Differential Influence of DOPG and DOPA Anionic Phospholipids on Single Channel Conductance of KcsA

Abstract

Binding of anionic phospholipids to non-annular binding site of KcsA has been shown with crystallographic and biochemical methods. To gain insight into the functional importance of these specific protein-lipid interactions we studied the single channel properties of KcsA in planar lipid bilayer by varying the anionic phospholipid content of the membrane. Phospholipids with identical acyl chains and equivalent headgroup charges have been chosen for this purpose. Single channel properties of KcsA were unchanged, when the content of anionic phospholipids was kept below 30% in planar lipid bilayers formed from DOPC neutral phospholipid as remainder component. Upon gradual increase of membrane DOPG content up to 100%, a significant increase in single channel conductance was observed only at positive potentials (from 82,2±1,4 pS to 116,3±0,7 pS at +100mV in symmetrical 150 mM KCl solution). Surprisingly, equimolar increase of DOPA content of the membrane had no influence on single channel conductance of KscA. Differential influence of DOPG and DOPA phospholipids was preserved at varying K+ concentrations of recording solutions ranging from 50 to 250 mM. Using KcsA-Kv1.3 chimera we could show that the stretch of amino acids between E51 and A65 in KcsA was responsible for differential sensitivity of KcsA for DOPA and DOPG. Taken together, our data show that the structure of the phospholipid headgroup is important for phospholipid-specific modulation of single channel conductance in KcsA.