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Review
. 2020 Jan 1;1862(1):183091.
doi: 10.1016/j.bbamem.2019.183091. Epub 2019 Oct 28.

Disruption of palmitate-mediated localization; a shared pathway of force and anesthetic activation of TREK-1 channels

E Nicholas Petersen  1 , Mahmud Arif Pavel  1 , Hao Wang  1 , Scott B Hansen  2
Affiliations
Review

Disruption of palmitate-mediated localization; a shared pathway of force and anesthetic activation of TREK-1 channels

E Nicholas Petersen et al. Biochim Biophys Acta Biomembr. .

Abstract

TWIK related K+ channel (TREK-1) is a mechano- and anesthetic sensitive channel that when activated attenuates pain and causes anesthesia. Recently the enzyme phospholipase D2 (PLD2) was shown to bind to the channel and generate a local high concentration of phosphatidic acid (PA), an anionic signaling lipid that gates TREK-1. In a biological membrane, the cell harnesses lipid heterogeneity (lipid compartments) to control gating of TREK-1 using palmitate-mediated localization of PLD2. Here we discuss the ability of mechanical force and anesthetics to disrupt palmitate-mediated localization of PLD2 giving rise to TREK-1's mechano- and anesthetic-sensitive properties. The likely consequences of this indirect lipid-based mechanism of activation are discussed in terms of a putative model for excitatory and inhibitory mechano-effectors and anesthetic sensitive ion channels in a biological context. Lastly, we discuss the ability of locally generated PA to reach mM concentrations near TREK-1 and the biophysics of localized signaling. Palmitate-mediated localization of PLD2 emerges as a central control mechanism of TREK-1 responding to mechanical force and anesthetic action. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

Keywords: Anesthesia; Lipid; Mechanosensation; Palmitoylation; Rafts; Super resolution microscopy.

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Conflict of interest statement

Authors declare no conflicts of interests.

Figures

Figure 1.
Figure 1.. Palmitate-mediated localization, a central component of TREK-1 mechanosensation and anesthesia.
(i) Phospholipase D2 (PLD2, green) is shown with two palmitoylation sites that localize the enzyme to a GM1 lipids (blue spheres). (ii) Mechanical force disrupts lipid domains resulting in smaller dispersed domains within the plasma membrane (indicated by disordered blue bars). (iii) Anesthetics (orange hexagon) disrupt lipid domains causing them to expand and become larger while remaining relatively ordered (indicated by intact blue rectangles). Both types of raft disruption perturb palmitate mediated localization of PLD2 to GM1 lipids resulting in PLD2 translocation, PLD2 binding to PIP2 lipids (red hexagon), substrate (phosphatidylcholine, grey) presentation, and phosphatidic acid (PA, yellow) production. PA then activates TREK-1 (not shown). The horizontal grey rectangle indicates clustered lipids on the inner leaflet (PIP2 domain).
Figure 2.
Figure 2.. A putative role for anionic lipid regulation in mechanosensation and anesthesia.
(AB) A model of graded mechanical response based on lipid activation. (A) A lipid (red circle) is shown activating inhibitory K+ channel (grey cylinders) (i) and excitatory Ca2+ and Na+ channels (ii). Inhibitory K+ ions (purple spheres) are shown flowing out of the cell. Inhibitory Cl− ions flow into the cell (not shown). Excitatory Ca2+ and Na+ (green spheres) are shown flowing into and depolarizing the cell. A ‘?’ indicates ion channels that are known to be regulated by an anionic signaling lipid but the role of the lipid has not been directly linked to the mechano- or anesthetic-sensitivity of the channel. (B) Four putative ratios of inhibitory and excitatory mechano-effector channels. The amount of conductance for each ion is indicated by the number spheres (1–5). Mechanosensory currents are elicited when more excitatory effectors are active than inhibitory. Having many channels present and open leads to faster and stronger firing. (C-D) A model of an anesthetic response based on lipid activation. An anionic signaling lipid, produced in response to anesthetic, is shown activating a potassium channel (e.g. TREK-1, i) and inhibiting neurotransmitter-gated (brown square) Ca2+ and Na+ channels (iii) (all channels are shown as grey cylinders. A second class of anesthetic sensitive excitatory channels are shown activated by anesthetics (e.g. TRPV1, iv). (D) A lipid driven binary response to anesthetics is shown where lipid dependent external K+ is high and internal Ca2+ and Na+ remains low resulting in anesthesia. (D) Lipid activation of a Ca2+ and Na+ conducting pain channel by anesthetic is shown counteracting lipid activation of K+ channels. (E-F) Traces of membrane potentials from neuroblastoma 2a (N2a) cells measured under current clamp with mechanical poke by a glass rod. The application and distance of the mechanical poke is shown underneath the trace. Data are taken from Brohawn et. al., PNAS, 111 (2014) pg. 3614–9. (F) The same conditions in (E) with over expressed TRAAK channel, from the same source.
Figure 3.
Figure 3.. Generation of mM lipid concentrations by local production. Generation of mM lipid concentrations by local production.
(A) Cartoon illustrating the proximity of phospholipase D2 (PLD2) and phosphatidic acid (PA) production (red lipid) to a TREK-1 channel (grey cylinder) in the plasma membrane. PLD2 bound to the C-terminus of TREK-1 will produce PA within 8 nm of the TREK-1 lipid binding site (red scale bar). Since TREK-1 is a homodimer, an identical setup would exist on the opposite side as well (not shown). (B) The effective molar concentration and mole percent of a single signaling lipid (PA) within select radii from the channel. The former is calculated by the formula 1/*πr2hNA, where NA is Avogadro’s number, the height, h, of a lipid leaflet is estimated to be 15 Å, and the distance, r, from TREK-1 is indicated. Mole percent were calculated using the assumption that each um2 of lipid contain 1×105 lipids**. Only at radii ~130 nm from the channel are concentrations estimated below the measured Kd of TREK-1 for PA (~20 μM).
Figure 4.
Figure 4.. Acquisition and analysis considerations of super-resolution images.
(A) Comparison of the size of CTxB labeled GM1 domains from three cell types with antibody labeled proteins known to localized within the GM1 domains. The potential added diameter from antibody or CTxB labeling is indicated by white stripes. Fixed proteins within the GM1 lipids form clusters approximately the same size as CTxB labeled GM1 clusters, suggesting CTxB does not cluster unfixed lipids after treatment. (Antibodies used: 13891, ab15272, ab41927, MAB5232; secondary Cy3b antibodies and CTxB, along with the protocols used can be found in [18,28]). (B) Fluorescence recovery after photobleaching (FRAP) quantification showing movement of CTxB labeled lipids after fixation. C2C12 cells show negligible recovery in fixed cells vs. live cells while N2a showed a slight increase indicated some diffusion of lipids in N2a cells. (C) Images from C2C12 cells showing changes in lipid and protein clustering upon application of the cholesterol-sequestering molecule methyl-β-cyclodextrin (mβCD). Removal of cholesterol leads to a reduction in diameter in CTxB (80 to 58 nm, red) and PIP2 clusters (73 to 57 nm, blue). Data taken from Petersen et al. Nature Comm. 7 (2016) 13873 with permission. (D) A hypothetical schematic showing how the selection of the maximum particle distance (MPD) parameter using DBSCAN can change the apparent diameter of a cluster in identical images or across treatments. In the two ‘samples’ shown, comparison of cluster sizes can lead to either shrinking (Cluster 2), enlargement (Cluster 3), or no apparent change (Cluster 1) in overall size in identical images. Care should be taken in both the selection of MPD used and the resulting interpretation of such data.
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