholipid PI(4,5)P2 (referred to as PIP2)[10,11,12,13,14] This interaction was confirmed in an atomic resolution structure based on x-ray crystallography of GIRK2 in complex with Gβγ[15]. We demonstrate that alcohol and cholesterol directly activate neuronal GIRK2 channels, in the absence of G protein Gβγ subunits but requiring PIP2. Determining the mechanism of activation of GIRK channels by modulators that bypass G protein-gating could provide a new therapeutic strategy for treating a variety of human diseases
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
