Evolution of the α-Subunit of Na/K-ATPase from Paramecium to Homo sapiens: Invariance of Transmembrane Helix Topology.

Na/K-ATPase is a key plasma membrane enzyme involved in cell signaling, volume regulation, and maintenance of electrochemical gradients. The α-subunit, central to these functions, belongs to a large family of P-type ATPases. Differences in transmembrane (TM) helix topology, sequence homology, helix-helix contacts, cell signaling, and protein domains of Na/K-ATPase α-subunit were compared in fungi (Beauveria), unicellular organisms (Paramecia), primitive multicellular organisms (Hydra), and vertebrates (Xenopus, Homo sapiens), and correlated with evolution of physiological functions in the α-subunit. All α-subunits are of similar length, with groupings of four and six helices in the N- and C-terminal regions, respectively. Minimal homology was seen for protein domain patterns in Paramecium and Hydra, with high correlation between Hydra and vertebrates. Paramecium α-subunits display extensive disorder, with minimal helix contacts. Increases in helix contacts in Hydra approached vertebrates. Protein motifs known to be associated with membrane lipid rafts and cell signaling reveal significant positional shifts between Paramecium and Hydra vulgaris, indicating that regional membrane fluidity changes occur during evolution. Putative steroid binding sites overlapping TM-3 occurred in all species. Sites associated with G-protein-receptor stimulation occur both in vertebrates and amphibia but not in Hydra or Paramecia. The C-terminus moiety "KETYY," necessary for the Na(+) activation of pump phosphorylation, is not present in unicellular species indicating the absence of classical Na(+)/K(+)-pumps. The basic protein topology evolved earliest, followed by increases in protein domains and ordered helical arrays, correlated with appearance of α-subunit regions known to involve cell signaling, membrane recycling, and ion channel formation.

The role of receptor topology in the vitamin D3 uptake and Ca(2+) response systems.

The steroid hormone, vitamin D3, regulates gene transcription via at least two receptors and initiates putative rapid response systems at the plasma membrane. The vitamin D receptor (VDR) binds vitamin D3 and a second receptor, importin-4, imports the VDR-vitamin D3 complex into the nucleus via nuclear pores. Here we present evidence that the Homo sapiens VDR homodimer contains two transmembrane (TM) helices ((327)E - D(342)), two TM "half-helix" ((264)K N(276)), one or more large channels, and 16 cholesterol binding (CRAC/CARC) domains. The importin-4 monomer exhibits 3 pore-lining regions ((226)E - L(251); (768)V - G(783); (876)S - A(891)) and 16 CRAC/CARC domains. The MEMSAT algorithm indicates that VDR and importin-4 may not be restricted to cytoplasm and nucleus. VDR homodimer TM helix-topology predicts insertion into the plasma membrane, with two 84 residue C-terminal regions being extracellular. Similarly, MEMSAT predicts importin-4 insertion into the plasma membrane with 226 residue extracellular N-terminal regions and 96 residue C-terminal extracellular loops; with the pore-lining regions contributing gated Ca(2+) channels. The PoreWalker algorithm indicates that, of the 427 residues in each VDR monomer, 91 line the largest channel, including two vitamin D3 binding sites and residues from both the TM helix and "half-helix". Cholesterol-binding domains also extend into the channel within the ligand binding region. Programmed changes in bound cholesterol may regulate both membrane Ca(2+) response systems and vitamin D3 uptake as well as receptor internalization by the endomembrane system culminating in uptake of the vitamin D3-VDR-importin-4 complex into the nucleus.

The pore-lining regions in cytochrome c oxidases: A computational analysis of caveolin, cholesterol and transmembrane helix contributions to proton movement.

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain. CcO catalyzes a four electron reduction of O2 to water at a catalytic site formed by high-spin heme (a3) and copper atoms (CuB). While it is recognized that proton movement is coupled to oxygen reduction, the proton channel(s) have not been well defined. Using computational methods developed to study protein topology, membrane channels and 3D packing arrangements within transmembrane (TM) helix arrays, we find that subunit-1 (COX-1), subunit-2 (COX-2) and subunit-3 (COX-3) contribute 139, 46 and 25 residues, respectively, to channel formation between the mitochondrial matrix and intermembrane space. Nine of 12 TM helices in COX-1, both helices in COX-2 and 5 of the 6 TM helices in COX-3 are pore-lining regions (possible channel formers). Heme a3 and the CuB sites (as well as the CuA center of COX-2) are located within the channel that includes TM-6, TM-7, TM-10 and TM-11 of COX-1 and are associated with multiple cholesterol and caveolin-binding (CB) motifs. Sequence analysis identifies five CB motifs within COX-1, two within COX-2 and four within COX-3; each caveolin containing a pore-lining helix C-terminal to a TM helix-turn-helix. Channel formation involves interaction between multiple pore-lining regions within protein subunits and/or dimers. PoreWalker analysis lends support to the D-channel model of proton translocation. Under physiological conditions, caveolins may introduce channel formers juxtaposed to those in COX-1, COX-2 and COX-3, which together with cholesterol may form channel(s) essential for proton translocation through the inner mitochondrial membrane.

Computational analysis of the extracellular domain of the Ca²âº-sensing receptor: an alternate model for the Ca²âº sensing region.

The extracellular Ca(2+) sensing receptor (CaSR) belongs to Class C G-protein-coupled receptors (GPCRs) which include receptors for amino acids, γ-aminobutyric acid and glutamate neurotransmitters. CaSR has been described as having an extended sequence containing a Ca(2+) binding pocket within an extracellular amino (N)-terminal domain, called a Venus Fly Trap (VFT) module. CaSR is thought to consist of three domains: 1) a Ca(2+-)sensory domain, 2) a region containing 7 transmembrane (TM) helices, and 3) a carboxy (C)-terminal tail. We find that SPOCTOPUS (a combination of hidden Markov models and artificial neural networks) predicts that Homo sapiens CaSR contains two additional TM helices ((190)D - G(210); (262)S-E(282)), with the second TM helix containing a pore-lining region ((265)K - I(280)). This predicts that the putative Ca(2+) sensory domain is within an extracellular loop, N-terminal to the highly conserved heptahelical bundle. This loop contains both the cysteine-rich domain ((537)V - C(598)) and a 14 residue "linker" sequence ((599)I - F(612)) thought to support signal transmission to the heptahelical bundle. Thus domain 1 may contain a 189 residue N-terminal extracellular region followed successively by TM-1, a short intracellular loop, TM-2 and a 329 residue extracellular loop; rather than the proposed 620 residue VFT module based on crystallography of the N-terminal region of mGluR1. Since the topologies of the two proteins differ, the published CaSR VFT model is questionable. CaSR also contains multiple caveolin-binding motifs and cholesterol-binding (CRAC/CARC) domains, facilitating localization to plasma membrane lipid rafts. Ion sensing may involve combination of pore-lining regions from CaSR dimers and CaSR-bound caveolins to form ion channels capable of monitoring ionized Ca(2+) levels.

Plasma membrane events associated with the meiotic divisions in the amphibian oocyte: insights into the evolution of insulin transduction systems and cell signaling.

BACKGROUND: Insulin and its plasma membrane receptor constitute an ancient response system critical to cell growth and differentiation. Studies using intact Rana pipiens oocytes have shown that insulin can act at receptors on the oocyte surface to initiate resumption of the first meiotic division. We have reexamined the insulin-induced cascade of electrical and ion transport-related plasma membrane events using both oocytes and intact plasma membranes in order to characterize the insulin receptor-steroid response system associated with the meiotic divisions. RESULTS: [(125)I]Insulin binding (K(d) = 54 ± 6 nM) at the oocyte plasma membrane activates membrane serine protease(s), followed by the loss of low affinity ouabain binding sites, with a concomitant 3-4 fold increase in high affinity ouabain binding sites. The changes in protease activity and ouabain binding are associated with increased Na(+)/Ca2(+) exchange, increased endocytosis, decreased Na(+) conductance resulting in membrane hyperpolarization, increased 2-deoxy-D-glucose uptake and a sustained elevation of intracellular pH (pHi). Hyperpolarization is largely due to Na(+)-channel inactivation and is the main driving force for glucose uptake by the oocyte via Na(+)/glucose cotransport. The Na(+) sym- and antiporter systems are driven by the Na(+) free energy gradient generated by Na(+)/K(+)-ATPase. Shifts in α and/or ß Na(+)-pump subunits to caveolar (lipid raft) membrane regions may activate Na/K-ATPase and contribute to the Na(+) free energy gradient and the increase in both Na(+)/glucose co-transport and pHi. CONCLUSIONS: Under physiological conditions, resumption of meiosis results from the concerted action of insulin and progesterone at the cell membrane. Insulin inactivates Na(+) channels and mobilizes fully functional Na(+)-pumps, generating a Na(+) free energy gradient which serves as the energy source for several membrane anti- and symporter systems.

Comparative properties of caveolar and noncaveolar preparations of kidney Na+/K+-ATPase.

To evaluate previously proposed functions of renal caveolar Na(+)/K(+)-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na(+)/K(+)-ATPase (α,ß,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na(+)/K(+)-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme. As minor constituents, both caveolar and noncaveolar pools also contained Src, EGFR, PI3K, and several other proteins known to be involved in stimulous-induced signaling by Na(+)/K(+)-ATPase, indicating that signaling function is not limited to the caveolar pool. Endogenous Src was active in both pools but was not further activated by ouabain, calling into question direct interaction of Src with native Na(+)/K(+)-ATPase. Chemical cross-linking, co-immunoprecipitation, and immunodetection studies showed that in the caveolar pool, caveolin-1 oligomers, annexin-2 tetramers, and oligomers of the α,ß,γ-protomers of Na(+)/K(+)-ATPase form a large multiprotein complex. In conjunction with known roles of E-cadherin and the ß-subunit of Na(+)/K(+)-ATPase in cell adhesion and noted intercellular ß,ß-contacts within the structure of Na(+)/K(+)-ATPase, our findings suggest that interacting caveolar Na(+)/K(+)-ATPases located at renal adherens junctions maintain contact of two adjacent cells, conduct essential ion pumping, and are capable of locus-specific signaling in junctional cells.

Progesterone-induced changes in the phosphoryl potential during the meiotic divisions in amphibian oocytes: role of Na/K-ATPase.

BACKGROUND: Progesterone triggers resumption of the first meiotic division in the Rana pipiens oocyte by binding to the N-terminal external loop of the catalytic subunit of Na/K-ATPase, releasing a cascade of lipid second messengers. This is followed by internalization of specific membrane proteins, plasma membrane depolarization and nuclear membrane breakdown, culminating in arrest at second metaphase. RESULTS: Progesterone initiates an increase in phosphoryl potential during the first meiotic division, resulting in the accumulation of high energy protein phosphate by second metaphase arrest. 31P-NMR, with saturation transfer, demonstrates that the phosphocreatine level rises ~2 fold and that the "pseudo" first order rate constant for the creatine kinase reaction falls to ~20% of the control by the onset of nuclear membrane breakdown. 32PO4 pulse-labeling reveals a net increase in phosphorylation of yolk protein phosvitin during this period. The increased yolk protein phosphorylation coincides with internalization of membrane Na/K-ATPase and membrane depolarizatio CONCLUSIONS: These results indicate that progesterone binding to the catalytic subunit of the Na-pump diverts ATP from cation regulation at the plasma membrane to storage of high energy phosphate in yolk protein. Phosvitin serves as a major energy source during fertilization and early cleavage stages and is also a storage site for cations (e.g. Na+, K+, Ca2+, Fe2+/3+) essential for embryonic development.

Progesterone modulation of transmembrane helix-helix interactions between the alpha-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane.

BACKGROUND: Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the alpha-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the alpha1-subunit. RESULTS: Preparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020) successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT) and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME), can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the alpha1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT. CONCLUSION: We propose that progesterone binding to the first external loop of the alpha1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions.

Progesterone and subsequent polar metabolites are essential for completion of the first meiotic division in amphibian oocytes.

We find that completion of the first meiotic division in Rana pipiens oocytes requires the sequential action of at least two steroids: progesterone and one or more subsequent polar metabolites of progesterone. Progesterone binding in vitro to oocyte surface receptors increases during the first 4-5h of exposure, followed by internalization of plasma membrane together with membrane-bound progesterone over the next hour. The internalized progesterone is metabolized to highly polar polyhydroxylated steroid(s) prior to nuclear membrane disappearance at 8-9h. Polar steroids alone cannot induce meiosis, but do so in oocytes pretreated with progesterone for 1h. Similarly, the non-metabolizable progestin R5020 cannot induce meiosis but does if oocytes are subsequently exposed to polar steroids. An inhibitor of steroid alpha-reductase (4-MA) prevents both progesterone metabolism and progesterone-induced meiosis. However, meiosis does occur if 4-MA is followed by a polar steroid. Thus, progesterone binding at the oocyte surface initiates a process which requires polar progesterone metabolites for completion of the first meiotic division.

Progesterone binding to the alpha1-subunit of the Na/K-ATPase on the cell surface: insights from computational modeling.

Progesterone triggers the resumption of meiosis in the amphibian oocyte through a signaling system at the plasma membrane. Analysis of [(3)H]ouabain and [(3)H]progesterone binding to the plasma membrane of the Rana pipiens oocyte indicates that progesterone competes with ouabain for a low affinity ouabain binding site on a 112kDa alpha1-subunit of the membrane Na/K-ATPase. Published amino acid sequences from both low and high affinity ouabain binding alpha1-subunits are compared, together with published site-directed mutagenesis studies of ouabain binding. We propose that the progesterone binding site is located in the external loop (23 amino acids) between the M1-M2 transmembrane helices. Analysis of loop topology and the countercurrent hydrophobicity/polarity gradients within the M1-M2 loop further suggest that the polar beta and hydrophobic alpha surfaces of the planar progesterone molecule interact with opposite sides of the amino acid loop. The 19-angular methyl group of progesterone is essential for activity; it could bind to the C-terminal region of the M1-M2 loop. Maximum biological activity requires formation of hydrogen-bond networks between the 3-keto group of progesterone and Arg(118), Asp(129) and possibly Glu(122-124) in the C-terminal region of the loop. The 20-keto group hydrogen may in turn hydrogen bond to Cys(111) near the M1 helix. Peptide flexibility undergoes a maximal transition near the midway point in the M1-M2 loop, suggesting that folding occurs within the loop, which further stabilizes progesterone binding.

Computational comparison of a calcium-dependent jellyfish protein (apoaequorin) and calmodulin-cholesterol in short-term memory maintenance.

Memory reconsolidation and maintenance depend on calcium channels and on calcium/calmodulin-dependent kinases regulating protein turnover in the hippocampus. Ingestion of a jellyfish protein, apoaequorin, reportedly protects and/or improves verbal learning in adults and is currently widely advertised for use by the elderly. Apoaequorin is a member of the EF-hand calcium binding family of proteins that includes calmodulin. Calmodulin-1 (148 residues) differs from Apoaequorin (195 residues) in that it contains four rather than three Ca2+-binding sites and three rather than four cholesterol-binding (CRAC, CARC) domains. All three cholesterol-binding CARC domains in calmodulin have a high interaction affinity for cholesterol compared to only two high affinity CARC domains in apoaequorin. Both calmodulin and apoaequorin can form dimers with a potential of eight bound Ca2+ ions and six high affinity-bound cholesterol molecules in calmodulin with six bound Ca2+ ions and a mixed population of eight cholesterols bound to both CARC and CRAC domains in apoaqueorin. MEMSAT-SVM analysis indicates that both calmodulin and apoaqueorin have a pore-lining region. The Peptide-Cutter algorithm predicts that calmodulin-1 contains 11 trypsin-specific cleavage sites (compared to 21 in apoaqueorin), four of which are potentially blocked by cholesterol and three are within the Ca-binding domains and/or the pore-lining region. Three are clustered between the third and fourth Ca2+-binding sites. Only calmodulin pore-lining regions contain Ca2+ binding sites and as dimers may insert into the plasma membrane of neural cells and act as Ca2+ channels. In a dietary supplement, bound cholesterol may protect both apoaequorin and calmodulin from proteolysis in the gut as well as facilitate uptake across the blood-brain barrier. Our results suggest that a physiological calmodulin-cholesterol complex, not cholesterol-free jellyfish protein, may better serve as a dietary supplement to facilitate memory maintenance.

Gonadotropin stimulation of steroid synthesis and metabolism in the Rana pipiens ovarian follicle: sequential changes in endogenous steroids during ovulation, fertilization and cleavage stages.

Steroid synthesis and metabolism have been followed in Rana pipiens ovarian follicles, denuded oocytes and eggs during ovulation, fertilization and cleavage stages (blastula formation). Under physiological conditions, gonadotropin stimulation of the fully grown follicle leads to progesterone synthesis from [(3)H]acetate as well as formation of much smaller amounts of 17alpha-hydroxyprogesterone, androstenedione, pregnanedione and pregnanediol. Progesterone levels increase during completion of the first meiotic division, but by ovulation progesterone disappears from the egg. Plasma membrane-bound progesterone is taken up into the oocyte cortical granules and is largely metabolized to 5alpha-pregnane-3alphaol,20-one and 5beta-pregnane-3alpha,17alpha,20beta-triol coincident with internalization of 60% of the oocyte surface (and >90% of bound progesterone) by the end of the hormone-dependent period. The principal steroid in the ovulated egg is 5beta-pregnane-3alpha,17alpha,20beta-triol. There is a rapid efflux of 5beta-pregnane-3alpha,17alpha,20beta-triol into the medium immediately following fertilization and residual steroid levels remain low in the developing blastula. Dissociated blastulae cells prepared from stage 9 1/2 embryos concentrate both pregnenolone and progesterone from the medium with minimal metabolism. The results indicate that the ovarian follicle has the ability to synthesize and metabolize progesterone but that this ability disappears in the ovulated egg. The progesterone metabolites formed during meiosis are largely released at fertilization.

Molecular Properties of Globin Channels and Pores: Role of Cholesterol in Ligand Binding and Movement.

Globins contain one or more cavities that control or affect such functions as ligand movement and ligand binding. Here we report that the extended globin family [cytoglobin (Cygb); neuroglobin (Ngb); myoglobin (Mb); hemoglobin (Hb) subunits Hba(α); and Hbb(ß)] contain either a transmembrane (TM) helix or pore-lining region as well as internal cavities. Protein motif/domain analyses indicate that Ngb and Hbb each contain 5 cholesterol- binding (CRAC/CARC) domains and 1 caveolin binding motif, whereas the Cygb dimer has 6 cholesterol-binding domains but lacks caveolin-binding motifs. Mb and Hba each exhibit 2 cholesterol-binding domains and also lack caveolin-binding motifs. The Hb αß-tetramer contains 14 cholesterol-binding domains. Computer algorithms indicate that Cygb and Ngb cavities display multiple partitions and C-terminal pore-lining regions, whereas Mb has three major cavities plus a C-terminal pore-lining region. The Hb tetramer exhibits a large internal cavity but the subunits differ in that they contain a C-terminal TM helix (Hba) and pore-lining region (Hbb). The cavities include 43 of 190 Cygb residues, 38 of 151 of Ngb residues, 55 of 154 Mb residues, and 137 of 688 residues in the Hb tetramer. Each cavity complex includes 6 to 8 residues of the TM helix or pore-lining region and CRAC/CARC domains exist within all cavities. Erythrocyte Hb αß-tetramers are largely cytosolic but also bind to a membrane anion exchange protein, "band 3," which contains a large internal cavity and 12 TM helices (5 being pore-lining regions). The Hba TM helix may be the erythrocyte membrane "band 3" attachment site. "Band 3" contributes 4 caveolin binding motifs and 10 CRAC/CARC domains. Cholesterol binding may create lipid-disordered phases that alter globin cavities and facilitate ligand movement, permitting ion channel formation and conformational changes that orchestrate anion and ligand (O2, CO2, NO) movement within the large internal cavities and channels of the globins.

The steroid-binding subunit of the Na/K-ATPase as a progesterone receptor on the amphibian oocyte plasma membrane.

Progesterone acts at a plasma membrane receptor on the Rana oocyte to initiate meiosis. A cascade of lipid messengers occurs within seconds, followed by sequential changes in membrane phospholipid composition. We now show that progesterone binding to the plasma membrane increases continuously over the first 4 h. Subsequently, about 60% of the total plasma membrane and > 90% of membrane-bound progesterone, ouabain binding sites, and Na/K-ATPase activity are internalized. Until the completion of membrane internalization, oocytes must be continuously exposed to nanomolar concentrations of exogenous progesterone for meiosis to continue. The membrane-bound progesterone remains unchanged, whereas microinjected [(3)H]progesterone is rapidly metabolized. We find that progesterone and the plant steroid ouabain compete for one of two ouabain binding sites on the oocyte surface. Ouabain blocks progesterone action and inhibits subsequent meiosis if added at any time during the first 4-5 h. Western blots of SDS/PAGE extracts of isolated oocyte plasma membranes contain a -110 kDa band which binds an antibody to the steroid-binding c-terminal domain in rat and human PR. The number of binding sites and K(d) for progesterone binding to the plasma membrane is comparable to those for low-affinity ouabain binding to the alpha-subunit of the Na/K-ATPase (112 kDa). Our results suggest that progesterone binding to the ouabain binding site on the N-terminal region of the alpha-subunit of Na/K-ATPase may modulate early plasma membrane events over the first 4-6 h. Progesterone may thus act in part through the plasma membrane Na/K-ATPase signaling system.

Transmembrane helices in "classical" nuclear reproductive steroid receptors: a perspective.

Steroid receptors of the nuclear receptor superfamily are proposed to be either: 1) located in the cytosol and moved to the cell nucleus upon activation, 2) tethered to the inside of the plasma membrane, or 3) retained in the nucleus until free steroid hormone enters and activates specific receptors. Using computational methods to analyze peptide receptor topology, we find that the "classical" nuclear receptors for progesterone (PRB/PGR), androgen (ARB/AR) and estrogen (ER1/ESR1) contain two transmembrane helices (TMH) within their ligand-binding domains (LBD).The MEMSAT-SVM algorithm indicates that ARB and ER2 (but not PRB or ER1) contain a pore-lining (channel-forming) region which may merge with other pore-lining regions to form a membrane channel. ER2 lacks a TMH, but contains a single pore-lining region. The MemBrain algorithm predicts that PRB, ARB and ER1 each contain one TMH plus a half TMH separated by 51 amino acids.ER2 contains two half helices. The TM-2 helices of ARB, ER1 and ER2 each contain 9-13 amino acid motifs reported to translocate the receptor to the plasma membrane, as well as cysteine palmitoylation sites. PoreWalker analysis of X-ray crystallographic data identifies a pore or channel within the LBDs of ARB and ER1 and predicts that 70 and 72 residues are pore-lining residues, respectively. The data suggest that (except for ER2), cytosolic receptors become anchored to the plasma membrane following synthesis. Half-helices and pore-lining regions in turn form functional ion channels and/or facilitate passive steroid uptake into the cell. In perspective, steroid-dependent insertion of "classical" receptors containing pore-lining regions into the plasma membrane may regulate permeability to ions such as Ca(2+), Na(+) or K(+), as well as facilitate steroid translocation into the nucleus.

Iron-catalyzed lipid peroxidation in aortic cells in vitro: protective effect of extracellular magnesium.

Low serum Mg2+ has been associated with an increased incidence of cardiovascular pathology in human populations. We investigated the effect of extracellular Mg2+ on Fe-catalyzed lipid peroxidation in rat aortic segments and in human aortic smooth muscle cells. Products of phospholipid oxidation [malonaldehyde (MDA) and 4-hydroxyalkenals (4-HA)], loss of fatty acyl double bonds (by proton-NMR) and glutathione levels indicated that exogenous ferric ions were several-fold more effective than ferrous ions in causing lipid peroxidation. Increased peroxidation was detectable at <1.0 microM Fe3+. Exogenous ferric iron-ionophore, 8-hydroxyquinoline, did not increase peroxidation by ferric ion, suggesting that Fe-catalyzed lipid peroxidation occurred at the cell surface. As ionized serum [Mg2+](o) was lowered from the physiological (0.7-0.96 mM) into the pathophysiological range (0.3-0.5mM) in Fe3+-containing medium, MDA/4-HA levels increased two to three-fold, with a concomitant loss of fatty acyl double bonds and decreased extracellular glutathione. Conversely, MDA/4-HA decreased as ionized Mg2+ was increased, accompanied by a rise in extracellular glutathione. The results indicate that Mg2+ protects aortic cell plasma membranes from ferric iron-catalyzed lipid peroxidation and that this is a contributing factor in the protective action of ionized Mg2+ on the cardiovascular system.

Increased Potassium Conductance in Rana Follicles after Stimulation by Pituitary Extract: (amphibian follicle/meiotic maturation/gonadotropin/K+ conductance/gap junctions).

Pituitary gonadotropins are believed to induce the somatic cell portion of the amphibian follicle to synthesize and release progesterone which, in turn, induces the resumption of the meiotic divisions in the follicular oocyte. We report here that pituitary extract, at concentrations that induce ovulation and meiosis, causes a rapid hyperpolarization of the follicular oocyte. A similar hyperpolarization is seen in response to porcine LH but not FSH. Voltage clamp studies indicate that this is due to an increase in follicle K+ conductance. An electrical model of the amphibian follicle suggests that pituitary factors act by increasing the K+ conductance of the oolemma, by increasing the extent of oocyte-follicle cell ionic coupling, or by increasing the conductance of follicle cell plasma membrane. The conductance change does not occur in the absence of follicle cells, is not mediated by progesterone, and is not necessary for meiotic maturation, per se, but may play a role in processes which accompany or follow maturation.

Expression of the nuclear factor-kappaB and proto-oncogenes c-fos and c-jun are induced by low extracellular Mg2+ in aortic and cerebral vascular smooth muscle cells: possible links to hypertension, atherogenesis, and stroke.

Proto-oncogene (c-fos, c-jun) and nuclear factor-kappa B (NF-kappaB) expression, as well as DNA synthesis, in aortic and cerebral vascular smooth muscle cells (VSMCs) were upregulated by a decrease in extracellular magnesium ions ([Mg2+]o). Upregulation of these transcriptional factors was inversely proportional to the [Mg2+]o and occurred over the pathophysiologic range of serum Mg2+ found in patients presenting with hypertension, ischemic heart disease, and stroke. Removal of extracellular Ca2+ ([Ca2+]o), use of nifedipine or protein kinase C (PKC) inhibitors prevented the upregulation of the proto-oncogenes and DNA synthesis in VSMCs. These data show that [Mg2+]o may be an important, heretofore, overlooked natural modulator of proto-oncogene and NF-kappaB expression in VSMCs and that Ca2+ and PKC may play critical roles in induction of c-fos and c-jun in VSMCs induced by a decrease in [Mg2+]o. These results point to a role for low serum Mg2+ in potential development of hypertension, atherogenesis, vascular disease, and stroke.

Interaction between ferric ions, phospholipid hydroperoxides, and the lipid phosphate moiety at physiological pH.

Iron-catalyzed lipid peroxidation was examined using 1H NMR in a biphasic aqueous-chloroform system. At physiological pH (7.4), mole ratios of phospholipids/Fe3+ as low as 1300:1 catalyzed the rapid disappearance of endogenous lipid hydroperoxides with a loss of two of the four double bonds in PC containing palmitic (16:0) and arachidonic (20:4) acids in the sn-1 and sn-2 positions, respectively. The predominant phospholipid products after 1 h at 20 degrees C were a 9-carbon mono-unsaturated carbonyl and a phospholipid with an 11-carbon delta5,8 FA in the sn-2 position. PC with linoleic acid (18:2) in the sn-2 position lost one double bond and formed a phospholipid with a 9-carbon FA. Cardiolipin (linoleic acid-rich) also lost about 40% of its double bonds. No detectable loss was seen for PC containing oleic acid (18:1) or neutral lipids with PUFA. At arachidonyl PC/Fe3+ ratios less than 20:1, significant broadening of the choline methyl proton peak was evident, indicating that Fe3+ may form a complex with the adjacent phosphate group and that the complex involves both the phosphate and the hydroperoxide adjacent to the delta11 double bond. The results demonstrate that, at physiological pH, Fe3+-catalyzed peroxidation in polyunsaturated phospholipids occurs selectively adjacent to specific double bonds (delta9 or delta11). These PC-derived products have been shown to activate components of the inflammatory system. This suggests that the episodic release of ferric ions may play a significant role in generating inflammatory mediators.

A computational analysis of non-genomic plasma membrane progestin binding proteins: signaling through ion channel-linked cell surface receptors.

A number of plasma membrane progestin receptors linked to non-genomic events have been identified. These include: (1) α1-subunit of the Na(+)/K(+)-ATPase (ATP1A1), (2) progestin binding PAQR proteins, (3) membrane progestin receptor alpha (mPRα), (4) progesterone receptor MAPR proteins and (5) the association of nuclear receptor (PRB) with the plasma membrane. This study compares: the pore-lining regions (ion channels), transmembrane (TM) helices, caveolin binding (CB) motifs and leucine-rich repeats (LRRs) of putative progesterone receptors. ATP1A1 contains 10 TM helices (TM-2, 4, 5, 6 and 8 are pores) and 4 CB motifs; whereas PAQR5, PAQR6, PAQR7, PAQRB8 and fish mPRα each contain 8 TM helices (TM-3 is a pore) and 2-4 CB motifs. MAPR proteins contain a single TM helix but lack pore-lining regions and CB motifs. PRB contains one or more TM helices in the steroid binding region, one of which is a pore. ATP1A1, PAQR5/7/8, mPRα, and MAPR-1 contain highly conserved leucine-rich repeats (LRR, common to plant membrane proteins) that are ligand binding sites for ouabain-like steroids associated with LRR kinases. LRR domains are within or overlap TM helices predicted to be ion channels (pore-lining regions), with the variable LRR sequence either at the C-terminus (PAQR and MAPR-1) or within an external loop (ATP1A1). Since ouabain-like steroids are produced by animal cells, our findings suggest that ATP1A1, PAQR5/7/8 and mPRα represent ion channel-linked receptors that respond physiologically to ouabain-like steroids (not progestin) similar to those known to regulate developmental and defense-related processes in plants.