MEMBRANE SURFACE-CHARGE TITRATION PROBED BY GRAMICIDIN-A CHANNEL CONDUCTANCE

We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the l ipid charge through bulk solution pH and dilution of a charged lipid b y neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsC l aqueous solutions, we show that the effects of lipid charge titratio n on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentrat ion when the bathing solution pH is less than 3. A smeared charge mode l permits us to separate different contributions to the channel conduc tance and to introduce a new method for ''bilayer pKa'' determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To cal culate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the pl anar Poisson-Boltzmann equation, as well as the construction of a ''Gi bbs dividing surface'' between salt bath and charged membrane. All app roximations yield the intrinsic pK(a)(in) of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admired neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the elec trostatic model. This observation is in accord with the different cond uctance saturation values for PS and PC lipids reported earlier (Apell et al. 1979, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also i nfluenced by lipid-dependent structural factors.