Fatty acid flip-flop and proton transport determined by short-circuit current in planar bilayers

Abstract

The effect of palmitic acid (PA) and oleic acid (OA) on electrical parameters of planar membranes was studied. We found a substantial difference between the effects of PA and OA on proton transfer. PA induced a small increase in conductance, requiring a new technique for estimating proton-mediated currents across low-conductance planar bilayers in which an electrometer is used to measure the transmembrane current under virtual short circuit (SCC). Open-circuit voltage and SCC were used to determine proton and leak conductances. OA caused a marked increase in membrane conductance, allowing the use of a voltage-clamp technique. From SCC data, we were able to estimate the flip-flop rate constants for palmitate (1 ؋ 10 ؊ 6 s ؊ 1 ) and oleate (49 ؋ 10 ؊ 6 s ؊ 1 ) anions. Cholesterol, included in the membrane-forming solution, decreased importantly the leak conductance both in membranes unmodified by FA and in membranes modified by PA added to the bath. Long-chain FA transport across the cell membranes is still a controversial subject. There are two lines of evidence pointing to either a simple diffusional mechanism (1) or a protein-mediated process (2). Through the diffusional mechanism, FA transport is described as being coupled to proton transport, as the protonated (uncharged) form of the FA diffuses better in the hydrophobic interior of the lipid bilayer (3). In this way, FA flip-flop would act as a proton shuttle.On the other hand, the presence of FA in the membrane may enhance proton transport independently of this shuttle mechanism, mainly in the case of unsaturated fatty acids, which reduce membrane order (4), and so could increase proton transport through aqueous defects. Cholesterol, through its membrane-ordering effect (5), could modulate this shuttle-independent proton pathway.The issue of long-chain FA translocation across cell membranes has been dealt with using many approaches (6-8). In biomimetic systems, indirect evidence of FAmediated proton transfer has been obtained mainly from unilamellar vesicles by acidification studies using fluorescent pH-sensitive probes (1).Despite being very sensitive regarding time resolution of the acidification process, studies using vesicles lack information concerning electrical parameters such as transmembrane potential difference, proton-associated electrical currents, and electrical conductance. On the other hand, electrical determinations of FA effects on proton transfer are scant and have been based on membrane conductance measurements (9), suggesting that FAs increase proton transport across the lipid bilayer. In such studies, the membrane-unspecific or leak conductance contributes importantly to the measured conductance and constitutes a major source of indeterminacy.The problem in measuring total membrane conductance arises when the membrane is subjected to an externally applied field, which drives protons, through a protonselective pathway, and other ions, through a nonspecific (leak) pathway. Identifying separately the proton and leak conductances i...

Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid bilayers

“…Figure 3 demonstrates that increased membrane conductance was measured in the presence of all FA and depended on the FA/lipid ratio (10 mol% FA-gray bars, 15 mol% FA-black bars). White bars demonstrate similar low conductivities for all FA (15 mol%) measured at zero voltage in the absence of UCP, which supports earlier observations (5, 54) but contradicts results obtained in decane-containing membranes (55). UCP2-mediated membrane conductance, G, differed in the presence of various FA: it was very small in the presence of palmitic acid in comparison with FA-free membranes, increased significantly in the presence of FAs having one, two, and three double bonds (oleic, linoleic, and eicosatrienoic acids, respectively), and reached maximum values in the presence of polyunsaturated arachidonic FA (Fig.…”

Different Mechanisms of Free Fatty Acid Flip-Flop and Dissociation Revealed by Temperature and Molecular Species Dependence of Transport across Lipid Vesicles

“…7B. We suggest that, from the aqueous phase (Step A), the FFA inserts tail first into the bilayer because Ͼ98% of the FFA is ionized at pH 7.4, and a carboxylate-first insertion would require an enthalpic activation energy of ϳ26 kcal/mol (37). However, dissociation (Figs.…”

“…(Flip-flop of the anionic form of the FFA is between 4 and 6 orders of magnitude slower than that of the protonated form (37,41).) We speculate that, by slipping farther into the bilayer and rotating in a folded conformation (Fig.…”

Fatty acid flux in adipocytes: The in's and out's of fat cell lipid trafficking

“…This concept is based on studies that demonstrate rapid fatty acid flip-flop rates in small unilamellar vesicles [5]. However, cellular membranes have complex lipid and protein structures on their surfaces as compared to smaller membrane vesicles [6] and together with lower substrate availability due to non-saturating physiological LCFA u concentrations could argue that lipid uptake is predominately protein-mediated. Fatty acid transporters could bind LCFA u and facilitate influx thereby overriding slower rates of fatty acid flip-flop and dissociation from the membrane bilayer [7].…”