Review
Do sterols reduce proton and sodium leaks through lipid bilayers?

https://doi.org/10.1016/S0163-7827(01)00009-1Get rights and content

Abstract

Proton and/or sodium electrochemical gradients are critical to energy handling at the plasma membranes of all living cells. Sodium gradients are used for animal plasma membranes, all other living organisms use proton gradients. These chemical and electrical gradients are either created by a cation pumping ATPase or are created by photons or redox, used to make ATP. It has been established that both hydrogen and sodium ions leak through lipid bilayers at approximately the same rate at the concentration they occur in living organisms. Although the gradients are achieved by pumping the cations out of the cell, the plasma membrane potential enhances the leakage rate of these cations into the cell because of the orientation of the potential. This review proposes that cells use certain lipids to inhibit cation leakage through the membrane bilayers. It assumes that Na+ leaks through the bilayer by a defect mechanism. For Na+ leakage in animal plasma membranes, the evidence suggests that cholesterol is a key inhibitor of Na+ leakage. Here I put forth a novel mechanism for proton leakage through lipid bilayers. The mechanism assumes water forms protonated and deprotonated clusters in the lipid bilayer. The model suggests how two features of lipid structures may inhibit H+ leakage. One feature is the fused ring structure of sterols, hopanoids and tetrahymenol which extrude water and therefore clusters from the bilayer. The second feature is lipid structures that crowd the center of the bilayer with hydrocarbon. This can be accomplished either by separating the two monolayers with hydrocarbons such as isoprenes or isopranes in the bilayer's cleavage plane or by branching the lipid chains in the center of the bilayers with hydrocarbon. The natural distribution of lipids that contain these features are examined. Data in the literature shows that plasma membranes exposed to extreme concentrations of cations are particularly rich in the lipids containing the predicted qualities. Prokaryote plasma membranes that reside in extreme acids (acidophiles) contain both hopanoids and iso/anteiso- terminal lipid branching. Plasma membranes that reside in extreme base (alkaliphiles) contain both squalene and iso/anteiso- lipids. The mole fraction of squalene in alkaliphile bilayers increases, as they are cultured at higher pH. In eukaryotes, cation leak inhibition is here attributed to sterols and certain isoprenes, dolichol for lysosomes and peroxysomes, ubiquinone for these in addition to mitochondrion, and plastoquinone for the chloroplast. Phytosterols differ from cholesterol because they contain methyl and ethyl branches on the side chain. The proposal provides a structure-function rationale for distinguishing the structures of the phytosterols as inhibitors of proton leaks from that of cholesterol which is proposed to inhibit leaks of Na+. The most extensively studied of sterols, cholesterol, occurs only in animal cells where there is a sodium gradient across the plasma membrane. In mammals, nearly 100 proteins participate in cholesterol's biosynthetic and degradation pathway, its regulatory mechanisms and cell-delivery system. Although a fat, cholesterol yields no energy on degradation. Experiments have shown that it reduces Na+ and K+ leakage through lipid bilayers to approximately one third of bilayers that lack the sterol. If sterols significantly inhibit cation leakage through the lipids of the plasma membrane, then the general role of all sterols is to save metabolic ATP energy, which is the penalty for cation leaks into the cytosol. The regulation of cholesterol's appearance in the plasma membrane and the evolution of sterols is discussed in light of this proposed role.

Introduction

This review addresses the structure of certain lipids in those biological membranes that maintain a cationic (H+, Na+) electrochemical gradient. These membranes are central to energy transduction and cellular function. To the extent that the cations leak through the lipid bilayer domains of plasma membranes, to that extent energy stored in the gradient is lost, i.e. ATP must be consumed to pump out the cations. Furthermore, the leakage is always enhanced by the electrochemical gradient because the cations are pumped to the positive side of the gradient and the membrane potential is so oriented as to enhance cation leakage into the cell. Lipid structures such as cholesterol have been shown to decrease the permeability of these membranes to cations.

For the handling of life's energy, living cells have invested in two fundamental mechanisms: chemical energy (primarily ATP), and cation electrochemical gradients (principally H+ and Na+) across membrane bilayers. These two mechanisms are intimately, and largely reversibly, entwined. The two principal sources of energy for life, photons and redox, produce electrochemical gradients. These in turn produce proton gradients that subsequently produce ATP. This occurs at mitochondrial, chloroplast and bacterial plasma membranes. Thus the source of energy increases the cation concentration on one side (positive) of the membrane bilayer whereas the ATP synthesis occurs on the other side (negative). In these membranes, the stored cations pass down the gradient through the ATPase's proton pore. Thus the electrochemical cation gradient is used to synthesize ATP. However, this is only one use of the electrochemical cation gradient. It is used for a myriad of activities such as transport of nutrients, excretion, bacterial flagellar motion, rejection of toxins from the cell and many other membrane functions. (Under some circumstances, i.e. anaerobic conditions, prokaryotes or eukaryotes produce ATP metabolically, and use it to create an electrochemical gradient in the plasma membrane.) Proton leakage though the bacterial plasma membrane is especially important for those organisms that live in hostile pH environments, the acidophiles and the alkaliphiles. These organisms appear to have unique lipid structures that may inhibit the proton leakage.

Eukaryote cells contain “prokaryote organelles” that produce ATP, much of which is used to generate an electrochemical gradient across the eukaryote plasma membrane. In animals, the cation gradient uses sodium; in prokaryotes and in all other eukaryotes, plants, yeast, fungi, etc., a proton gradient is used. All cells use K+ as the counterion to adjust the osmolality and the overall potential across the cell membrane. Cells maintain a relatively high and relatively constant internal [K+]. The extracellular [Na+] together with the sodium channels permits controlled lateral signals along the membrane, and therefore motion in animals. Nature does not contain gated H+ channels that permit signaling along membranes. Gated channels, which permit such signaling, are exclusively for metal cations, principally Na+, K+, and Ca++ or anions, principally Cl. The latter are used by plants. These channels permit signaling and therefore neural activity [1]. The switch to sodium taken together with the evolution of the sodium-gated channel served animals by granting them motion. Meanwhile, the use of the Na+ electrochemical gradient, with its energy supplied by mitochondrial ATP also served all the purposes in the animal eukaryote plasma membrane for which microbes and plants use the H+ gradient.

The first measurements of K+, Na+ leakage across defined lipid bilayers were conducted in 1972 by Papahadjopoulos [2], [3]. His group examined the diffusion of many cations across a variety of defined lipid bilayers without a membrane potential. They found that the permeability of all of the phospholipid bilayers they tested to either Na+ or K+ was approximately 10−12 cm/s. They, and most workers at the time, considered this too low to be of any biological significance. In those experiments where the cholesterol concentration was in the range of that found in living animal membranes the Na+ leakage was reduced to one third that of the control cholesterol-free bilayers.

Following up Nicholls' [4], [5] early studies on proton leakage in brown fat mitochondrial inner membrane (which turned out to be due to uncoupling proteins), Hinkle [6], [7], measured the H+ leakage through the lipids of heart mitochondrial inner membrane. He found that the rate of proton leakage was such that, in order to get accurate measurements of H+ utilization for ATP synthesis (the P/O ratio), he had to measure the proton leakage through the lipid bilayer. He was also motivated by the then recent measurements [8], [9] of the proton permeability of vesicular phospholipid bilayers (10−5 cm/s), Hinkle made another important observation, that mitochondrial membranes display about the same permeability for K+ as they do for H+. He noted that the [K+] is 10−1 M, seven orders of magnitude greater than [H+], which is about 10−7 M. Because measurements [2], [3] of lipid bilayers display virtually the same permeabilities (10−12) to Na+ and K+, and because that permeability is seven orders of magnitude smaller than the observed H+ leakage, the leakage of the two cations is approximately the same in plasma membranes. In summary, the lipid bilayer domains of living plasma membranes have approximately the same permeabilities to H+ and Na+. In all plasma membranes, the electrochemical gradient is so oriented to enhance the leakage of these cations into the cell. Potassium leakage need not be considered since it is on the negative side of the gradient.

There are four factors that affect the leakage rate of the cations, H+ or Na+, across lipid bilayers. These are

  • 1.

    The structures of the lipids in the membrane.

  • 2.

    The relative concentration of the cations on the two sides the bilayer.

  • 3.

    The temperature.

  • 4.

    The electrochemical potential of the membrane, the magnitude and direction of which provide a driving force for leakage in membranes containing it.

This review focuses on a hypothesis that explains how protons leak across lipid bilayers. The hypothesis explains a wide variety of lipid structures including the distinction between the molecular structures of cholesterol and that of the phytosterols. It provides a rational additional role for the ubiquitous occurrence in cells of isopranes and isoprenes, hopanoids and iso/anteiso lipids in bacteria. But perhaps most important, it provides a rational role for the sterol requirements by eukaryotes.

Included in the discussion is the lipid structures and lipid content of those membranes that, according to the above factors would be expected to protect the cell from lost energy. Some of the membranes discussed are resident in high (or low) cation concentrations. Such membranes would be rich in lipids that inhibit cation leaks. The permeability of lipid bilayers to H+ is discussed in the context of a proposed mechanism for proton leakage. We examine the lipids found in the cell membranes of organisms exposed to extreme of pHs in the context of that model. A change in the cation concentration outside the plasma membrane, a change in the temperature, or a change in the membrane potential, must trigger a signal to regulate those lipids that inhibit the cation leakage. The proposal suggests many experiments. It also suggests why many of the unique and unusual lipids in natural membranes are designed the way they are.

Section snippets

Cation (Na+, H+) leaks across lipid bilayers

Interest in the permeability of phospholipid bilayers to cations began in the late 1960s and early 1970s as chemically defined bilayer films made such research possible [10]. The classical quantitative measurements of sodium leakage across lipid bilayers were made by Dimitri Papahadjopoulos [2], [3]. In 1971 he established the permeability of a wide variety of lipid bilayer vesicles to K+, Na+ and other common metal cations found in biological systems. His results have been confirmed by many

Sterols in membranes

Despite nearly a century of intensive research on cholesterol and the phytosterols their role in the plasma membranes of eukaryotes remains a mystery. Most authors, in introductory texts [21], advanced texts [22], research articles [23] and reviews [24] have explained cholesterol's role as affecting membrane rigidity or fluidity, although this has been questioned more recently [25]. Prokaryotes do not need sterols, albeit some contain a likely substitute — the hopanoids [26], [27]. Finally,

Protons leak across lipid bilayers

Experimental evidence that protons leak across simple lipid bilayers [8], [9] at rates that are biologically relevant has been confirmed by many laboratories. Two molecular models have been proposed for proton leaks across lipid bilayers: the “defect” mechanism and the “water wire” mechanism. A third, the “cluster” mechanism will be proposed herein.

According to the “defect” mechanism [79] protons permeate bilayers via defects, or transient pores, as is widely presumed for monovalent metal

Sterols and energy

For aerobic prokaryotes, photosynthetic prokaryotes, mitochondria and chloroplasts the production of the proton gradient is remarkably efficient since it is direct. A transmembrane protein pumps the protons and the cell both produces and uses the pmf across that membrane to produce ATP. This is in contrast to the energy consumption required to maintain a membrane potential at the eukaryote plasma membrane. Here the production of ATP is either by mitochondria, etc., or by metabolic enzymes. The

A note on sterol evolution

In a proposal on the evolution of sterols, Bloch noted [125] that sterol evolution stopped at squalene in prokaryotes, which lack sterols. This was prior to the appearance of eukaryotes and of oxygen in the atmosphere. The prokaryote hopanoids are cyclized from squalene in the absence of oxygen. Sterol synthesis from squalene on the other hand requires oxygen. If squalene inhibits proton leaks in alkaliphiles and other prokaryotes, then a cation leakage feedback control mechanism exists to

Summary and conclusions

A model is proposed for the leakage of protons across the lipids of cellular membranes. The model assumes water forms clusters in the low dielectric. It also assumes that certain clusters may be charged according to the pH of the water facing the bilayer. This model contrasts with the commonly accepted proton wire model. Either model implies that proton leaks may be inhibited by (1) extruding the water from the lipid bilayer or (2) blocking the contact between the clusters with hydrocarbon in

Acknowledgements

The author thanks Randy Scheckman, Daniel Koshland and the University of California, Berkeley for providing facilities. The author has appreciated insightful discussions with Gunther Blobel, Robert Bittman, Martin Blank, Sanda Clejan, David Deamer, Howard Goldfine, Russell Jones, Terry Krulwich, Kim Lewis, Hiroshi Nikaido, Jasper Rine, Theodore Steck, Alan Verkman, and Mary M. Cleveland, who also edited the manuscript.

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