Cupric Ions Selectively Modulate TRAAK–Phosphatidylserine Interactions

Abstract

TRAAK and TREK2 are two-pore domain K+ (K2P) channels and are modulated by diverse factors including temperature, membrane stretching, and lipids, such as phosphatidic acid. In addition, copper and zinc, both of which are essential for life, are known to regulate TREK2 and a number of other ion channels. However, the role of ions in the association of lipids with integral membrane proteins is poorly understood. Here, we discover cupric ions selectively modulate the binding of phosphatidylserine (PS) to TRAAK but not TREK2. Other divalent cations (Ca2+, Mg2+, and Zn2+) bind both channels but have no impact on binding PS and other lipids. Additionally, TRAAK binds more avidly to Cu2+ and Zn2+ than TREK2. In the presence of Cu2+, TRAAK binds similarly to PS with different acyl chains, indicating a crucial role of the serine headgroup in coordinating Cu2+. High-resolution native mass spectrometry (MS) enables the determination of equilibrium binding constants for distinct Cu2+-bound stoichiometries and uncovered the highest coupling factor corresponds to a 1:1 PS-to-Cu2+ ratio. Interestingly, the next three highest coupling factors had a ∼1.5:1 PS-to-Cu2+ ratio. Our findings bring forth the role of cupric ions as an essential cofactor in selective TRAAK–PS interactions.

Combining native mass spectrometry and lipidomics to uncover specific membrane protein–lipid interactions from natural lipid sources

“…Here, a mix of unsaturated and saturated PS species is identied across multiple wash steps. 33 While TG and plasmalogen PE species are not actively emphasized for their role in cell membranes, both classes have a presence in cellular membranes and are linked to increased membrane uidity due to increased curvature. [69][70][71] We also note a possibility of lipid-lipid interactions that may be inuencing the retention of these lipids.…”

Native mass spectrometry of proteoliposomes containing integral and peripheral membrane proteins

“…5F ). In our recent work, we have discovered that copper( ii ) binds TRAAK, 8 and the adducts observed here are likely copper( ii ) binding to the channel.…”

“…Although TREK2 shares ∼45% sequence identity with TRAAK, 59,61 copper( ii ) does not modulate TREK2–lipid interactions. 62 The ejected TREK2 complexes were bound to a near equal abundance of POPC and SOPS (Fig. S7†).…”

“…These results further corroborate our observations for TRAAK and TREK2 solubilized in detergent. 62 More complex lipid environments can be explored, such as TRAAK ejected from proteoliposomes formed from a brain polar lipid extract. However, future directions will be the development of methods to further dissect adduct heterogeneity.…”

“…TREK2 (Uniprot P57789-1, residues 55–335 with T58A mutation) was expressed and purified as previously described. 8 Generation of recombinant baculovirus and expression of TREK2 was identical to that described for G β1γ2 with the exception that 5 μM kifunensine was added to the culture during expression.…”