Effect of phosphatidylserine on unitary conductance and Ba²⁺ block of the BK Ca²⁺-activated K⁺ channel: Re-examination of the surface charge hypothesis

Incorporation of BK Ca²⁺-activated K⁺ channels into planar bilayers composed of negatively charged phospholipids such as phosphatidylserine (PS) or phosphatidylinositol (PI) results in a large enhancement of unitary conductance (g_ch) in comparison to BK channels in bilayers formed from the neutral zwitterionic lipid, phospatidylethanolamine (PE). Enhancement of g_ch by PS or PI is inversely dependent on KCl concentration, decreasing from 70% at 10 mM KCl to 8% at 1,000 mM KCl. This effect was explained previously by a surface charge hypothesis (Moczydlowski, E., O. Alvarez, C. Vergara, and R. Latorre. 1985. J. Membr. Biol. 83:273-282), which attributed the conductance enhancement to an increase in local K⁺ concentration near the entryways of the channel. To test this hypothesis, we measured the kinetics of block by external and internal Ba²⁺, a divalent cation that is expected to respond strongly to changes in surface electrostatics. We observed little or no effect of PS on discrete blocking kinetics by external and internal Ba²⁺ at 100 mM KCl and only a small enhancement of discrete and fast block by external Ba²⁺ in PS-containing membranes at 20 mM KCl. Model calculations of effective surface potential sensed by the K⁺ conduction and Ba²⁺-blocking reactions using the Gouy-Chapman-Stern theory of lipid surface charge do not lend support to a simple electrostatic mechanism that predicts valence-dependent increase of local cation concentration. The results imply that the conduction pore of the BK channel is electrostatically insulated from the lipid surface, presumably by a lateral distance of separation (>20 Å) from the lipid head groups. The lack of effect of PS on apparent association and dissociation rates of Ba²⁺ suggest that lipid modulation of K⁺ conductance is preferentially coupled through conformational changes of the selectivity filter region that determine the high K⁺ flux rate of this channel relative to other cations. We discuss possible mechanisms for the effect of anionic lipids in the context of specific molecular interactions of phospholipids documented for the KcsA bacterial potassium channel and general membrane physical properties proposed to regulate membrane protein conformation via energetics of bilayer stress.