Docosahexaenoic acid alters bilayer elastic properties Michael J. Bruno*, Roger E. Koeppe II†, and Olaf S. Andersen*‡ *Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY 10021; and †Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701 Edited by Alexander Leaf, Harvard University, Charlestown, MA, and approved April 24, 2007 (received for review February 2, 2007) At low micromolar concentrations, polyunsaturated fatty acids Table 1. Membrane proteins that are modulated by DHA and (PUFAs) alter the function of many membrane proteins. PUFAs other PUFAs exert their effects on unrelated proteins at similar concentrations, Protein PUFA Action Ref. suggesting a common mode of action. Because lipid bilayers serve as the common ‘‘solvent’’ for membrane proteins, the common Cardiac Naϩ channel AA, EPA, DHA Inhibit 10 mechanism could be that PUFAs adsorb to the bilayer/solution L-type Ca2ϩ channel ALA, LA, AA, EPA, Inhibit 11 interface to promote a negative-going change in lipid intrinsic DHA curvature and, like other reversibly adsorbing amphiphiles, in- Kv1.5 channel AA, DHA Inhibit 12 crease bilayer elasticity. PUFA adsorption thus would alter the HERG channel AA, DHA Inhibit 13 bilayer deformation energy associated with protein conforma- TRAAK-1 channel AA, EPA, DHA Activate 14 tional changes involving the protein/bilayer boundary, which TRPV1 EPA, DHA Activate 15 1 would alter protein function. To explore the feasibility of such a nAChR channel DHA Desensitization 16 1 mechanism, we used gramicidin (gA) analogues of different GABAa channel DHA Desensitization 17 lengths together with bilayers of different thicknesses to assess GluR6 glutamate AA, DHA Inhibit 18 whether docosahexaenoic acid (DHA) could exert its effects receptor through a bilayer-mediated mechanism. Indeed, DHA increases gA Connexin43 channel GLA, AA, EPA, Inhibit 19 DHA channel appearance rates and lifetimes and decreases the free ϩ ϩ energy of channel formation. The appearance rate and lifetime Na ,K -ATPase EPA, DHA Inhibit 20 changes increase with increasing channel-bilayer hydrophobic mis- AA, arachidonic acid; ALA, ␣-linolenic acid; EPA, eicosapentaenoic acid; match and are not related to differing DHA bilayer absorption GLA, ␥-linolenic acid coefficients. DHA thus alters bilayer elastic properties, not just lipid intrinsic curvature; the elasticity changes are important for DHA’s bilayer-modifying actions. Oleic acid (OA), which has little effect on ⌬ 0 For a given protein, Gdef varies as a function of the mismatch membrane protein function, exerts no such effects despite OA’s between the protein’s hydrophobic length (l) and the bilayer’s adsorption coefficient being an order of magnitude greater than hydrophobic thickness (d ), the intrinsic curvature (c )ofthe DHA’s. These results suggest that DHA (and other PUFAs) may 0 0 bilayer-forming lipids, and the bilayer compression and bending modulate membrane protein function by bilayer-mediated mech- moduli. To a first approximation, ⌬G0 can be expressed as a anisms that do not involve specific protein binding but rather def biquadratic form in the hydrophobic mismatch, d Ϫ l, and changes in bilayer material properties. 0 c0 (28): bilayer material properties ͉ bilayer stiffness ͉ gramicidin channels ͉ ⌬G0 ϭ H ⅐͑d Ϫ l͒2 ϩ H ⅐͑d Ϫ l͒⅐c ϩ H ⅐c2, [1] hydrophobic mismatch ͉ polyunsaturated fatty acid def B 0 X 0 0 C 0 where the coefficients HB, Hx, and Hc are determined by the olyunsaturated fatty acids (PUFAs) modulate a wide variety protein geometry, the bilayer thickness and elastic moduli (29). Pof biological processes (1–3), and alter the function of a The elastic moduli (30–32) and intrinsic curvature (33, 34) may diverse group of unrelated membrane proteins (Table 1, for be altered by reversibly adsorbing amphiphiles, which would additional examples, see ref. 4), whereas saturated or mono- provide a basis for the acute effects of PUFAs on membrane unsaturated fatty acids such as oleic acid (OA) are relatively protein function, although the relative importance of changes in inert. Among the acute effects of PUFAs is the reversal of the elastic moduli and curvature would need to be established. arrhythmias underlying sudden cardiac death in rats (5), dogs PUFAs and micelle-forming amphiphiles, for example, have (6), and humans (7), most likely due to inhibition of cardiac opposite effects on lipid intrinsic curvature (34, 35), yet both sodium and L-type calcium channels. The mechanism(s) under- shift the steady-state inactivation curve for voltage-dependent lying the reversal remain unclear, but it occurs at the low micromolar PUFA concentrations where PUFAs are general sodium channels in the same (hyperpolarizing) direction (35, 36), modulators of membrane protein function. Because PUFAs which would suggest that changes in elastic moduli dominate avidly adsorb to biological membranes (8, 9), and the common- over changes in curvature. ality among the proteins in Table 1 is that they are imbedded in lipid bilayers, PUFAs may act through some common, bilayer- Author contributions: M.J.B. and O.S.A. designed research; R.E.K. and O.S.A. designed the mediated mechanism. gramicidin analogues; M.J.B. performed research; M.J.B. analyzed data; and M.J.B., R.E.K., Bilayer-dependent regulation of membrane function can oc- and O.S.A. wrote the paper. cur when membrane proteins undergo conformational changes The authors declare no conflict of interest. that involve the protein/bilayer boundary (for example, see ref. This article is a PNAS Direct Submission. 21). Because lipid bilayers are elastic bodies (22) and bilayer- Abbreviations: PUFA, polyunsaturated fatty acid; OA, oleic acid; gA, gramicidin; DHA, spanning proteins are coupled to the host bilayer through docosahexaenoic acid. hydrophobic interactions (23), membrane protein conforma- ‡To whom correspondence should be addressed. E-mail: [email protected]. tional changes incur an energetic cost (24–26), the bilayer This article contains supporting information online at www.pnas.org/cgi/content/full/ ⌬ 0 deformation energy ( Gdef), which causes protein function to be 0701015104/DC1. modulated by the lipid bilayer (27, 28). © 2007 by The National Academy of Sciences of the USA 9638–9643 ͉ PNAS ͉ June 5, 2007 ͉ vol. 104 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0701015104 Downloaded by guest on September 30, 2021 Fig. 1. Effect of OA and DHA on gA channel activity. Current traces before and after addition of 10 ␮M DHA (top two traces) or OA (bottom two traces) to Ϫ both sides of a DC18:1PC/n-decane bilayer containing gA(13) and AgA (15). (Results from two different experiments.) The interrupted lines denote the current levels for gA(13) (short dash) and AgAϪ(15) (long dash). 1 M NaCl, 10 mM Hepes, pH 7, Ϯ 200 mV, 500 Hz. The cartoons at the bottom of the figure illustrate the differences in bilayer deformation with differing hydrophobic mismatch between channel (shaded blocks) and lipid bilayer (represented by springs). To address these questions we used gramicidin (gA) channels channel population. (In the absence of gA, neither DHA nor OA of different lengths to monitor how PUFAs modulate lipid caused channel-like activity.) bilayer properties. gA channels are formed by transbilayer When DHA was added to only one side of a bilayer, the association of two monomers (37). When the bilayer’s hydro- single-channel currents and lifetimes were similar at both po- phobic thickness, thickness to match the protein’s hydrophobic larities (results not shown), indicating that transmembrane DHA length, differs from the channel’s hydrophobic length, l, the flux (for example, see refs. 39 and 40) caused the DHA mole fraction to be similar in the two leaflets. bilayer will adjust its d0 resulting in a local bilayer deformation ⌬ 0 As will be important below, DHA is more active in altering the with energetic cost Gdef (cf. Eq. 1). The bilayer responds by applying a disjoining (restoring) force to the channel dimer: function of the shorter gA(13) channels. This difference is not ϭ ⅐ ⅐͑ Ϫ ͒ ϩ ⅐ Fdis 2 HB d0 l HX c0. [2] A B Changes in this disjoining force are reflected as changes in channel lifetime (␶), meaning that gA channels can be used as force transducers (38) to report changes in bilayer elasticity and lipid curvature. It is thus possible to assess whether docosa- hexaenoic acid (DHA) alters bilayer properties and thus might exert its effects on membrane protein function through an indirect (bilayer-mediated) mechanism. Results DHA is a potent modifier of gramicidin channel activity, whereas OA is not (Fig. 1). One can examine the role of channel-bilayer hydrophobic mismatch by comparing the relative BIOPHYSICS changes in lifetimes and appearance rates for the shorter and longer channels (Fig. 1 Lower). 10 ␮MDHA§, but not 10 ␮M OA, increases the appearance rates of both gA(13) and AgAϪ(15) channels with the larger effect on the shorter channels (Figs. 2 and 3). [The enantiomeric gA(13) and AgAϪ(15) were used to prevent hybrid channel formation, which simplifies the analysis.] DHA also increases the single-channel current transition amplitudes (i), whereas OA has little or no effect (Fig. 2A). The lifetime distributions in the absence or presence of DHA (or Fig. 2. Effect of OA and DHA on gA single-channel current transitions and OA) can be fit by single exponential distributions (Fig. 2B), lifetimes. (A) Current transition amplitude histograms of gA(13) (left peak) Ϫ meaning that DHA modulates the function of an existing and AgA (15) (right peak) channels in DC18:1PC bilayers in the absence or channel type, rather than promoting the appearance of a new presence of 10 ␮M DHA or OA. (B) Normalized single-channel survivor histo- grams for gA(13) (Upper) and AgAϪ(15) (Lower) fitted with single exponential distributions; note the 10-fold difference in the scale of the abscissae.