Cholesterol Regulation of Membrane Proteins Revealed by Two-Color Super-Resolution Imaging

Abstract

Cholesterol and phosphatidyl inositol 4,5-bisphosphate (PIP2) are hydrophobic molecules that regulate protein function in the plasma membrane of all cells. In this review, we discuss how changes in cholesterol concentration cause nanoscopic (<200 nm) movements of membrane proteins to regulate their function. Cholesterol is known to cluster many membrane proteins (often palmitoylated proteins) with long-chain saturated lipids. Although PIP2 is better known for gating ion channels, in this review, we will discuss a second independent function as a regulator of nanoscopic protein movement that opposes cholesterol clustering. The understanding of the movement of proteins between nanoscopic lipid domains emerged largely through the recent advent of super-resolution imaging and the establishment of two-color techniques to label lipids separate from proteins. We discuss the labeling techniques for imaging, their strengths and weakness, and how they are used to reveal novel mechanisms for an ion channel, transporter, and enzyme function. Among the mechanisms, we describe substrate and ligand presentation and their ability to activate enzymes, gate channels, and transporters rapidly and potently. Finally, we define cholesterol-regulated proteins (CRP) and discuss the role of PIP2 in opposing the regulation of cholesterol, as seen through super-resolution imaging.

Super-resolution imaging of potassium channels with genetically encoded EGFP

“…ACM, as described in Figure 1E, was introduced to cultured cells. Following fixation, permeabilization, and staining for GM1 and PIP 2 clusters with CTxB and anti-PIP 2 antibody respectively, we gauged the proximity of the two fluorophores using a pair correlation function 5 . Molecules in close proximity yielded a curve with a high pair correlation (as shown in Fig.…”

“…PIP 2 lipid clusters emerge from charged protein interactions, while GM1 lipid clusters spontaneously form due to optimized hydrophobic packing. Cholesterol (yellow shading) partners with GM1 lipids, promoting the increased order of GM1 clusters 5,7,22,23 . ( B ) Breakdown of GM1 lipid components.…”

“…Proteins typically segregate into compartments of cholesterol and ganglioside lipids (GM1) or charged unsaturated lipids like PIP2 and PIP3 (as illustrated in Fig. S1A) [4][5][6][7] . To visualize the spatial organization of proteins alongside lipids, these lipids must also be fluorescently labeled.…”

GABA and astrocytic cholesterol determine the lipid environment of GABAAR in cultured cortical neurons

“…But similar experiments could be done in the presence of well-defined synapses. A second limitation is that of using multidentate antibodies and toxin during live imaging, this could cause clustering and increase the size of lipid rafts 24,33 . Since we utilized localization, not cluster size, this should not affect our conclusion.…”

“…Within the synapse, GABAAR is further localized to gephyrin containing synaptic sub domains (SSDs) [15][16][17][18][19] . The lipid composition of the SSDs is not well defined, but both GABAAR and gephyrin are palmitoylated (a saturated 16 carbon lipid covalently attached to the protein) and palmitoylation regulates raft affinity of most proteins 20 and specifically enhances GABAAR's presence within lipid rafts and with monosialotetrahexosylgangliosides (GM1) [20][21][22] , a common marker of lipid rafts 20,23,24 (see Fig. S1).…”