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.

Figure 1.

High-resolution native MS reveals distinct metal and brain PS lipid bound states of TRAAK and TREK2. A) Mass spectrum of TRAAK with enriched brain PS lipids. B) Deconvolution of the mass spectrum shown in A. The first PS binding event is annotated. C) TREK2 bound to enriched brain PS lipids. D) Deconvolution of the mass spectrum shown in C.

Figure 2.

Cupric ions distinctly enhance POPS interactions with TRAAK but not TREK2. A-C) Native mass spectra of A) TRAAK with 10 μM POPS and 5 μM of B) Cu²⁺ or C) Zn²⁺. The inset annotates the lipid binding event along with metal ion bound states. D-F) Native mass spectra of D) TREK2 with 12 μM POPS and 6 μM of E) Cu²⁺ or F) Zn²⁺. The inset annotates the highlighted lipid binding event along with metal ion bound states. Each lipid binding event is annotated with the number of POPS and Cu²⁺.

Figure 3.

Allosteric coupling of PS and Cu²⁺ binding to TRAAK. A-D) Equilibrium binding constants for TRAAK binding Cu²⁺ and B) POPS, C) dOPS, and D) SOPS. Illustrated in panel A is the number of cupric ions and PS lipids bound to TRAAK per box and the respective KD value plotted. E) Coupling factors (α) for TRAAK binding x PS lipids and y cupric ions (α_x,y). Reported are from repeated measurements (n = 3).