Influence of lipids on membrane assembly and stability of the potassium channel KcsA

Abstract

Recently we observed in an in vitro system that newly synthesized KcsA assembles e⁄ciently into a tetramer in lipid vesicles [van Dalen et al. (2002) FEBS Lett. 511, 51^58]. Here we used this system to get insight into the importance of the lipid composition for KcsA membrane association and tetramerization and we compared this to the lipid dependency of the thermo-stability of the KcsA tetramer. It was found that a large amount of phosphatidylethanolamine ( s 40 mol%) and a lower amount of phosphatidylglycerol (V V20^30 mol%) were optimal for e⁄cient KcsA membrane association and tetramerization. Strikingly, vesicles of the abundant and commonly used membrane lipid phosphatidylcholine did not support assembly, further demonstrating the importance of membrane lipid composition for KcsA assembly. The in vitro assembled KcsA tetramer showed similar thermo-stability in biological and pure lipid membranes, demonstrating that both tetramers are alike. In addition, we show that solubilization of the membrane with detergent reduces the thermo-stability of the tetramer. The highest KcsA tetramer stability was observed in intact bilayers in the presence of anionic lipids. ß

Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K + channel: The power of native nanodiscs

“…SDS/PAGE analysis of purified KcsA in native nanodiscs shows a pronounced band at a molecular weight of ∼60 kDa ( Fig. 2A, lane 2), similar to that observed with tetrameric KcsA purified in a standard protocol, using the nonionic detergent n-dodecyl-β-D-maltoside (DDM) (lane 4) (22). Exposing the samples to 95°C in the presence of SDS results in a virtually complete transition to the monomeric form of KcsA, as evident from the appearance of a band at ∼17 kDa ( Fig.…”

“…Thus, the more rigid structure of the particles could convey the higher stability of KcsA. However, it should also be noted that the mere presence of lipids can increase the stability of KcsA, as suggested by studies on reconstituted protein (20,22). Conserving and stabilizing a lipid environment around MPs during their extraction by SMA polymers thus directly yields particles that maintain a higher stability of the trapped protein than detergent micelles.…”

Functional Equilibrium of the KcsA Structure Revealed by NMR

“…At neutral pH, the tetrameric assembly of KcsA is reportedly influenced, whether it is reconstituted in the detergent micelles or in the lipid bilayers (23). We then reconstituted KcsA into rHDL, where KcsA is surrounded by the lipid bilayer, to investigate the effect of the surrounding environment on the structural equilibrium under acidic conditions.…”

“…Numbers indicate the names of the KcsA variants. detergent micelles or in the lipid bilayer (23). We then investigated the p perm values in the lipid bilayer under acidic conditions by embedding KcsA in rHDL, in which the KcsA reconstituted in the lipid bilayer is surrounded by membrane scaffold proteins to form a disc-shaped particle (17,19 S2).…”

Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

“…In addition to protein-protein interaction between monomers, protein-lipid and protein-ion interactions play important roles in stabilizing the KcsA tetramer (17)(18)(19)(20). The selectivity filter of KcsA, coordinated with K ϩ ions, can serve as a bridge between the four monomers to maintain the structure of the selectivity filter and the tetrameric architecture of the channel as a whole (11,21).…”