–activated K Channel: Re-examination of the Surface Charge Hypothesis

Incorporation of BK Ca 2 –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 Ba 2 , 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 Ba 2 at 100 mM KCl and only a small enhancement of discrete and fast block by external Ba 2 in PS-containing membranes at 20 mM KCl. Model calculations of effective surface potential sensed by the K conduction and Ba 2 -blocking reactions using the Gouy-Chapman-Stern theory of lipid surface charge do not lend support to a simple electrostatic mechanism that predicts valencedependent 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 Ba 2 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. key words: electrostatics • Gouy-Chapman theory • lipid modulation • phospholipid • Slowpoke I N T R O D U C T I O N Ion transport proteins are responsible for movement of ions across a cell membrane that separates the external and internal aqueous milieu by 40 Å. The two membrane interfaces on the outside and inside of the cell exist as charged surfaces due to the presence of ionized chemical groups on protein, carbohydrate, and lipid. Discrete charges located at the membrane–water interface give rise to an electrostatic surface potential that varies in a complex fashion according to the distribution and density of fixed charges, electrolyte composition, and distance from the charged groups (McLaughlin, 1989; Honig and Nicholls, 1995). Because local concentrations of ionic substrates (e.g., Na , K , H , Ca 2 , Cl ) near the membrane are determined by this complex surface potential map, surface charge is considered to play an important role in modulating ion channel function (Green and Andersen, 1991; Latorre et al., 1992; Hille, 2001). One of the first experiments to identify functional effects of lipid surface charge showed that the unitary conductance of the gramicidin cation channel is increased 4.3-fold, from 15 pS in phosphatidylcholine (PC)* to 65 pS in a phosphatidylserine (PS) membrane under conditions of 100 mM CsCl (Apell et al., 1979). According to electrostatic theory, the radius of the cylindrical gramicidin channel, 6–13 Å (Killian, 1992; Jin Bong Park’s present address is Department of Physiology, College of Medicine, Chungnam National University, Taejeon 301-131, Re-