Transient Ca2+ entry by plasmalogen-mediated activation of receptor potential cation channel promotes AMPK activity.

Honsho M, Mawatari S, and Fujino T. (2022) Transient Ca2+ entry by plasmalogen-mediated activation of receptor potential cation channel promotes AMPK activity. Frontiers in Molecular Biosciences

Plasmalogens are a unique class of phospholipid that contain a vinyl-ether bond at the sn-1 position, which causes these lipids to have a more compact structure. This double bond gives plasmalogens a range of roles within cells including in cell membrane fluidity and function, scavenging radical oxygen species, lipid raft formation, cholesterol transportation, and vesicular fusion. Supplementation with plasmalogens has also been shown in mice and cultured cells to activate signaling pathways mediated by protein kinase B (AKT), extracellular signal-regulated kinases (ERK), and brain-derived neurotrophic factors (BDNF). In mice this was found to improve learning and memory, suggesting that plasmalogens have a role in regulating signaling pathways. Honsho et al were interested in confirming the theory that plasmalogens activate cellular pathways using primary human fibroblasts and specifically looked at AMP-activated protein kinase (AMPK), an enzyme that has a role in cellular energy homeostasis.

To perform this study, primary human dermal fibroblast (HDF-a) cells were treated with plasmalogens derived from scallop. The activation of cellular signaling pathways was accomplished by detecting the phosphorylation status of proteins involved in signaling pathways. In the plasmalogen supplemented cells, phosphorylation of threonine 172 was significantly upregulated but neither ERK nor AKT at serine 473 showed increased phosphorylation with plasmalogen supplementation. This would suggest that plasmalogens can enhance phosphorylation of AMPK independent from ERK and AKT. Interestingly, this was also found in other cells including cervical cancer HeLa cells, neuroblastoma SH-SY5Y cells, human hair outer sheath cells (HHORSC), and human hair dermal papilla cells (HHDPC) which indicates that this phenomenon is not specific to human dermal fibroblasts.

As increased intracellular Ca2+ leads to AMPK activation, Honsho et al were also interested in whether plasmalogens have a role in calcium influx. This was accomplished by recording intracellular Ca2+ levels with a fluorescent calcium indicator, Fluo-4 AM. They found that quickly after adding the plasmalogen supplement to the culture medium, Ca2+ levels were elevated, suggesting that plasmalogens influence plasma membrane localized channels. Extracellular Ca2+ influx is mediated by different transporters than intracellular Ca2+ movement. These include transient receptor potential channels and voltage-gated Ca2+ channels. To determine whether extracellular transport of Ca2+ is mediated by plasmalogens, and if so which channels, cells were treated with channel inhibitors. The L-type voltage-gated calcium channel blocker (verapamil) did not suppress the influx of Ca2+ after plasmalogen supplementation, but this Ca2+ influx was blocked by an inhibitor for the canonical transient receptor potential (TRPC) channel. There are five different TRPC channels in humans and while TRPC3 and TRPC6 are known to be activated by diacylglycerol the author’s wanted to determine which of the other three may be activated by plasmalogens. When a selective TRPC4 channel inhibitor, ML204, was used Ca2+ influx was suppressed, suggesting that plasmalogen-mediated influx of Ca2+ occurs through activating TRPC4 channels. To support these findings, when ML204 was present or TRPC4 was knocked down, plasmalogen-mediated phosphorylation of AMPK was suppressed.

Honsho et al were interested in determining the role of plasmalogens in activating signaling pathways, specifically looking at the AMPK pathway. They showed that plasmalogen supplementation increases Ca2+ influx by activating the TRPC4 channel. Also, they demonstrated that phosphorylation of AMPK is activated by plasmalogen supplementation in many different cell lines, suggesting this is a general phenomenon, but this is not seen with other pathways like ERK and AKT. This was supported by the findings that the TRPC4 channel inhibitor, ML204, reduced Ca2+ influx. It was also confirmed that this was specific to plasmalogens because neither phosphatidylethanolamine or lyso-plasmalogens, the product from hydrolyzing plasmalogens, supplementation elicited the same activation. The authors suggest that the vinyl-ether bond and fatty acid at sn-2 is necessary for TRPC4 activation. This work reinforces the importance of plasmalogens, however plasmalogens are typically located in the inner leaflet of the plasma membrane and are less likely to be involved in TRPC4 activation which is an extracellular channel. Because of this, more work is needed to fully determine the role for plasmalogen-mediated TRPC4 activation.