Stability of KcsA Tetramer Depends on Membrane Lateral Pressure

Abstract

The potassium channel KcsA forms an extremely stable tetramer. Despite this high stability, it has been shown that the membrane-mimicking solvent 2,2,2-trifluoroethanol (TFE) can induce tetramer dissociation [Valiyaveetil, F. I., et al. (2002) Biochemistry 41, 10771-7, and Demmers, J. A. A., et al. (2003) FEBS Lett. 541, 69-77]. Here we have studied the effect of TFE on the structure and oligomeric state of the KcsA tetramer, reconstituted in different lipid systems. It was found that TFE changes the secondary and tertiary structure of KcsA and that it can dissociate the KcsA tetramer in all systems used. The tetramer is stabilized by a lipid bilayer as compared to detergent micelles. The extent of stabilization was found to depend on the nature of the lipids: a strong stabilizing effect of the nonbilayer lipid phosphatidylethanolamine (PE) was observed, but no effect of the charged phoshosphatidylglycerol (PG) as compared to phosphatidylcholine (PC) was found. To understand how lipids stabilize KcsA against TFE-induced tetramer dissociation, we also studied the effect of TFE on the bilayer organization in the various lipid systems, using (31)P and (2)H NMR. The observed lipid dependency was similar as was found for tetramer stabilization: PE increased the bilayer stability as compared to PC, while PG behaved similar to PC. Furthermore, it was found that TFE has a large effect on the acyl chain ordering. The results indicate that TFE inserts primarily in the membrane interface. We suggest that the lipid bilayer stabilizes the KcsA tetramer by the lateral pressure in the acyl chain region and that this stabilizing effect increases when a nonbilayer lipid like PE is present.

Functional Equilibrium of the KcsA Structure Revealed by NMR

“…The effect of TFE on the structural equilibrium is seemingly inconsistent with the reported effect of TFE to destabilize the tetramer assembly of KcsA (25), which would lead to the decrease in the p perm value. However, the shift of the structural equilibrium was observed at the lower TFE concentrations (2-3%) as compared with the disruption of the tetrameric assembly (20%) (25), suggesting that the mechanism of the structural equilibrium shift by TFE may not be due to the destabilization of KcsA.…”

“…However, the shift of the structural equilibrium was observed at the lower TFE concentrations (2-3%) as compared with the disruption of the tetrameric assembly (20%) (25), suggesting that the mechanism of the structural equilibrium shift by TFE may not be due to the destabilization of KcsA.…”

“…TFE Shifts the Structural Equilibrium of KcsA-In the lipid bilayer, the tetrameric stability of KcsA is reportedly perturbed by the surrounding membrane environments such as lipid composition (23) and partition of alcohol, such as TFE (24,25). We then acquired the methyl-TROSY spectra of KcsA160 in rHDL upon sequential additions of TFE (Fig.…”

Effects of Conducting and Blocking Ions on the Structure and Stability of the Potassium Channel KcsA

“…In these experiments, because the homotetrameric KcsA is very stable in SDS (24,37,38), analysis by SDS-PAGE provides a simple means to study the effects of destabilizing agents such as TFE on the oligomerization state of KcsA (39). The gels in Fig.…”

Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS

“…SDS-stable complexes were shown to dissociate under the influence of small, fluorinated, alcohols such as 2,2,2-trifluoroethanol (TFE) that are thought to disturb the local interactions between hydrophobic protein segments, and/or between the hydrophobic protein segments and the surrounding membrane lipids or SDS-molecules [5][6][7][8]. In addition, TFE has been used at high concentrations (≥50%) to improve the separation of membrane proteins from E. coli [9] and Streptococcus mutans [10] in the classic 2D iso-electric focusing (IEF)-SDS-PAGE approach, illustrating its potential to solubilize membrane proteins.…”