Acta Neurobiol Exp 2007, 67: 219–233

Steroids and ion channels in evolution: From bacteria to synapses and mind

Evolutionary role of steroid regulation of GABAA receptors

Maria Dorota Majewska

Marie Curie Chair (European Commission), Department of Pharmacology, Institute of Psychiatry and Neurology, 9 Sobieskiego St,, 02-957 Warsaw, Poland, Email: majewska@ipin,edu,pl

Review Abstract" Ion channels are vital components of plasma membranes, This article presents an evolutionary view of the biochemical mechanism of controlling activity of ion channels by rigid lipids, such as steroids or biophysically similar molecules, which were instrumental in formation and control of ion channels in cell membranes at the very origin of life, Such regulatory mechanisms exist in all cellular forms of life from ancient bacteria to humans and participate in a diversity of biological functions, from the most basic, such as maintenance of cell shape, homeostasis, feeding, cell fusion, and reproduction to the most intricate, such as the mind, Learning about the regulation of membrane ion channels by steroids and like molecules is important for understanding the evolution of life and various aspects of cell and organism physiology, for unraveling the mysteries of mind, and for practical purposes such as developing new pharmacotherapies,

Key words: steroids, neurosteroids, ion channels, GABAA receptors, regulation, evolution, bacteria, reproduction, brain, mind 220 M,D, Majewska

INTRODUCTION mechano/pressure- or thermo-sensitive, and LGICs, The latter group, which in multicellular metazoans In past two decades endogenous steroids have been plays a critical role in inter-cellular communication, shown to be potent bimodal controllers of ionotropic constitutes a family of cationic and anionic channels receptors (ligand-gated ion channels) in the central (Ortells and Lunt 1995, Tasneem et al, 2005), In nervous system (CNS), Originally this type of regula- mature mammalian nervous system they encompass tion was documented for the GABAA receptors fast acting ionotropic receptor-channel complexes,

(GABAAR), subsequently also for glycine, NMDA, such as GABAA, GABAC and glycine receptors that – nACh and 5HT3 receptors, and it was shown to partic- conduct Cl , and nACh, 5HT3, glutamate (NMDA, ipate in numerous CNS functions, Because ligand- AMPA, kainite) and purinergic P2X receptors, which gated ion channels (LGICs) are evolutionarily very conduct cations (Na+, K+, and Ca++), ancient and they participate in many vital cellular The LGICs found in contemporary eukaryotic cells activities in unicellular and multicellular organisms, manifest a high degree of structural homology I was curious to explore the steroid interaction with (Langosch et al, 1988), They are oligomers, which can such channels in evolutionary perspective and reveal be divided into three superfamilies based on their sub- here its origin at the very beginning of life on Earth, unit quaternary structure, In mammalian cells they can

be pentamers (GABAA, glycine, nACh, and 5HT3 ION CHANNELS receptors), tetramers (glutamate receptors), or trimers (P2X receptors) composed of homologous or heterolo- Separation of the intracellular molecular content from gous subunits, Each of these subunits spans the plasma extracellular milieu is an essential attribute of all living membrane several times (two times in the trimeric cells, whether they exist as unicellular organisms or as family, three times in tetrameric, or four times in pen- tissue components, The intracellular-extracellular elec- tameric family of receptors), In the best characterized trochemical gradient of ions is a driving force for numer- pentameric LGICs superfamily, the membrane-span- ous cellular activities, The ion gradient is generated by ning domains (M1–M4) form the channel, where the their active transport by specialized pumps located in M2 domain mainly contributes to lining of the pores, plasma membranes, which utilize cellular energy, and is The N and C termini of each subunit are extracellular, sustained by the lipid bilayers largely impermeable to and the intracellular loop usually exists between M3 inorganic ions, Ions can diffuse into or out of cells down and M4 domains, Characteristic for this family of their gradient via ion- and charge-selective protein chan- channels is conserved 15 aminoacid-spaced disulfide nels embedded in cell membranes, The transmembrane loop in their extracellular ligand-binding domain flux of inorganic ions (primarily Na+, K+, Ca++, and Cl–) (Cascio 2004, Schofield et al, 2003), regulates many aspects of cellular physiology, including The ligand (neurotransmitter, hormone, drug) bind- excitability, metabolism, osmolarity, volume, shape, ing to the extracellular receptor domain alters the con- movement, fusion, and other functions, formation of the receptor/channel complex from closed The ion channels of prokaryotic and eukaryotic cells to open (Karlin and Akabas 1995, O’Shea and Harrison share many common features, They are ion-selective 2000, Taly et al, 2005, Wagner and Czajkowski 2001), homo- or oligomeric protein structures, containing It is believed to cause lateral twisting of the M2 several integral transmembrane domains, which form domain, which exposes different residues to the pore water-filled ionic pores, The hydrophobic surfaces of lumen and enlarges its size (Keramidas et al, 2000, the pore interact with lipids of the membrane bilayer, Lynch et al, 1997, Unwin 1995), The principal ion-gat- whereas the charged hydrophilic pore lumen serves as ing mechanism is thought to be located in the narrow- an ion selector and gating mechanism, Lumen’s charge est region of the pore, close to its cytoplasmic end of these channels, created by their lining with charged (Keramidas et al, 2000, Wilson and Karlin 1998, Xu residues of amino acids, facilitates the selective attrac- and Akabas 1996), whereas a cloud of charges located tion of different ions and their rapid transport (Cascio at the channel’s mouth serves as the ion selector (Imoto 2004, Taly et al, 2005), et al, 1988, Keramidas et al, 2000), The high degree of The ion channels set in plasma membranes can be structural and functional similarity between different divided into the categories of voltage-dependent, LGICs is evidenced by the fact that replacement of just Ion channels steroids neurosteroids evolution 221 a few aminoacid residues in critical regions of the pore sterols, but they are furnished with a variety of com- results in conversion of their ion selectivity from pounds, which play analogous roles, Haines (2001) cations to anions and vice versa (Galzi et al, 1992, proposed that the principal role of rigid lipid mole- Gunthorpe and Lummis 2001, Keramidas et al, 2000), cules, like sterols, in biological membranes is to reduce Molecular evolutionary analysis of LGICs based on proton and sodium leaks through lipid bilayers and in DNA and aminoacid data revealed their heritage from this way to conserve biological energy, a common ancestor, which existed at least 2,5 billion Sterol-like molecules emerged early in evolution, years ago (Ortells and Lunt 1995), before origin of Archaebacteria do not contain such molecules in their eukaryotes (Hedges et al, 2001), It is hypothesized that simple cellular membranes, but have a class of diphy- ancient LGICs receptors existed on cell membranes of tanyl lipids, which inhibits proton leakage, Eubacteria early living organisms and served nutrient seeking and may contain sterol-like molecules, such as non-cycli- osmotic regulation functions (Ortells and Lunt 1995), cal squalene, or cyclical hopanoids and tetrahymanol, With growth of organismal complexity, these LGICs synthesized anaerobically from squalene, as well as have changed their function to serve predominantly lipids with branched-chain fatty acids (Barenholz intercellular communication, 2002), which effectively inhibit water and In prokaryotes the lipid bilayer serves not only as proton/sodium leaks across membranes, In mem- a physical barrier separating intracellular and extracel- branes of eukaryotes this function is served by various lular milieus, but also as a mechanism controlling sterols – by isoprenes in organellar membranes functions of membrane proteins, which protects cell’s (dolichol in peroxisomes and lysosomes, ubiquinone osmotic homeostasis and permit movement, Cell mem- in mitochondria), and by plastoquinone in chloroplas- branes of prokaryotes are equipped in a diversity of ts (Haines 2001), Membranes of yeasts contain ergos- cationic and anionic channels, which function as terols and plants have a diversity of phytosterols, osmoregulators/volume protectors and mechanosen- Sensu stricto sterols appeared in evolution together sors (Koprowski and Kubalski 2001, Martinac 2001), with eukaryotic cells circa 2,5 billion years ago, con- The gating mechanism, which detects the deformation temporarily with rise of atmospheric oxygen levels, of lipid bilayer, seems to be evolutionarily the oldest required for sterol synthesis (Hedges et al, 2001), mode of operating ion channels, as mechano-sensitive Oxygenation or hydroxylation of steroid molecules at channels are present already in bacteria Archea various positions resulted in the formation of a diversi- (Martinac 2001), which originated close to the dawn of ty of compounds, endowed with a remarkable variety life on Earth about 4 billion years ago (Hedges et al, of cellular functions – from regulators of membrane 2001), Some bacteria possess in their membrane also properties to genome controllers, growth and repro- LIGCs similar to those found in metazoan cells, which ductive controllers, signal messengers, stress adaptors, serve chemotactic functions (Tasneem et al, 2005), and sculptors of evolution, In animals, sterols play roles as cell membrane stabilizers and regulators, hor- EVOLUTION OF STEROLS mones, parahormones, autohormones, agents of inter- cellular communication, transducers of intracellular The lipid bilayer of mammalian plasma membranes signals, and metabolic and reproductive regulators, is a complex structure, containing more than 2000 lipid molecular species (Barenholz 2002), Sterols are vital STEROLS INSTRUMENTAL IN THE elements of these membranes, They dynamically mod- FORMATION OF ION CHANNELS ulate physicochemical properties of membranes, restrict the permeability of water and solutes, control Amphiphilic sterol molecules partition easily to the cellular volume and govern functions of membrane phospholipid bilayer and orient perpendicularly to its embedded proteins, Deficit of sterols is pathogenic and plane, with the rigid nucleus of steroid rings positioned in extreme cases – lethal to eukaryotes, The critical in the region of the hydrocarbon core, and the significance of sterols in these cells is best illustrated hydrophilic groups – usually hydroxyl or sulfate – by the fact that in mammals nearly a 100 proteins sup- oriented toward phospholipid head groups at the mem- port the metabolism and transport of cholesterol, brane surface (Barenholz 2002, de Kruijff 1990, Membranes of prokaryotes do not contain classical Golden et al, 1998), Such orientation obstructs the 222 M,D, Majewska intrinsic movement of fatty acids chains and allows I found it in biochemical experiments, while studying steroid molecules to interact with both the aqueous the influence of cholesterol and endogenous steroid milieu of cell surface and the hydrophobic membrane hormones on GABAAR function (Majewska et al, interior, Presence of cholesterol in the membrane 1985), whereas Harrison with colleagues observed it increases separation of phospholipid head groups and while examining the electrophysiological effects of thus increases water’s freedom of movement on the steroidal anesthetics on the GABAAR (Harrison and membrane surface (Huang 1976), The amphiphilic Simmonds 1984), Subsequently, while collaborating nature of the sterol molecules also makes them ideal we made further discoveries (Harrison et al, 1987, partners of membrane proteins, Majewska et al, 1986), This work was discussed in de Kruijff and subsequently other investigators several reviews (Majewska 1987, Majewska 1992, noticed that cholesterol is a target of polyene antibi- 1999), Soon it became evident that other LGICs are otics (filipin, amphothericin B) and bacterial toxins, also subjects of steroid control, which was not surpris- and that interaction of these molecules with sterols ing, considering their structural homology, results in the formation of aqueous pores in the lipid Our research established that different endogenous bilayer (rev, de Kruijff 1990, Dufourc and Smith steroids modulate the function of the GABAAR associ- 1985), He proposed a model of ion channel creation, in ated Cl- ionophore in the opposite directions, At phys- which molecules of an antibiotic or a peptide of bacte- iological, nanomolar, concentrations the steroids with rial toxin partition to membrane, so that their a reduced A ring at 5a or 5b position, hydroxyl at 3a hydrophobic sites interact with the hydrophobic core position and electronegative atom (usually oxygen) at of sterols, which orients them perpendicularly to the C-17 or C-20, such as 3a,5a-tetrahydroprogesterone membrane plane, (THP; allopregnanolone; 3a-OH-DHP), 3a,5a- A hydrophilic pore can be cooperatively shaped by tetrahydrodeoxycorticosterone (THDOC), or andros- several molecules of toxins or antibiotics interacting terone (5a-androstan-3a-ol-17-one), promote channel with sterols in the lipid bilayer (de Kruijff 1990, opening and increase Cl– flux (Harrison et al, 1997, Dufourc and Smith 1985), In his model of channel for- Majewska 1992, Majewska et al, 1986), In contrast, mation de Kruijff points to the functional stereoselec- the steroids which have sulfate at 3b position and D5 tivity of sterol’s 3-OH group, 3b-OH groups (as in configuration, such as pregnenolone sulfate (PS) and native cholesterol) favor the formation of pores, dehydroepiandrosterone sulfate (DHEAS), at physio- because they can form stable hydrogen bonds with logical, low micromolar, concentrations act primarily – carbonyl oxygen of phospholipids, proteins, or antibi- as allosteric antagonists of the GABAAR-associated Cl otic molecules, In contrast, 3a-OH may not form such channel and facilitate its closing (Demirgoren et al, bonds, because sterol isomers with such a group have 1991, Majewska 1992, 1995, Majewska and Schwartz a tilted, energetically unfavorable orientation in the 1987, Majewska et al, 1988, 1990a,b), Figure 1 illus- membrane, and interact differently with phospholipids trates mechanistic model of THP-mediated potentia- – and proteins (Huang 1976), It is likely, that interactions tion of Cl transport via GABAAR and PS-induced of different sterols with peptides/proteins were critical closing of Cl– channels linked to this receptor, in the evolution, stabilization, and regulation of ion In vertebrates steroid modulators of the GABAAR channels in cell membranes, The above model may and of other LGICs are synthesized in steroidogenic explain some aspects of the stereoselective regulation glands, such as adrenals, gonads and placenta, but also of these channels by different steroids, in the liver, skin and in the CNS (Corpechot et al, 1981, 1983), The steroids, which originate in the brain STEROLS AS ‘DOORMEN’ OF ION and(or) are neuroactive, have been named neuros- CHANNELS teroids, While the steroid regulation of LGICs has been The body of research accumulated over the past proven beyond any doubt and pharmacodynamics and 20 years documented that certain endogenous steroids structure-function relationships of this regulation have are allosteric modulators of LGICs, This phenomenon been established in biochemical, electrophysiological was first observed for Cl– channels associated with and behavioral experiments, the specific domains of

GABAA receptors by two independent approaches, steroid action at these channels are still a subject of Ion channels steroids neurosteroids evolution 223

Fig, 1, The mechanistic model of interaction of the inhibitory neurosteroid, 3a-OH-THP, and the stimulatory steroid, PS, with the GABAAR (gray; shown in open and Fig, 2, The schematic representation of interaction sites of closed conformation) and the phospholipid bilayer, 3a- the inhibitory neurosteroids (3a-THP, 3a-THDOC) with the

OH-THP works as allosteric receptor agonist – it GABAAR complex according to Hosie and others (2006), changes conformation of the receptor complex in favor The planar view of the GABAAR shows five receptor sub- of widely open channel, allowing more intense Cl– influx units: two a, two b, and one g, Within each subunit are into the neuron (or other cell) than permitted by action of shown four transmembrane domains M1, M2, M3 and M4, GABA alone, The functional groups of this steroid may where M2 domain is facing ion-conducting areas of the Cl– disturb hydrogen bonding between the receptor subunits lumen, BDZ determines a benzodiazepine, In this model and phospholipids, promoting open conformation of the four steroid molecules (shown as triangles) interact with one Cl– channel, The excitatory neurosteroid PS interacts receptor pentamer, One steroid molecule binds within the with the GABAAR complex as allosteric antagonist to hydrophobic cavity of a subunit to cause potentiation of close its channel, The 3b-sulfate group of this steroid is GABA effect, and another steroid molecule interacts at the shown to interact with the external surface of the mem- interface between a and b subunits to cause direct ion chan- brane to increase water trapping, This swelling of mem- nel activation, Typically each receptor complex contains 2a brane surface may promote slow conformational change and 2b subunits, hence maximally 4 inhibitory neurosteroid of the receptor complex, which facilitates channel clos- molecules may interact with one receptor to cause the poten- ing, tiation of GABA effect and direct receptor activation,

investigations and discussions, The main difficulty nists of the GABAAR-ionophore, Of these two, the with identification of these sites ensues from the larger molecule of PS seems to be more deeply rooted amphiphilic nature of steroids, capable of interacting in the lipid bilayer, as its binding sites were insensitive both with the lipid bilayer and protein receptors, Our to thermal and proteolytic denaturing, In contrast the early biochemical studies indicated existence of dis- binding sites for DHEAS were obliterated by such tinct binding sites for different steroids at the treatment (Demirgoren et al, 1991, Majewska 1992,

GABAAR-Cl-ionophore complex (Demirgoren et al, 1999), which suggested theirs location closer to mem- 1991, Majewska 1992, Majewska et al, 1990a,b), The brane surface, Such a conclusion was confirmed by the conclusions of others were similar (Covey et al, 2001, observations of others (Morissette et al, 1999), Park-Chung et al, 1999), Based on these observations The location of binding site for GABA and its ago-

I proposed that the steroids interact at the interface of nists at the GABAAR has been characterized with help proteins and lipids and this interaction promotes the of aminoacid substitution studies (Amin and Weiss conformational change of the receptor, facilitating 1993, O’Shea and Harrison 2000, Boileau et al, 2002), channel’s opening or closing (Majewska 1992), It is thought to be situated in the pocket between a and Originally we identified two water-soluble steroid b subunits of the receptor complex, Ligand binding to sulfates, DHEAS and PS, which at physiological this site has been proposed to cause conformational (micromolar) concentrations act as allosteric antago- change and opening of the Cl– channel, 224 M,D, Majewska

Studies attempted to explain in a similar fashion the Morissette et al, 1999), At quasi-physiological temper- locus and the mechanism of allosteric modulation of atures DHEA behaved like cholesterol and it increased the GABAAR by the steroids, but this problem has been the order of fatty acid chains in membrane bilayer, tantalizing, Chang and coauthors (2003) proposed that whereas more hydrophilic DHEAS, which binds clos- the anesthetic steroid bind at the hydrophobic pockets er to the membrane surface, decreased the order (Dicko of the M1 transmembrane domains of the b subunits, et al, 1998, Morissette et al, 1999), A possible expla- However, in recently published study, Hosie and col- nation for this phenomenon may derive from the study leagues (2006) identified two types of discrete binding of Faure and others (1996), who examined with bio- sites for agonistic steroids in the transmembrane area physical methods the effects of cholesterol and its sul- of the GABAAR, which are required for high efficacy fate on phospholipid membranes, They found that the receptor activation, The site positioned in the presence of sulfate at the 3b position in cholesterol hydrophobic cavity of a subunit (involving Gln241 molecule increased water trapping at the surface of and Asn407) mediates GABA-potentiating effect of membrane in the region of phospholipid head groups allosteric steroid agonists such as THP and THDOC, (Faure et al, 1996), Theoretically, such swelling at the whereas the site located in the pocket at interface membrane surface may contribute to the slow confor- between a and b subunits (involving aThr 236 and mational modification of the receptor-ionophore, bTyr 284) mediates direct activation effect of the Cl– resulting in channel closing, It is conceivable therefore channels (Hossie et al, 2006), The hypothetical inter- that DHEAS and PS, bound at the interface of the action sites of inhibitory steroids with the GABAAR are receptor protein and phospholipids, may be responsi- illustrated on Fig, 2, ble for such effects, The ring of negative charges of Likewise, it was not easy to identify loci and exact steroid sulfate groups located near external mouth of mechanism of action of allosteric steroid antagonists at the GABAAR-associated channels may additionally act – the GABAAR, The functional antagonism of this recep- to reduce Cl attraction to the positively charged chan- tor by PS and DHEAS does not seem to be due to nel interior, blockage of the Cl– channel in its open state, but rather Some studies suggest that membrane phospholipids from conformational change of the receptor that facili- may participate also in the channel-opening effect of tates channel’s closing (Akk et al, 2001, Covey et al, steroid agonists of the GABAAR, The functional 2001, Majewska et al, 1990b, Spivak 1994), Electro- 3a-OH group of these steroids, which is necessary for physiological experiments conducted with recombi- their allosteric GABA-agonistic properties (Harrison nant GABAAR with mutated aminoacid residues et al, 1987, Majewska et al, 1986), disturbs the hydro- (V256S) in the channel lining suggested that a residues gen bonding between sterols and phospholipids or near intracellular end of the channel of the a1 subunit proteins in the membrane (Huang 1976), Such an are critical for the channel closing by PS (Akk et al, affect may facilitate conformational alteration of the 2001), Because this region seems to function as a chan- channel promoting its opening, Quaternary structure nel gating mechanism, the authors speculated that it of the proteins implanted in lipid bilayer is shaped by participates in conformational receptor alteration biophysical properties of the membrane, Because induced by PS, sterols interact with both proteins and lipids, they have One universally observed feature of the channel the capacity to alter membrane order and hydration, blockade by PS and DHEAS is its temperature depend- and in this way to change conformation of membrane ency and relatively slow action kinetics (Akk, et al, proteins, Collectively data suggest that steroids gov- 2001, Demirgoren al, 1991, Majewska and Schwartz ern activity of LGICs through dual domains – the 1987, Park-Chung et al, 1999), Such characteristics receptor proteins and their lipid milieu, suggest involvement of the membrane compartment or the site of the receptor that is deep-rooted in the mem- STEROID REGULATION OF ION brane, Infrared spectroscopy investigation of the influ- CHANNELS IN EVOLUTION ence of steroids on biophysical properties of artificial phospholipid membranes or natural membranes from Eukaryotic cells are assumed to have emerged in rat brains revealed a difference in the pattern of inter- evolution through symbiogenesis by syntrophic union action between DHEA and DHEAS (Dicko et al, 1998, between several prokaryotes, such as anaerobic Ion channels steroids neurosteroids evolution 225 archaebacteria and eubacteria before the biogenic rise mechanism seems to be primeval, It also exists in mito- of atmospheric oxygen on Earth (Lopez-Garcia and chondria from different organisms, where sterols are Moreira 1999, Margulis et al, 2000), While different essential for proper folding of porin polypeptides to hypotheses stress distinct nature of the primary create functional channels (Popp et al, 1995), The eukaryotic organizing principle, most point to cell hydroxyl groups of sterols, which form hydrogen fusion as its vital element, Chimeras of loosely asso- bonds with porin aminoacids, stabilize their tertiary ciated prokaryotes probably existed as intermediate and quaternary structures, Protozoa, such as Ciliates cellular formations, which eventually fused forming and other singe-celled eukaryotes (e,g, Paramecium), a composite nucleus and organelles, typical for are auxotrophic for phytosterols, which support eukaryotes, In the model of Margulis, the earliest growth, movement and thermal sensoring in these protoeukaryotes originated from the union of archae- organisms via membrane mediated phenomena bacteria forming the “cell body” and several spiro- (Hennessey et al, 1983, Whitaker and Nelson 1988), chete-like eubacteria forming protokinetosomes, Membranes of the primitive metazoan Hydra vulgaris which equipped the symbionts with motility (Margulis possess the GABAAR, which control feeding, The et al, 2000), function of these receptors is potentiated by the Cellular fusion thus lays at the basis of the develop- steroids THP and THDOC, which prolong the duration ment of complex forms of life, Biophysical experi- of the mouth opening and enhance feeding in this ani- ments show that efficient fusion of phospholipid vesi- mal (Concas et al, 1998), cles in aqueous environment requires presence of ion Many species of nematodes, which are plant and channels (Cohen et al, 1989, Woodbury and Hall animal parasites, are incapable of de novo sterol syn- 1988), Translocation of ions trough those channels thesis, but have nutritional requirements for these com- generates the hyperosmotic environment inside the pounds and are capable of their metabolism to different vesicles and their subsequent swelling, which pro- hormones (Chitwood 1999), The function of sterols in motes fusion with the membrane of another vesicle, nematodes is poorly understood, but their cellular

Since sterol and sterol-like molecules control the activ- membranes possess ionotropic GABAA and nACh ity of ion channels in cell membranes, they likely have receptors, which participate in their movement and been involved in the bacterial fusion at the dawn of feeding, and are sensitive to sterols (Bamber et al, eukaryotes’ origin, Perhaps it is not incidental that 2003), In a mollusk, the land snail Cepaea nemoralis, classical sterols are present only in eukaryotes, but not THP acting via GABAAR exerts antinociceptive effect in prokaryotes, and that they appeared in evolution similar to that in mammals (Kavaliers et al, 2000), concurrently with rise of atmospheric oxygen, which Also the function of GABAAR from Drosophila coincide with emergence of mitochondria from bacter- melanogaster was potentiated by anesthetic neuros- ial endosymbionts (Haines 2001, Hedges et al, 2001, teroids (Hosie and Sattelle 1996), These examples pro- Lopez-Garcia and Moreira 1999), The oxidized sterols vide evidence that regulation of membrane ion chan- synthesized in the eubacterial symbiont, which became nels by steroids is a primordial phenomenon, which the mitochondria, may have efficiently controlled ion governs many different cellular and organismal func- channels in membranes of archaebacterial hosts, thus tions, facilitating the cellular fusion and genesis of eukary- otes and complex forms of life, STEROIDS AND ION CHANNELS IN It is interesting to trace the regulation of LGICs and FERTILIZATION AND REPRODUCTION other membrane channels by steroids in evolution, Oliver and coauthors (1991) examined the sensitivity Fertilization, i,e, union of two gametes, in some of different animal species to the anesthetic effects of sense recapitulates cellular fusion, which took place

THP, which potentiates GABAAR function in mam- early in evolution to create complex forms of life, mals, and he concluded that it appeared late in evolu- Steroid-ion channel duets are prominently involved in tion in the phylum Chordata, but was absent in evolu- the genesis of diploid cells from haploid gametes, tionarily more ancient animals, However, other studies GABA and its receptors are present in abundance in documented control of the GABAAR and other LGICs female reproductive tracts of mammals, They are found by steroids also in invertebrates, In fact, this biological in the ovaries, fallopian tubes and the uterus, GABA 226 M,D, Majewska changes the polarity and membrane conductance of GABA, STEROIDS, AND FEEDING unfertilized human oocytes (but not in fertilized ones), BEHAVIORS indicating that it directly participates in fertilization (Dolci et al, 1985), THP, whose concentration increas- In many animal species GABA system participates es along with progesterone during the ovulation, may in feeding behaviors and GABA generally increases support this process by potentiating GABAAR func- food consumption, The GABA agonistic steroids act- tion, ing via GABAAR enhance feeding in a primitive organ-

GABA and GABAAR are also present in various tis- ism such as Hydra vulgaris (Concas et al, 1998) and in sues of the male reproductive tract (Napoleone et al, mammals via the hypothalamic neurons (Chen et al,

1990), Mammalian spermatozoa possess GABAAR 1996, Reddy and Kulkarni 1998), In contrasts, GABA and the GABA transport system, GABA increases antagonists, including excitatory neurosteroids, gener- sperm motility and initiates its capacitation – an ally inhibit feeding in mammals (Chen et al, 1996, essential step of fertilization (Burrello et al, 2004, Hu Reddy and Kulkarni 1998), However, in the worm et al, 2002, Kuroda et al, 1999, Shi et al, 1997), In the Caenorhabdis elegans, GABA agonists reduce food fallopian tubes, GABA, whose gradient increases intake (Avery and Horvitz 1990), which may be due to toward the ampula (Erdo 1989), acting via the the fact that GABA neurotransmission is excitatory in

GABAAR induces hyperactivated state of the sperm these animals (Reiner and Thomas 1995), (Calogero et al, 1996), After traversing the cumulus The mechanism of feeding control by steroids acting oophorus, the spermatozoon reacts with the egg enve- via GABAAR may come prominently into play during lope, zona pellucida, inducing acrosome reaction in physiological states, which require alteration of food the sperm (Roldan et al, 1994, Vacquier 1998), which ingestion, For example, during pregnancy prolonged is coactivated by progesterone and its metabolites exposure to GABA-agonistic metabolites of proges- present in large quantities in cumulus oophorus (Baldi terone may be responsible for increased appetite and et al, 2002, Calogero et al, 2000, Roldan et al, 1994), eating, necessary for fetal growth, Plasma levels of

This process involves the activation of GABAAR- various neuroactive steroid hormones also dramatical- linked Cl– channels on the sperm, leading to Cl– efflux, ly change during stress, which distinctly influences which triggers an influx of Na+ and Ca++, and subse- appetite in different individuals, Because normal indi- quent activation of phospholipases and other metabol- viduals markedly vary in their expression of steroid ic events leading to exocytosis and transfer of sperm synthesizing enzymes, it is not difficult to imagine, that genetic material to oocyte (Baldi et al, 2002, Vacquier the persons, whose adrenals secrete more GABA-ago-

1998), Participation of the GABAAR regulated by nistic steroids, may manifest increased appetite, steroids in the oocyte fertilization and initiation of whereas those, producing more neuroexcitatory new life shows how fundamental is this biological steroids, may have suppressed appetite during stress, phenomenon,

We have documented that steroid regulation of the NEUROSTEROIDS AND GABAA RECEPTOR

GABAAR also plays a critical role in pregnancy and CHANNELS IN THE CNS birth, The reduced metabolite of progesterone, THP, by enhancing the activity of uterine GABAAR, inhibits Direct interaction of steroids with the GABAAR- uterine contractions, whereas the GABA antagonistic chloride channel in neurons has been studied most steroids, such as PS, activate contractions (Majewska extensively, Because GABA is a ubiquitous neuro- and Vaupel 1991), Because during pregnancy the plas- transmitter in the vertebrate CNS and the GABAAR are ma levels of progesterone and its metabolites are very present on most neurons in the brain, regulation of high, these hormones play a vital quieting role on uter- these receptors by steroids is of paramount neurophys- ine motility, In contrast, during parturition relatively iological importance for all vertebrate species, Since lowered plasma or tissue levels of the inhibitory our discovery of the interaction of steroids with the steroids, combined with increased secretion of stimu- GABAAR and later with other LGICs by other investi- latory ones, such as DHEAS and PS, promote uterine gators, a great body of evidence has been accumulated, contractions and delivery (Doganay et al, 2004, Hill et documenting its physiological and pathological signif- al, 2002, Majewska and Vaupel 1991), icance, These issues were discussed in many review Ion channels steroids neurosteroids evolution 227 articles (Majewska 1987, 1992, 1999, 2002, Rupprecht indicating that the CNS is equipped with its own neu- et al, 1996, Schumacher et al, 2003), hence I will only roprotective mechanisms, which utilize steroids (di briefly outline its significance here, Michele et al, 2000, Meffre et al, 2007, Schumacher et Research generally supported my early theoretical al, 2000), Such mechanisms may also be responsible predictions regarding biological implications of the for the protection of fetuses and newborns from hypox- mechanism of rapid regulation of neurotransmission ic insults (Nguyen et al, 2003), The neuroprotective by steroids interacting with membrane ionotropic effects of progesterone and its inhibitory metabolites in receptors, Synthesis of GABA-agonistic (inhibitory) animals and humans (Wright et al, 2007) resemble steroids, such as THP and THDOC, has been shown to those of general anesthetics (Majewska et al, 1978), be markedly increased in women during the luteal which have been used with some success for many phase of the menstrual cycle and pregnancy, in both years to reduce brain damage after trauma (Hoffman sexes during stress and in some other physiological WE et al, 1998, Koerner and Brambrink 2006), Such states, when they exert anxiolytic and hypnotic effects steroid effects may be due to synergistic GABA-ago- (Barbaccia et al, 2001, Gilbert Evans et al, 2005, nistic and NMDA-antagonistic actions of inhibitory Paoletti et al 2006, Parizek et al, 2005), These steroids neurosteroids, resulting in decreased neuronal metabo- also possess neuroprotective and anticonvulsive prop- lism and activity, erties (Frye 1995, Landgren et al, 1987), Lowered lev- In contrast, the neuroexcitatory steroids, PS and els of progesterone and its inhibitory metabolites or DHEAS, exert analeptic and invigorating effects dur- their sharp decline before and during menstruation ing daily cycles and stress, and may have antidepres- may be contributing to catamenial seizures in women, sant actions in some situations (reviewed in Majewska whereas pharmacotherapy with such steroids might be 2002), Due to their neurostimulant actions as GABAAR beneficial in treating these forms of epilepsy antagonists and NMDAR agonists, and interactions (Backstrom et al, 2003, Nohria and Giller 2007, Reddy with the sigma receptors, these steroids seem to 2004, Rosciszewska et al, 1986), GABA-agonistic improve learning and memory in experimental animals steroids also may be efficacious in treating intractable (Farr et al, 2004, Flood et al, 1995, Maurice et al, 2001, infantile spasms and other forms of epilepsy (Kerrigan Sliwinski et al, 2004), However, prospective clinical et al, 2000), studies did not show consistent effects of DHEA Inhibitory steroids may participate in the regulation improving cognition in a healthy aging population of sleep cycles (Lancel et al, 1997, Soderpalm et al, (Grimley-Evans et al, 2006), As expected, excessive 2004) and influence mood, They may potentiate or concentrations of the excitatory steroid DHEAS in alleviate depression, and their withdrawal before men- plasma were shown to exert an anxiogenic and pro- struation or post partum may induce anxiety and other convulsant influence (Jacobs et al, 1999, Steffensen mood disturbances in some women (Andreen et al, 1995), DHEA also appears to have a therapeutic utility 2006, Rasmusson et al, 2006), Inhibitory steroids may promoting recovery from traumatic brain injury (Fiore be responsible for relaxation, sedation and increased et al, 2004, Hoffman SW et al, 2003, Lur et al, 2006, appetite during pregnancy, and may have proamnesic Malik et al, 2003), due to a complex mechanism of effect in some stress situations (Johansson et al, 2002), action, which combine mild neurostimulant, trophic

Interactions of steroids with the GABAAR may also and antiapoptotic effects (Suzuki et al, 2004, Zhang et come into play in the regulation of peripheral systems, al, 2002), particularly the reproductive system, For example, dur- DHEA is of particular interest for neurophysiology ing pregnancy THP acting via GABAAR quiets uterine and neuropsychiatry, because it is promoted and used activity (Majewska and Vaupel 1991) and may produce by millions of people around the world as antiaging antinociceptive effects (Winfree et al, 1992), panacea without a solid proof of its therapeutic effica- Inhibitory metabolites of progesterone are neuropro- cy, DHEA is often called a mother hormone, because tective during brain trauma or hypoxia (Djebaili et al, in addition to its own physiological and neuronal 2005, Rhodes et al, 2004) and regulate neuronal sur- activity, it serves as a precursor of other steroid hor- vival (Leskiewicz et al, 2006), It is interesting to note mones, including estrogens and androgens, Several that the synthesis of these steroids in the brain and the studies indicated that higher levels of endogenous spinal cord rapidly increases after ischemia or trauma, DHEA and DHEAS in blood correlate with physical 228 M,D, Majewska

Table I addicts with high pharmacological doses of DHEA was not therapeutic and even resulted in increased cocaine Cellular and organismal functions, in which interactions use (Shoptaw et al, 2004), These apparently discordant between steroids and LGICs may come into play results suggest the existence of a complex relationship between the levels of different neuroactive steroids in Evolution the blood (and presumably in the CSF) and intricate mental phenomena such as drug dependence, craving • Endosymbiosis, Eukaryote origin and use, • Cell fusion Wealth of accumulated scientific and medical data • Creation of multicellular organisms shows that alterations of the biosynthesis and release of diverse and metabolically linked neuroactive General Functions steroids as hormones, parahormones or autohormones during development, aging and in different physiolog- • Intercellular communication ical states serves to shape brain architecture and tune • Integration of tissue and organ functions brain functions and the mind to those of the whole • Feeding • Mineral and energy balance body in order to harmonize an organism’s complex • Maintenance of cell volume and integrity responses to environmental or physiopathological • Movement challenges, • Homeostasis • Stress survival CONCLUSIONS • Physiological cycles (diurnal, monthly, seasonal) Modulation of ion channels in plasma membranes Reproduction and development by endogenous and exogenous steroids or steroid-like molecules is a biological mechanism, which seems to • Gamete movement have emerged at the dawn of life, It is implicated in • Gamete fusion many basic cell functions, which guarantee mainte- • Development nance of the cell’s shape, volume, homeostasis and • Reproductive cycles survival, This mechanism is involved in feeding, • Adaptation of maternal organism to pregnancy waste removal, cell fusion and division, reproduction, movement, defense, cellular communication and other Brain functions functions in both simple monocellular and complex organisms, This primeval mechanism, which supports • Cell integrity • Neurotransmission and neuromodulation an amazing variety of organismal functions from the • Neuroplasticity very basic to the most intricate, such as the mind, rep- • Neurodevelopment resents a true evolutionary wonder and is intimately • Cognition interwoven in the very definition of life, Table I shows • Emotions examples of cellular and organismal activities in • Sleep/diurnal cycles which steroid-GABAAR interactions may come into • Pain play, • Behavior • Neuroprotection and neuroregeneration REFERENCES and psychological resiliency, In this regard we have Akk G, Bracamontes J, Steinbach JH (2001) Pregnenolone sul- shown that cocaine addicts, who had higher concentra- fate block of GABAA receptors: mechanism and involve- tions of endogenous DHEAS in blood during therapy ment of a residue in the M2 region of the alpha subunit, J for drug dependence, were more likely to succeed in Physiol 532: 673–684, maintaining prolonged abstinence after the therapy Amin J, Weiss DS (1993) GABAA receptor needs two homolo- than those, who had lower levels of this hormone gous domains of the beta-subunit for activation by GABA (Wilkins et al, 2005), However, treatment of cocaine but not by pentobarbital, Nature 366: 565–569, Ion channels steroids neurosteroids evolution 229

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