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PubMed1998

Magnetically aligned phospholipid bilayers with positive ordering: a new model membrane system.

A stable smectic phospholipid bilayer phase aligned with the director parallel to the magnetic field can be generated by the addition of certain trivalent paramagnetic lanthanide ions to a bicellar solution of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in water. Suitable lanthanide ions are those with positive anisotropy of their magnetic susceptibility, namely Eu3+, Er3+, Tm3+, and Yb3+. For samples doped with Tm3+, this phase extends over a wide range of Tm3+ concentrations (6-40 mM) and temperatures (35-90 degrees C) and appears to undergo a transition from a fluid nematic discotic to a fluid, but highly ordered, smectic phase at a temperature that depends on the thulium concentration. As a membrane mimetic, these new, positively ordered phospholipid phases have high potential for structural studies using a variety of techniques such as magnetic resonance (EMR and NMR), small-angle x-ray and neutron diffraction, as well as optical and infrared spectroscopy.

Dimyristoylphosphatidylcholine↗

PubMed2002

Identification of binding sites of prazosin, tamsulosin and KMD-3213 with alpha(1)-adrenergic receptor subtypes by molecular modeling.

This investigation was performed to assess the importance of interaction in the bindings of selective and nonselective alpha(1)-antagonists to alpha(1)-adrenergic receptor (alpha(1)-AR) subtypes using molecular modeling. The alpha(1)-antagonists used in this study were prazosin, tamsulosin and KMD-3213. Molecular modeling was performed on Octane 2 workstation (Silicon Graphics) using Discover/Insight II software (Molecular Simulations Inc.). Through molecular modeling, possible binding sites for these drugs were suggested to lie between transmembrane domains (TM) 3, 4, 5 and 6 of the alpha(1)-AR subtypes. In prazosin, the 4-amino group, 1-nitrogen atom and two methoxy groups of quinazoline ring possibly interact with the amino acids in TM3, TM5 and TM6 of alpha(1)-ARs. In tamsulosin, amine group of ethanyl amine chain, methoxy group of benzene ring and sulfonamide nitrogen of benzene ring interacts in TM3, TM4 and TM5 of alpha(1)-ARs. In KMD-3213, amine of ethyl amine chain and indoline nitrogen of this compound possibly interact within TM3 and TM5 of alpha(1)-ARs. Amide nitrogen of KMD-3213 also interacts within TM4 of alpha(1A)-AR. The results of the present study suggested that prazosin has similar binding sites in all the alpha(1)-AR subtypes while tamsulosin interacts at higher number of sites with alpha(1D)-subtype than other alpha(1)-AR subtypes. KMD-3213 being an alpha(1A)-AR selective ligand, binds to higher number of sites of alpha(1A) subtype than to other subtypes. All these amino acids are located near the extracellular loop. These findings are consistent with the previous studies that antagonists bind higher in the pocket closer to the extracellular surface unlike agonist binding.

Adrenergic alpha-Antagonists↗

PubMed1997

Modeling of the three-dimensional structure of the human melanocortin 1 receptor, using an automated method and docking of a rigid cyclic melanocyte-stimulating hormone core peptide.

A model is presented of the melanocortin 1 receptor (MC1R), constructed by use of an unbiased, objective method. The model is created directly from data derived from multiple sequence analysis, a low-resolution EM-projection map of rhodopsin, and the approximate membrane thickness. The model agrees well with available data concerning natural mutations of MC1Rs occurring in different species. A model is also presented of the most rigid ligand for this receptor, the cyclic pentapeptide cHFRWG, shown docked in the receptor model. The receptor-ligand complex model agrees well with available experimental data. The ligand is located between transmembrane region 1 (TM1), TM2, TM3, TM6, and TM7 of the receptor. Multiple interactions occur between ligand and receptor, including interactions with Leu-48 (TM1), Ser-52 (TM1), Glu-55 (TM1), Asn-91 (TM2), Glu-94 (TM2), Thr-95 (TM2) Ile-98 (TM2), Asp-121 (TM3), Thr-124 (TM3), Phe-257 (TM6), Phe-283 (TM7), Asn-290 (TM7), and Asp-294 (TM7) of the receptor.

Amino Acid Sequence↗

PubMed1994

Lysine 182 of endothelin B receptor modulates agonist selectivity and antagonist affinity: evidence for the overlap of peptide and non-peptide ligand binding sites.

The potent vasoactive peptide hormone endothelin (ET) binds to receptors which belong to the G-protein coupled receptor family. The availability of non-peptide antagonists for ET receptors allows investigation of the relationship among the binding sites for peptide and non-peptide ligands. In this study, a lysine residue, conserved within transmembrane domain 3 (TM3) of the ETA and ETB receptor subtypes, is implicated in agonist and antagonist binding by its analogous position within TM3 to a binding site aspartate residue conserved within bioactive amine receptors. Replacement of this lysine within hETB by arginine, alanine, methionine, aspartate, or glutamate results in hETB variants with unaltered affinities for agonist peptide ET-1 but which have affinities for peptide agonists ET-2, ET-3, sarafotoxin 6C, and TRL 1736 which are between 1-3 orders of magnitude lower than their corresponding wild-type hETB values. Significantly, the affinities of non-peptide antagonists, (+/-)-SB 209670 and its analogs as well as Ro 46-2005, are abrogated. The results suggest that an interaction of K182 of hETB with the indan 2-carboxyl of (+/-)-SB 209670 may contribute to the high-affinity binding of the diarylindan antagonists. The results indicate that TM3 of hETB is a region of overlap among the binding sites of non-peptide antagonists and the affected peptide agonists.

Amino Acid Sequence↗

PubMed2001

Wnt-1 and int-2 mammary oncogene effects on the beta-catenin pathway in immortalized mouse mammary epithelial cells are not sufficient for tumorigenesis.

Development of strategies for prevention of breast cancer development requires an understanding of the effects of mammary oncogenes on mammary cells at early stages in neoplastic transformation. As mammary oncogenes wnt-1 and int-2 affect different signal transduction pathways, we investigated their effects on established mouse mammary epithelial cell lines (MMECLs) reflecting early stages in tumorigenesis. Normal interactions between beta-catenin and E-cadherin were abrogated in all three immortalized MMECLs and the cells lacked beta-catenin-mediated transactivation activity, detectable using a reporter assay, suggesting that alterations in cell adhesion may be very early events in mammary tumorigenesis. Immortalized FSK4 and EL12 cells and hyperplastic TM3 cells were stably transfected with expression vectors encoding wnt-1 or int-2 or the control vector, and drug-selected pooled cells from each line were confirmed by reverse transcription-polymerase chain reaction to express the transfected oncogene; this expression persisted in the cells analysed in vitro and in vivo. Resultant phenotypic changes depended both on the oncogene and the target mammary cell line. In FSK4 cells, expression of wnt-1 or int-2 resulted in proliferative changes in vitro, including reduced contact inhibition, increased beta-catenin expression, and decreased p53 transcriptional activity, but neither oncogene conferred upon those cells the ability to produce tumors in vivo. EL12 cells were highly refractory to the effects of both oncogenes, with the only measurable changes being increased E-cadherin levels induced by both oncogenes and increased proliferation of the int-2-transfected cells in the absence of serum. Parental TM3 cells were phenotypically similar to wnt-1- or int-2-transfected FSK4 cells and displayed an increased rate of proliferation in vitro and markedly increased tumorigenicity in vivo following transfection with int-2 but not with wnt-1. These results suggest that wnt-1 signaling is redundant in the hyperplastic TM3 cells and indicate that wnt-1-induced effects in the immortalized FSK4 and EL12 cells were not sufficient to mediate a tumorigenic phenotype. This study showed that the wnt-1 and int-2 oncogenes have similar but distinguishable effects on immortalized MMECLs and that the genetic background of the mammary cells greatly influences the consequences of oncogene expression at early stages of cell transformation.

Animals↗

PubMed2000

Permeation and gating residues in serotonin transporter.

The third transmembrane domain (TM3) of serotonin transporter (SERT) contains two isoleucine residues previously proposed to be involved in binding and transport of serotonin. When Ile-172 was replaced with cysteine, SERT became sensitive to inactivation by externally added [2-(trimethylammonium)ethyl]methanethio-sulfonate (MTSET). The disulfide product of this inactivation was not sensitive to reduction by externally added sulfhydryl compounds, but apparently reacted with intracellular reducing agents to spontaneously regenerate active SERT. The apparent accessibility of this residue to both external and cytoplasmic reagents is consistent with its localization near a serotonin binding site that is alternately exposed to both internal and external media. In another SERT mutant, I179C, transport also was inactivated by MTSET but substrate binding was resistant. External substrate bound to the inactivated I179C and enhanced its reactivation by free thiols. In norepinephrine transporter (NET), cysteine replacement of Ile-155 (corresponding to SERT Ile-179) also rendered the transporter sensitive to MTSET inactivation. In NET I155C, cocaine enhanced this inactivation, and the substrate, dopamine, apparently protected against inactivation. The characteristics of this protection suggest that dopamine was transported, converting NET to a form in which Ile-155 was occluded. The results support the proposal that TM3 of SERT and NET constitute part of the substrate permeation pathway, and that Ile-172 in SERT resides close to the substrate binding site. They also suggest that Ile-179 in SERT (and Ile-155 in NET) is in a conformationally sensitive part of TM3, which may act as part of an external gate.

Allosteric Regulation↗

PubMed2004

Differential stability of biogenesis intermediates reveals a common pathway for aquaporin-1 topological maturation.

Topological studies of multi-spanning membrane proteins commonly use sequentially truncated proteins fused to a C-terminal translocation reporter to deduce transmembrane (TM) segment orientation and key biogenesis events. Because these truncated proteins represent an incomplete stage of synthesis, they transiently populate intermediate folding states that may or may not reflect topology of the mature protein. For example, in Xenopus oocytes, the aquaporin-1 (AQP1) water channel is cotranslationally directed into a four membrane-spanning intermediate, which matures into the six membrane-spanning topology at a late stage of synthesis (Skach, W. R., Shi, L. B., Calayag, M. C., Frigeri, A., Lingappa, V. R., and Verkman, A. S. (1994) J. Cell Biol. 125, 803-815 and Lu, Y., Turnbull, I. R., Bragin, A., Carveth, K., Verkman, A. S., and Skach, W. R. (2000) Mol. Biol. Cell 11, 2973-2985). The hallmark of this process is that TM3 initially acquires an Nexo/Ccyto (Type I) topology and must rotate 180 degrees to acquire its mature orientation. In contrast, recent studies in HEK-293 cells have suggested that TM3 acquires its mature topology cotranslationally without the need for reorientation (Dohke, Y., and Turner, R. J. (2002) J. Biol. Chem. 277, 15215-15219). Here we re-examine AQP1 biogenesis and show that irrespective of the reporter or fusion site used, oocytes and mammalian cells yielded similar topologic results. AQP1 intermediates containing the first three TM segments generated two distinct cohorts of polypeptides in which TM3 spanned the ER membrane in either an Ncyto/Cexo (mature) or Nexo/Ccyto (immature) topology. Pulse-chase analyses revealed that the immature form was predominant immediately after synthesis but that it was rapidly degraded via the proteasome-mediated endoplasmic reticulum associated degradation (ERAD) pathway with a half-life of less than 25 min in HEK cells. As a result, the mature topology predominated at later time points. We conclude that (i) differential stability of biogenesis intermediates is an important factor for in vivo topological analysis of truncated chimeric proteins and (ii) cotranslational events of AQP1 biogenesis reflect a common AQP1 folding pathway in diverse expression systems.

Animals↗

PubMed2005

Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants.

In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [(125)I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/I172M had a synergistic impact on (RS)-CIT recognition ( approximately 10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT.

Adrenergic Uptake Inhibitors↗

PubMed2005

Rat synapsin 1 promoter mediated transgene expression in testicular cell types.

Previous reports described the rat synapsin 1 promoter as primarily neuron selective. However, ectopic expression of a transgene under the rat synapsin 1 promoter was also detected in testis from some transgenic mouse lines. Here we investigate which cells within the testis express a transgene consisting of the rat synapsin 1 promoter fused with luciferase. Synapsin 1-luciferase expression vectors were introduced into HeLa cells, into TM3 cells derived from mouse testicular Leydig cells, and into one-cell embryos to make transgenic mice. Indirect immunofluorescence suggests that nontransfected TM3 cells do not express endogenous synapsin 1. TM3 stable transfectants, however, expressed luciferase under the direction of the synapsin 1 promoter, in both promoter orientations. HeLa cells displayed only low levels of activity. Transgenic mice carrying the synapsin 1-luciferase construct displayed high levels of luciferase activity in the brain, spinal cord, and testis. Enriched populations of prepuberal types A and B spermatogonia and adult Leydig cells, pachytene spermatocytes, and round spermatids prepared from transgenic mice all displayed substantial luciferase activity. Thus, the rat synapsin 1 promoter can mediate reporter gene expression in neurons and testicular cell types.

Animals↗

PubMed2000

Reorientation of aquaporin-1 topology during maturation in the endoplasmic reticulum.

The topology of most eukaryotic polytopic membrane proteins is established cotranslationally in the endoplasmic reticulum (ER) through a series of coordinated translocation and membrane integration events. For the human aquaporin water channel AQP1, however, the initial four-segment-spanning topology at the ER membrane differs from the mature six-segment-spanning topology at the plasma membrane. Here we use epitope-tagged AQP1 constructs to follow the transmembrane (TM) orientation of key internal peptide loops in Xenopus oocyte and cell-free systems. This analysis revealed that AQP1 maturation in the ER involves a novel topological reorientation of three internal TM segments and two peptide loops. After the synthesis of TMs 4-6, TM3 underwent a 180-degree rotation in which TM3 C-terminal flanking residues were translocated from their initial cytosolic location into the ER lumen and N-terminal flanking residues underwent retrograde translocation from the ER lumen to the cytosol. These events convert TM3 from a type I to a type II topology and reposition TM2 and TM4 into transmembrane conformations consistent with the predicted six-segment-spanning AQP1 topology. AQP1 topological reorientation was also associated with maturation from a protease-sensitive conformation to a protease-resistant structure with water channel function. These studies demonstrate that initial protein topology established via cotranslational translocation events in the ER is dynamic and may be modified by subsequent steps of folding and/or maturation.

Amino Acid Sequence↗

PubMed2005

Specific features of neuronal size and shape are regulated by tropomyosin isoforms.

Spatially distinct populations of microfilaments, characterized by different tropomyosin (Tm) isoforms, are present within a neuron. To investigate the impact of altered tropomyosin isoform expression on neuronal morphogenesis, embryonic cortical neurons from transgenic mice expressing the isoforms Tm3 and Tm5NM1, under the control of the beta-actin promoter, were cultured in vitro. Exogenously expressed Tm isoforms sorted to different subcellular compartments with Tm5NM1 enriched in filopodia and growth cones, whereas the Tm3 was more broadly localized. The Tm5NM1 neurons displayed significantly enlarged growth cones accompanied by an increase in the number of dendrites and axonal branching. In contrast, Tm3 neurons displayed inhibition of neurite outgrowth. Recruitment of Tm5a and myosin IIB was observed in the peripheral region of a significant number of Tm5NM1 growth cones. We propose that enrichment of myosin IIB increases filament stability, leading to the enlarged growth cones. Our observations support a role for different tropomyosin isoforms in regulating interactions with myosin and thereby regulating morphology in specific intracellular compartments.

Actin Cytoskeleton↗

PubMed1975

A genetic analysis of phototactic behavior in Drosophila melanogaster I. Selection in the presence of inversions.

The effectiveness of selection for positive and negative phototactic behavior in populations of Drosophila melanogaster heterozygous for various multiple inversions was compared using the method of realized heritability. Selection in the presence of FM6, SM1 or TM3 alone was as effective as in populations carrying no inversions. However, the presence of FM6 and TM3 together reduced the effectiveness of selection for photopositive behavior and FM6 and SM1 and TM3 restricted the response to selection for negative phototactic behavior. The results are discussed in terms of the organization of genes influencing phototactic behavior in this species.

Animals↗

PubMed1992

Localization and synthesis of entactin in seminiferous tubules of the mouse.

Localization and synthesis of entactin in seminiferous tubules of mouse testis was studied by immunocytochemistry. Frozen sections from adult mice testes were subjected to anti-entactin and anti-laminin immunofluorescence. Both entactin and laminin were localized within the seminiferous tubule basement membrane and intertubular region of the testis. The addition of excess amount of entactin (but not fibronectin), premixed with anti-entactin antiserum, abolished the immunostain. Western blotting showed that a protein extract from a seminiferous tubule basement membrane preparation was recognized by anti-entactin anti-serum and comigrated with recombinant entactin. Enriched fractions of isolated primary Sertoli cells and peritubular myoid cells cultured for 6 days on a glass coverslip were able to synthesize and secrete entactin as detected by immunofluorescence microscopy. Entactin was also produced by TM3 (Leydig-like) and TM4 (Sertoli-like) cell lines as detected by both immunofluorescence and Western blotting. The distribution of entactin vs. laminin within both the cultured primary cells and the TM3 and TM4 cell lines differed. Entactin appeared mainly localized extracellularly. In contrast, laminin was mainly localized intracellularly. The above findings suggested that 1) entactin existed in the seminiferous tubule basement membrane and intertubular region of adult mice testis, co-localized with laminin; 2) entactin was synthesized by the cultured primary Sertoli cells and peritubular myoid cells and the TM3 and TM4 cell lines; 3) entactin was exocytosed with little intracellular accumulation, in contrast to an intracellular accumulation of laminin.

Animals↗

PubMed1998

Physical conditioning attenuates respiratory responses to steady-state exercise in late gestation.

PURPOSE: To study the ventilatory effects of closely monitored cycle ergometer conditioning (HR target, 145-150 beats x min(-1); 25 min/session; three sessions per week) during the second and third pregnancy trimesters (TMs) in healthy human pregnancy. METHODS: Subjects were 27 previously sedentary pregnant women (exercised group, EG). A sedentary control group (CG, N = 20) was also studied. Subjects were tested at rest and during upright cycle ergometry at three steady-state power outputs (approximately 20, 45, and 65 W) at the start of the second TM (ENTRY), at the end of the second and third TMs (post-training), and 3 months postpartum (PP, nonpregnant control). RESULTS: Significant reductions in exercise HR were observed in the EG at each exercise level, confirming achievement of an aerobic conditioning effect. In both groups the ventilatory equivalent for oxygen (VE/O2) was significantly higher at all work rates during pregnancy compared with that during PP. This effect was caused by significantly higher exercise tidal volumes (VT) at each work rate in both groups during pregnancy versus PP. Respiratory sensitivity as reflected by the ventilatory equivalent for carbon dioxide (E/VECO2) and calculated arterial carbon dioxide tension (PaCO2) was significantly increased at all three work rates in late gestation versus PP and increased slightly with advancing gestational age in both groups. The main effects of physical conditioning were attenuation of increases in VE/VO2 observed at all exercise levels between the end of TM2 and TM3 and reductions in respiratory perception of effort (RPEr) between ENTRY and the end of TM3. Attenuation of increases in VE/VO2 during TM3 in the EG was attributed to the combined effects of enhanced fat versus carbohydrate utilization (resulting in reduced CO2 output and drive to ventilate) and attenuation of pregnancy-induced increases in dead space ventilation in late gestation. CONCLUSION: The study results support the hypothesis that physical conditioning reduces both ventilatory demand and respiratory perception of effort in late gestation.

Adult↗

PubMed2025

Single-cell profiling of trabecular meshwork identifies mitochondrial dysfunction in a glaucoma model that is protected by vitamin B3 treatment.

Since the trabecular meshwork (TM) is central to intraocular pressure (IOP) regulation and glaucoma, a deeper understanding of its genomic landscape is needed. We present a multimodal, single-cell resolution analysis of mouse limbal cells (includes TM). In total, we sequenced 9,394 wild-type TM cell transcriptomes. We discovered three TM cell subtypes with characteristic signature genes validated by immunofluorescence on tissue sections and whole-mounts. The subtypes are robust, being detected in datasets for two diverse mouse strains and in independent data from two institutions. Results show compartmentalized enrichment of critical pathways in specific TM cell subtypes. Distinctive signatures include increased expression of genes responsible for 1) extracellular matrix structure and metabolism (TM1 subtype), 2) secreted ligand signaling to support Schlemm's canal cells (TM2), and 3) contractile and mitochondrial/metabolic activity (TM3). ATAC-sequencing data identified active transcription factors in TM cells, including LMX1B. Mutations in LMX1B cause high IOP and glaucoma. LMX1B is emerging as a key transcription factor for normal mitochondrial function and its expression is much higher in TM3 cells than other limbal cells. To understand the role of LMX1B in TM function and glaucoma, we single-cell sequenced limbal cells from Lmx1b V265D/+ mutant mice (2,491 TM cells). In V265D/+ mice, TM3 cells were uniquely affected by pronounced mitochondrial pathway changes. Mitochondria in TM cells of V265D/+ mice are swollen with a reduced cristae area, further supporting a role for mitochondrial dysfunction in the initiation of IOP elevation in these mice. Importantly, treatment with vitamin B3 (nicotinamide), to enhance mitochondrial function and metabolic resilience, significantly protected Lmx1b mutant mice from IOP elevation.

Journal Article↗

PubMed2006

Structural requirements of transmembrane domain 3 for activation by the M1 muscarinic receptor agonists AC-42, AC-260584, clozapine, and N-desmethylclozapine: evidence for three distinct modes of receptor activation.

Transmembrane domain 3 (TM3) plays a crucial role mediating muscarinic acetylcholine receptor activation by acetylcholine, carbachol, and other muscarinic agonists. We compared the effects of point mutations throughout TM3 on the interactions of carbachol, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), a potent structural analog of AC-42 called 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), N-desmethylclozapine, and clozapine with the M(1) muscarinic receptor. The binding and activation profiles of these ligands fell into three distinct patterns; one exemplified by orthosteric compounds like carbachol, another by structural analogs of AC-42, and a third by structural analogs of N-desmethylclozapine. All mutations tested severely reduced carbachol binding and activation of M(1). In contrast, the agonist actions of AC-42 and AC-260584 were greatly potentiated by the W101A mutation, slightly reduced by Y106A, and slightly increased by S109A. Clozapine and N-desmethylclozapine displayed substantially increased maximum responses at the Y106A and W101A mutants, slightly lower activity at S109A, but no substantial changes in potency. At L102A and N110A, agonist responses to AC-42, AC-260584, clozapine, and N-desmethylclozapine were all substantially reduced, but usually less than carbachol. D105A showed no functional responses to all ligands. Displacement and dissociation rate experiments demonstrated clear allosteric properties of AC-42 and AC-260584 but not for N-desmethylclozapine and clozapine, indicating that they may contact different residues than carbachol to activate M(1) but occupy substantially overlapping spaces, in contrast to AC-42 and AC-260584, which occupy separable spaces. These results show that M(1) receptors can be activated in at least three distinct ways and that there is no requirement for potent muscarinic agonists to mimic acetylcholine interactions with TM3.

Benzoxazines↗

PubMed1989

Secretion of activin by interstitial cells in the testis.

Activin, a dimer formed by the beta subunits of inhibin, has an effect that is opposite to that of inhibin in a number of biological systems. Which cell types secrete activin in vivo is not known. TM3 cells, a Leydig-derived cell line, contained messenger RNAs that hybridized with human beta A and beta B complementary DNA probes and were similar in size to the porcine messenger RNA for the beta subunits of inhibin. No hybridization to the inhibin alpha subunit was detectable in the TM3 cells. Conditioned medium from TM3 cells and from primary cultures of rat and porcine interstitial cells stimulated the release of follicle-stimulating hormone in a pituitary cell culture assay. It is likely that, in the testis, the Leydig cells secrete activin and the Sertoli cells produce inhibin, or a combination of both.

Activins↗

PubMed2004

Interaction of PomB with the third transmembrane segment of PomA in the Na+-driven polar flagellum of Vibrio alginolyticus.

The marine bacterium Vibrio alginolyticus has four motor components, PomA, PomB, MotX, and MotY, responsible for its Na(+)-driven flagellar rotation. PomA and PomB are integral inner membrane proteins having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel complex. First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB, and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent. Secondly, the Cys substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34, Asp-170, and Ala-178) were combined. Cross-linked products were detected for the combination of PomB-S38C and PomA-D170C mutant proteins. The Cys substitutions in the periplasmic boundaries of PomA TM3 (from Met-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double mutants. Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it. Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it was recovered by reducing reagent. The strain with this combination showed a lower affinity for Na(+) than the wild-type combination. PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of PomA TM3 and the PomB TM, also formed the cross-linked product. From these lines of evidence, we infer that TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between these two TMs is required for coupling of Na(+) conduction to flagellar rotation.

Amino Acid Sequence↗