Biomedical subjects

Y Lu

Publications and source records attributed to Y Lu.

At least 487 records · Page 27Linked to original sources

PubMed1999

[Synthesis and spectroscopic characterization of O,O,O',O'-tetramethyl-S,S'-(2-N,N-dimethylamino-trimethylene)-bis-phosphonodithionate oxalate].

A new organophosphorus insecticide, shacanlin, was synthesized by 1-dimethylamino-2,3-dichloropropane reacting on O,O-dimethyl-dithiophosphate and its oxalate was prepared. The molecular structure of shacanlin oxalate was characterized by 1H and 13C NMR spectrometry, IR and MS. The results indicate that the reaction product is 1,3-disubstituted product. The molecular structure of shacanlin oxalate is (CH3)2N+ HCH[CH2SP(S)(OCH3)2]2 x C2O4H-[O,O,O',O'-tetramethyl-S,S'-(2-N,N-dimethylamino-trimethylene)-bis phosphonodithionate oxalate].

Magnetic Resonance Spectroscopy↗

PubMed1998

The ligamento-muscular stabilizing system of the spine.

STUDY DESIGN: Electrical and mechanical stimulation of the lumbar supraspinous ligament of three patients with L4-L5 spinal deficits and of the feline model, respectively, was applied while recording electromyography on the multifidus muscles. OBJECTIVES: To determine if mechanoreceptors in the human spine can reflexively recruit muscle force to stabilize the lumbar spine, and to demonstrate, in the feline model, that such ligamento-muscular synergy is elicited by mechanical deformation of the lumbar supraspinous ligament (and possibly of other spinal ligaments), the facet joint capsule, and the disc. SUMMARY OF BACKGROUND DATA: The literature repeatedly confirms that ligaments have only a minor mechanical role in maintaining spine stability, and that muscular co-contraction of anterior and posterior muscles is the major stabilizing mechanism of the spine. The literature also points out that various sensory receptors are present in spinal ligaments, and that the ligaments are innervated by spinal and autonomic nerves. Data that describe how ligaments and muscles interact to provide stability to the spine were not found. METHODS: The supraspinous ligament at L2-L3 and L3-L4 was electrically stimulated in three patients undergoing surgery to correct deficits at L4-L5. Electromyography was performed from the multifidus muscles at L2-L3 and L3-L4, bilaterally. In 12 cats, the supraspinous ligaments from L1-L2 to L6-L7 were mechanically deformed, sequentially, while electromyography was performed from the multifidus muscles of the six levels. Loading of the ligament was applied before and after each of the two vertebrae were externally fixed to prevent motion. RESULTS: Electromyograms were recorded from the multifidus muscles, bilaterally, in the two of the three patients, demonstrating a direct relationship to receptors in the supraspinous ligament. Electromyograms were recorded from the feline multifidus muscle with mechanical loading of the supraspinal ligament at each of the L1-L2 to L6-L7 motion segments. In the free-spine condition the largest electromyographic discharge was present in the level of ligament deformation, and lower electromyographic discharge was recorded in two rostral and caudal segments. After immobilizing any two vertebrae, loading of the ligament resulted in electromyographic discharge in the muscles of the same level and at least one level above and/or below. CONCLUSIONS: Deformation or stress in the supraspinous ligament, and possibly in other spinal ligaments, recruits multifidus muscle force to stiffen one to three lumbar motion segments and prevent instability. Strong muscular activity is seen when loads that can cause permanent damage to the ligament are applied, indicating that spastic muscle activity and possibly pain can be caused by ligament overloading.

Animals↗

PubMed1998

Adenoviral transgene expression of MMAC/PTEN in human glioma cells inhibits Akt activation and induces anoikis.

The MMAC/PTEN tumor suppressor gene encodes for a phosphatase that recently has been shown to have phosphotidylinositol phosphatase activity, implicating its possible involvement in phosphatidylinositol 3'-kinase-mediated signaling. To investigate possible alterations in growth factor-mediated signal transduction, an adenovirus containing MMAC/PTEN, Ad-MMAC, previously shown to inhibit growth and tumorigenicity in glioma cells, was used to acutely express the transgene. Human glioma cells infected with Ad-MMAC but not with control adenoviruses exhibited an inhibition of phosphorylation of both activating residues of Akt, Ser-473, and Thr-308, along with Akt's serine/threonine kinase activity, without significantly altering Akt expression. The effects of functional MMAC/PTEN expression were relatively specific, because members of several other growth factor-mediated signaling pathways showed no altered responses. The presence of MMAC/PTEN also inhibited phosphorylation of BAD, although no evidence of apoptosis in the in situ treated cells was observed. However, U251 glioma cells infected with Ad-MMAC were induced to undergo anoikis at a significantly higher rate than U251 cels treated with control viruses or mock infected with media. These results demonstrate that the acute administration of MMAC/PTEN results in the inhibition of Akt-mediated signaling, growth inhibition, and anoikis, implying that loss of MMAC/PTEN increases cellular proliferation and significantly augments a cell's survival potential during cellular processes that are associated with malignancy.

Adenoviridae↗

PubMed1998

Phosphatidylinositol 3-kinase is required for CD28 but not CD3 regulation of the TEC family tyrosine kinase EMT/ITK/TSK: functional and physical interaction of EMT with phosphatidylinositol 3-kinase.

Ligation of the TCR or CD28 induces activation of phosphatidylinositol 3-kinase (PI3K), the TEC family protein tyrosine kinase, EMT/ITK/TSK (EMT), and the SRC family tyrosine kinase, LCK. LCK is required for the activation and phosphorylation of EMT induced by ligation of the TCR or CD28 placing LCK upstream of EMT in T cell signaling cascades. We report herein that inhibition of PI3K activity with the specific inhibitors LY294002 and wortmannin markedly decreased EMT activation induced by CD28 cross-linking but not by CD3 cross-linking. Further, inhibition of PI3K markedly decreased EMT in vitro autokinase activity induced by activated LCK. In contrast, PI3K inhibitors did not alter CD28 or CD3 cross-linking or LCK-induced EMT phosphorylation. Consistent with the requirement of PI3K activity for CD28 but not CD3-induced stimulation of the EMT in vitro autokinase activity, a small but significant portion of cellular EMT associates with PI3K following CD28 cross-linking but not following CD3 cross-linking. CD28-induced association of EMT with PI3K also requires functional expression of LCK. Fusion proteins containing the SRC homology 2 domain of EMT interact with PI3K or a PI3K-associated molecule in a tyrosine phosphorylation-dependent manner. Taken together, the data suggest that EMT is differentially regulated and recruited to different signaling complexes following ligation of CD28 or the TCR complex, perhaps contributing to the disparate roles that EMT appears to play downstream of CD28 and the TCR.

Animals↗

PubMed1998

FTIR and UV-Vis Spectroscopic Studies of Black Soap Film.

FTIR and UV-vis spectroscopies have been employed to investigate the self-assembly features of a black soap film (BSF) prepared from aqueous solution consisting of 10(-2) M cetyltrimethylammonium bromide (CTAB) and 10(-3) M thiazole yellow (TY) in the liquid-crystalline and gel states, respectively. The construction of two surfactant monolayers of the soap film is independent of the thickness of aqueous core, and the thickness is approximately 1.7 nm at equilibrium. In the liquid-crystalline state, the methylene segments of the hydrocarbon chains are averagely oriented at an angle of 70 degrees with respect to the film normal, and the alkyl chains take a long-range interaction to improve the film elasticity. The TY molecules in the film are horizontally aligned at the monolayer interface in the J-aggregate form. The double-negatively charged TY aggregate greatly enhances the film stability. Dye neutral red is further introduced into the CTAB soap film, the film lifetime is related to the type of the added dyes, in the order of cationic < nonionic < anionic. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Myoblast city, the Drosophila homolog of DOCK180/CED-5, is required in a Rac signaling pathway utilized for multiple developmental processes.

The Rac and Cdc42 GTPases share several regulators and effectors, yet perform distinct biological functions. The factors determining such specificity in vivo have not been identified. In a mutational screen in Drosophila to identify Rac-specific signaling components, we isolated 11 alleles of myoblast city (mbc). mbc mutant embryos exhibit defects in dorsal closure, myogenesis, and neural development. DOCK180, the mammalian homolog of Mbc, associates with Rac, but not Cdc42, in a nucleotide-independent manner. These results suggest that Mbc is a specific upstream regulator of Rac activity that mediates several morphogenetic processes in Drosophila embryogenesis.

Animals↗

PubMed1998

Rhesus macaques that become systemically infected with pathogenic SHIV 89.6-PD after intravenous, rectal, or vaginal inoculation and fail to make an antiviral antibody response rapidly develop AIDS.

A new simian-human immunodeficiency virus (SHIV) stock (SHIV 89.6-PD), derived from plasma of a rhesus macaque used for in vivo serial passage of virulence-attenuated SHIV 89.6, produces systemic infection after intravenous, intravaginal, or intrarectal inoculation of rhesus macaques. Infection with this virus results in high levels of viral antigen in plasma, a precipitous decline in CD4+ T-cell counts, and a disease syndrome that is characteristic of AIDS. Rapid progression to disease was associated with failure to seroconvert to viral antigens, whereas longer survival was associated with production of antiviral antibodies. In intravenously inoculated animals, peak antigenemia occurred at 7 days postinjection (PI) and severe CD4+ depletion occurred at 14 days PI. In mucosally infected animals, peak antigenemia occurred at 14 days PI and severe CD4+ depletion was not evident until 21 days PI. The 1-week delay in both viral antigenemia and CD4+ T-cell decline in mucosally infected animals is consistent with the hypothesis that, following vaginal inoculation, virus dissemination proceeds in a stepwise manner from the mucosal surface to the draining lymph nodes and subsequently to the bloodstream. This animal model can be used to test the ability of HIV-1 envelope-based vaccines to prevent infection or disease after challenge by the three major routes of HIV transmission.

Acquired Immunodeficiency Syndrome↗

PubMed1998

The human homologue for the Caenorhabditis elegans cul-4 gene is amplified and overexpressed in primary breast cancers.

Amplification is a key mechanism whereby a cancer cell increases the message level of genes that confer a selective advantage when they are overexpressed. In breast cancer, there are many chromosome regions present in multiple copies relative to overall DNA copy number (amplicons), and their target genes are unknown. Using differential display, we have cloned and sequenced the full coding region of a candidate amplicon target gene located on chromosome 13. This candidate is the human homologue of the Caenorhabditis elegans cul-4 gene, cul-4A, a member of the novel cullin gene family, which is involved in cell cycle control of C. elegans. cul-4A was amplified and overexpressed in 3 of 14 breast cancer cell lines analyzed, and it was overexpressed in 8 additional cell lines in which it was not amplified. The latter observation, indicating that its overexpression can occur by mechanisms other than gene amplification, suggests that cul-4A plays a key role in carcinogenesis. Moreover, cul-4A was found to be amplified in 17 of 105 (16%) cases of untreated primary breast cancers, and 14 of 30 cases analyzed (47%) were shown by RNA in situ hybridization to overexpress cul-4A. These results suggest that up-regulation of cul-4A may play an important role in tumor progression.

Amino Acid Sequence↗

PubMed1998

Effects of the diuretic agent indapamide on Na+, transient outward, and delayed rectifier currents in canine atrial myocytes.

The diuretic agent indapamide has been reported to block the slow component of the delayed rectifier K+ current (IKs) without altering the rapid component (IKr) or the inward rectifier current and has been used as a pharmacological probe for IKs; however, the effects of indapamide on Na+ (INa), L-type Ca2+ (ICa), and transient outward K+ (Ito) currents have not been determined. We applied tight-seal, whole-cell, patch-clamp techniques to assess the effects of indapamide on INa, Ito, ICa, and IKs in canine atrial myocytes. Indapamide inhibited INa, Ito, and IKs in a concentration-dependent and reversible way, without altering ICa. Block increased with depolarization, with the 50% blocking concentration (EC50) decreasing from 129 +/- 26 micromol/L (at -60 mV) to 79 +/- 17 micromol/L (at -10 mV) for INa, from 174 +/- 19 micromol/L (at + 10 mV) to 98 +/- 7 micromol/L (at +60 mV) for Ito and from 148 +/- 28 micromol/L (at +10 mV) to 86 +/- 18 micromol/L (at +60 mV) for IKs. Significant inhibition was seen at concentrations as low as 10 micromol/L for all 3 currents. In addition, indapamide effectively inhibited the ultrarapid delayed rectifier current in a voltage-independent way, with an EC50 of 138 +/- 7 micromol/L at +10 mV. Standard microelectrode experiments showed the effects of indapamide on the action potential to be consistent with the ionic actions seen. We conclude that in addition to its well-recognized IKs-blocking action, indapamide also inhibits INa and Ito effectively and with similar potency. Thus, indapamide is not a reliable pharmacological probe with which to study the specific effects of IKs blockade, and INa and Ito block may contribute to the potential profile of cardiac actions of the compound.

Action Potentials↗

PubMed1998

Benzo[a]pyrene enhances the formation of 8-hydroxy-2'-deoxyguanosine by ultraviolet A radiation in calf thymus DNA and human epidermoid carcinoma cells.

The objective of this study is to investigate if benzo[a]pyrene (BaP) and ultraviolet (UV) radiation synergistically induce oxidative DNA damage. Calf thymus DNA was incubated with BaP and irradiated with UVB (280-320 nm) and UVA (335-400 nm). BaP substantially enhanced the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) by UVA, but only moderately increased the level of 8-OHdG by UVB. Formation of 8-OHdG proportionally correlated with both UV dose and BaP concentration. Human epidermoid carcinoma cells were incubated with 10 microg of BaP/mL for 24 h and then exposed to 10 kJ/m2 UVB and 25 kJ/m2 UVA. UVB plus BaP did not affect the level of 8-OHdG in cultured cells, whereas UVA plus BaP substantially increased 8-OHdG by over 4-fold compared to BaP and UVA controls. To confirm what reactive oxygen species (ROS) are involved in BaP plus UVA-induced oxidative DNA damage, less or more specific ROS quenchers were added to DNA solution. The results showed that only superoxide dismutase and genistein significantly quenched BaP plus UVA-induced 8-OHdG, whereas catalase, sodium azide, and mannitol exhibited no effect. Our studies suggest that BaP enhances the formation of 8-OHdG in purified DNA and cultured cells by UVA, but not by UVB, and that superoxide anion plays an important role in the synergistic induction of oxidative DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1998

Sensory cells determine afferent terminal morphology in cross-innervated electroreceptor organs: implications for hair cells.

Type I and type II hair cells of the vestibular system are innervated by afferents that form calyceal and bouton terminals, respectively. These cannot be experimentally cross-innervated in the inner ear to determine how they influence each other. However, analogous organs are accessible for transplantation and cross-innervation in the brown ghost electric fish. These fish possess three types of electroreceptor organs. Of these, the sensory receptors of the type I tuberous organ are S-100- and parvalbumin-positive with a calbindin-positive afferent that forms a large calyx around the organ. Neither the sensory receptors nor the afferents of the ampullary organs label with these antibodies, and the afferent branches form a single large bouton beneath each receptor cell. In controls, when cut ampullary afferents reinnervate transplanted ampullary organs, they have characteristic calbindin-negative terminals with large boutons. When type I tuberous afferents reinnervate ampullary organs, receptor cells remain S-100- and parvalbumin-negative, and the tuberous afferents still express calbindin. The nerve terminals, however, make large ampullary-like boutons on the receptor cells. These results suggest that (1) afferent terminal morphology is dictated by the receptor organ; (2) expression of calbindin by the afferent is not suppressed by innervation of the incorrect end organ; (3) ampullary organs generate ampullary receptor cells although innervated by tuberous afferents; and (4) ampullary receptor cells can be trophically supported by tuberous afferents.

Animals↗

PubMed1998

Antisense c-myc retroviral vector suppresses established human prostate cancer.

Prostate cancer eventually becomes androgen resistant, resumes growth, and kills the patient. Characterization of genetic events that lead to androgen refractory prostatic neoplasia has revealed the frequent overexpression of c-myc and uncontrolled prostate cancer proliferation. A novel strategy to combat advanced prostate cancer utilized a replication incompetent retrovirus that contained the mouse mammary tumor virus (MMTV) promoter within the retroviral vector to allow transcription of antisense c-myc gene within target prostate tumor cells. The transduction of cultured DU145 cells by XM6:MMTV-antisense c-myc RNA retrovirus did not affect cell proliferation in culture, yet a single direct injection of MMTV-antisense c-myc viral media into established DU145 tumors in nude mice produced a 94.5% reduction in tumor size compared to tumors treated with control virus MTMV sense fos and untreated tumor by 70 days. Two animals in the antisense c-myc-treated group had complete regression of their tumors. Histopathological examination of the tumors revealed that MMTV-antisense c-myc-transduced DU145 tumors had increased tumor cell differentiation, decreased invasion, and a marked stromal response. The mechanism for the antitumor effect of MMTV-antisense c-myc retrovirus appears to be suppression of c-myc mRNA and protein, and decreased bcl-2 protein. The in vivo transduction of prostate cancer cells with MMTV-antisense c-myc retroviruses reduced tumor growth by suppressing c-myc, resulting in the down-regulation of bcl-2 protein. Consequently, the MMTV-antisense c-myc retrovirus may be useful for gene therapy against advanced, hormone-refractory prostate cancer.

Animals↗

PubMed1998

Arginine residues in the C-terminus of HIV-1 Vpr are important for nuclear localization and cell cycle arrest.

HIV-1 viral protein R (Vpr) is predominantly localized to the nucleus and plays an important role for viral preintegration complex import into the nucleus. In this study, we investigated the influence on subcellular localization of Arg residues in the C-terminus of Vpr. Consistent with previous studies, about 90% of the cells manifested diffuse nuclear staining in the Vpr-expressed cells. Besides diffuse nuclear staining, punctate perinuclear staining, and punctate cytoplasmic staining were also observed in the immunofluorescence studies. Deletion of the Ser-Arg-lle-Gly residues (amino acids 79-82; SRIG) had no effect on the Vpr localization. However, deletion of the Arg-Gln-Arg-Arg residues (amino acids 85-88; RQRR) resulted in a smooth perinuclear staining pattern. Substitution of five Arg residues with Asn (amino acids 80, 85, 87, 88, and 90; R-->N5) resulted in a diffuse cytoplasmic staining. Subcellular fractionation analyses support the immunofluorescence staining results. These findings indicate that the C-terminal Arg residues of HIV-1 Vpr play an important role for Vpr nuclear localization. All the Vpr mutants were appropriately expressed, exhibited no significant defect on the protein stability, and were incorporated efficiently into virus-like particles. Both SRIG and R-->N5 mutants lost their cell cycle arrest activities and the RQRR deletion only exhibited a low level of cell arrest activity. Therefore, the Arg residues in the HIV-1 Vpr C-terminus are important for Vpr nuclear localization and cell cycle arrest, but had no effect on protein stability or Vpr incorporation into virus-like particles.

Amino Acid Sequence↗

PubMed1998

Efficient CD28 signalling leads to increases in the kinase activities of the TEC family tyrosine kinase EMT/ITK/TSK and the SRC family tyrosine kinase LCK.

Optimal T cell activation requires crosslinking of the T cell receptor (TCR) concurrently with an accessory receptor, most efficiently CD28. Crosslinking of CD28 leads to increased interleukin 2 (IL2) production, inhibition of anergy and prevention of programmed cell death. Crosslinking of CD28 leads to rapid increases in tyrosine phosphorylation of specific intracellular substrates including CD28 itself. Since CD28 does not encode an intrinsic tyrosine kinase domain, CD28 must activate an intracellular tyrosine kinase(s). Indeed, crosslinking of CD28 increases the activity of the intracellular tyrosine kinases EMT/ITK and LCK. The phosphatidylinositol 3-kinase (PI3K) and GRB2 binding site in CD28 is dispensable for optimal IL2 production in Jurkat T cells. We demonstrate herein that murine Y170 (equivalent to human Y173) in CD28 is also dispensable for activation of the SRC family tyrosine kinase LCK and the TEC family tyrosine kinase EMT/ITK. In contrast, the distal three tyrosines in CD28 are required for optimal IL2 production as well as for optimal activation of the LCK and EMT/ITK tyrosine kinases. The distal three tyrosines of CD28, however, are not required for recruitment of PI3K to CD28. Furthermore, PI3K is recruited to CD28 in JCaM1 cells which lack LCK and in which EMT/ITK is not activated by ligation of CD28. Thus optimal activation of LCK or EMT/ITK is not obligatory for recruitment of PI3K to CD28 and thus is also not required for tyrosine phosphorylation of the YMNM motif in CD28. Taken together the data indicate that the distal three tyrosines in CD28 are integral to the activation of LCK and EMT/ITK and for subsequent IL2 production.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Mutated p21(WAF1/CIP1/SDI1) lacking CDK-inhibitory activity fails to prevent apoptosis in human colorectal carcinoma cells.

Human colorectal tumor cell lines were established which express wildtype p21 or p21 with a mutation at codon 46 (Cys) or 140 (Gly) on IPTG treatment (LacSwitch). The IPTG-induced wildtype p21 bound to CDK2 and PCNA and inhibited CDK activity in the cells and reduced cell growth rate; whereas, both IPTG-induced mutated p21 proteins neither bound to CDK2 nor affected the CDK activity but did bind to PCNA, and they did not affect the cell growth rate. Wildtype p21 suppressed apoptosis and enhanced survival of X-ray-irradiated or adriamycin-treated cells; but, mutated p21 neither suppressed apoptosis nor affected cell survival. When cells were treated with mimosine, a p53-independent p21-inducer, or butyrolactone I, a specific inhibitor of CDK, cellular endogenous p21 was induced and X-ray or adriamycin-induced apoptosis was blocked. These results suggest that CDK-binding or CDK-inhibitory activity of p21 is required to prevent apoptosis, i.e., CDK is required for apoptosis in human tumor cells.

4-Butyrolactone↗

PubMed1998

Transcriptional activation of the p34cdc2 gene by cdc2 promoter binding factor/nuclear factor-Y in fetal rat ventricular myocytes.

To determine how myocardial terminal differentiation is regulated by cell cycle control genes, we studied cdc2 expression in rat cardiac muscle and found that cdc2 mRNA and protein levels were reduced in neonatal compared with fetal ventricles and became undetectable in juvenile and adult ventricles. To further determine whether cdc2 downregulation is attributed to a decrease in transcription, transient expression assay was performed using the progressively truncated 6.2-, 1.8-, 1.1-, 0.7-, and 0.1-kb human cdc2 5' flanking regions. All five fragments activated reporter expression in fetal myocytes and were significantly less active in neonatal myocytes. The 0.1-kb fragment showed 65% of the activity of the 6.2-kb fragment. A protein binding site that contains an inverted CCAAT box was identified within the 0.1-kb fragment by DNase I footprint assay and named the cdc2 promoter binding factor (CPBF) site. Point mutations within the CPBF site that abolish CPBF binding significantly decreased both 0.1- and 6.2-kb promoter activities. Competition and antibody supershift assays suggested that CPBF was identical or related to the transcription factor, nuclear factor Y (NF-Y). The 0.1-kb promoter activity was suppressed by a dominant-negative NF-Y mutant in fetal myocytes. Taken together, our results demonstrate that cardiac cdc2 expression is downregulated after birth and turned off when the juvenile stage is attained. A 0.1-kb promoter fragment of cdc2 contains major information for both cdc2 transcriptional activation and suppression in fetal and neonatal myocytes, respectively. NF-Y or its related factor plays a critical role in activating the 0.1-kb cdc2 promoter.

Animals↗

PubMed1998

Mechanosensitivity of the cardiac muscarinic potassium channel. A novel property conferred by Kir3.4 subunit.

Muscarinic potassium channels are heterotetramers of Kir3.1 and other Kir3 channel subunits and play major roles in regulating membrane excitability in cardiac atrial, neuronal, and neuroendocrine tissues. We report here that rabbit atrial muscarinic potassium channels are rapidly and reversibly inhibited by membrane stretch, possibly serving as a mechanoelectrical feedback pathway. To probe the molecular basis for this phenomenon, we heterologously expressed heteromeric Kir3.1/Kir3.4 channels in Xenopus oocytes and found that they possess similar mechanosensitivity in response to hypo-osmolar stress. This could be attributed in part, if not exclusively, to the Kir3.4 subunit, which reproduced the mechanosensitivity of the heteromeric channel when expressed as a homomeric channel in oocytes. Kir3.4 is the first stretch-inactivated potassium channel to be identified molecularly. Physiologically, this feature may be important in atrial volume-sensing and other responses to stretch.

Animals↗

PubMed1998

Topology of a functionally important region of the cardiac Na+/Ca2+ exchanger.

The cardiac Na+/Ca2+ exchanger, NCX1, has been modeled to consist of 11 transmembrane segments and a large cytoplasmic loop (loop f). Cysteine mutagenesis and sulfhydryl modification experiments demonstrate that the loop connecting transmembrane segments 1 and 2 (loop b) is located on the cytoplasmic side of the membrane, as previously modeled. A mutation in loop b, asparagine 101 to cysteine (N101C), renders the exchanger insensitive to regulation by cytoplasmic Na+ and Ca2+. Nearby mutations at residue threonine 103 (T103C or T103V) increase the apparent affinity of the exchanger for cytoplasmic Na+ and also produce a significant Li+ transport capacity. The evidence suggests that the region at the interface of cytoplasmic loop b and transmembrane segment 2 is important in Na+ transport and also in secondary regulation. Thus, this region may form part of the link between the ion translocation pathway formed by the transmembrane segments and regulatory sites that have previously been localized to loop f.

Animals↗