Phloretin Affects the Voltage Gating of Alpha-Hemolysin Channel

Abstract

We studied the effect of dipole modifiers on the channel forming activity of Staphylococcus aureus alpha-hemolysin in phosphocholine bilayers bathed in 1 M KCl (pH 7.5). We manipulate bilayer dipole potential (Vd) adding phloretin or phloridzin to reduce (Vd), and 6-ketocholestanol and RH 421 to increase Vd. In the absence of dipole modifiers, an alpha-hemolysin pore fluctuates between high (∼1 nS) and low (∼0.1nS) conductance states at transmembrane voltage V≥|100| mV. Addition of 20 µM phloretin after the channel formation induces transition of the channel to low-conductance state at V≥|25| mV. Adding phloretin before channel incorporation does not influence its voltage gating. Additions of phloridzin (20 µM) and RH 421 (10 µM) in the membrane bathing solution before or after channel formation, or addition of 6-ketocholestanol (50 mol.%) into the membrane composition do not affect the channel gating. We conclude that variation in Vd induced by dipole modifiers does not influence alpha-hemolysin pore gating and the effect of phloretin is likely to be attribute to specific interaction. It is also likely that the binding site for phloretin becomes accessible after alpha-toxin incorporation into the membrane and subsequent pore formation. We suggest that phloretin binding reduces the energy barrier for conformation transition of alpha-hemolysin pore to low-conductance state. The nature of specific interaction between phloretin and alpha-hemolysin channel is discussed. The work is supported in part by RFBR (project 09-04-00883), the Program of Presidium of the RAS “Molecular and Cell Biology”, the Grant of Administration of St.-Petersburg for young scientists and State Contract (FAE Π1372).