Biomedical subjects

Xiaoyan Ren

Publications and source records attributed to Xiaoyan Ren.

7 recordsLinked to original sources

PubMed2006

Fluorescent Na+-Ca+ exchangers: electrophysiological and optical characterization.

The cardiac sarcolemmal Na+-Ca2+ exchanger (NCX1) influences cardiac contractility by extruding Ca2+ from myocytes. As a Ca2+ efflux mechanism, the exchanger plays a prominent role in Ca2+ homeostasis. To track NCX1 and study changes in conformation, NCX1 was tagged with derivatives of green fluorescent protein. Cyan (CFP) and yellow (YFP) fluorescent proteins were used for both visualization of the protein in HEK cells and fluorescent resonance energy transfer (FRET). CFP or YFP was inserted at position 266, 371, 467, or 548 of the large intracellular loop of NCX1 located between transmembrane segments 5 and 6. These constructs were tested for functional activity and visualized for cell surface expression. All constructs were targeted to the plasma membrane. Transport properties were assessed by both 45Ca2+ uptake and electrophysiological measurements. The fluorescent-tagged exchangers had similar biophysical properties to the wild type NCX1. Unexpectedly, all constructs retain their sensitivity to regulation by cytoplasmic Na+ and Ca2+ ions. FRET analysis indicates the proximity of NCX1 to plasma membrane phosphatidylinositol 4,5-bisphosphate. These results indicate that insertion of CFP or YFP into the large intracellular loop of NCX1 protein does not impair exchanger properties. These constructs will be useful to further characterize the biological properties of the exchanger in intact cells.

Animals↗

PubMed2006

Mechanism of shortened action potential duration in Na+-Ca2+ exchanger knockout mice.

In cardiac-specific Na(+)-Ca(2+) exchanger (NCX) knockout (KO) mice, the ventricular action potential (AP) is shortened. The shortening of the AP, as well as a decrease of the L-type Ca(2+) current (I(Ca)), provides a critical mechanism for the maintenance of Ca(2+) homeostasis and contractility in the absence of NCX (Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na(+)-Ca(2+) exchanger knockout mice: reduced transsarcolemmal Ca(2+) flux. Circ Res 97: 1288-1295, 2005). To investigate the mechanism that underlies the accelerated AP repolarization, we recorded the transient outward current (I(to)) in patch-clamped myocytes isolated from wild-type (WT) and NCX KO mice. Peak I(to) was increased by 78% and decay kinetics were slowed in KO vs. WT. Consistent with increased I(to), ECGs from KO mice exhibited shortened QT intervals. Expression of the I(to)-generating K(+) channel subunit Kv4.2 and the K(+) channel interacting protein was increased in KO. We used a computer model of the murine AP (Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, and Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: 1378-1403, 2004) to determine the relative contributions of increased I(to), reduced I(Ca), and reduced NCX current (I(NCX)) on the shape and kinetics of the AP. Reduction of I(Ca) and elimination of I(NCX) had relatively small effects on the duration of the AP in the computer model. In contrast, AP repolarization was substantially accelerated when I(to) was increased in the computer model. Thus, the increase in I(to), and not the reduction of I(Ca) or I(NCX), is likely to be the major mechanism of AP shortening in KO myocytes. The upregulation of I(to) may comprise an important regulatory mechanism to limit Ca(2+) influx via a reduction of AP duration, thus preventing Ca(2+) overload in situations of reduced myocyte Ca(2+) extrusion capacity.

Action Potentials↗

PubMed2006

Helix packing of the cardiac Na+-Ca2+ exchanger: proximity of transmembrane segments 1, 2, and 6.

The cardiac Na+-Ca2+ exchanger (NCX1) is a membrane protein that extrudes Ca2+ from cells using the energy of the Na+ gradient and is a key protein in regulating intracellular Ca2+ and contractility. Based on the current topological model, NCX1 consists of nine transmembrane segments (TMSs). The N-terminal five TMSs are separated from the C-terminal four TMSs by a large intracellular loop. Cysteine 768 is modeled to be in TMS 6 close to the intracellular surface. In this study, the proximity of TMS 6 to TMSs 1 and 2 was examined. Insect High Five cells were transfected with cDNAs encoding mutant NCX1 proteins. Each mutant contained cysteine 768 and an introduced cysteine in TMS 1 or 2. Cross-linking between cysteines was determined after reaction with thiol-specific cross-linkers containing spacer arms of 6.5-12 A. The data indicate that residues in TMSs 1 and 2 are close to cysteine 768 in TMS 6. Cysteine 768 cross-linked with residues at both ends of TMSs 1 and 2 and is likely located toward the middle of TMS 6. Based on these results, we present an expanded helix-packing model for NCX1.

Animals↗

PubMed2005

Culinary archaeology: Millet noodles in Late Neolithic China.

Noodles have been a popular staple food in many parts of the world for at least 2,000 years, although it is debatable whether the Chinese, the Italians or the Arabs invented them first. Here we analyse a prehistoric sample of noodles contained in a well preserved, sealed earthenware bowl discovered in the Late Neolithic archaeological site of Lajia in northwestern China. We identify millet as the source of the abundant seed-husk phytoliths and starch grains present in the vessel. This shows that the conversion of ground millet flour into dough that could be repeatedly stretched into long, thin strands for the preparation of boiled noodles was already established in this region 4,000 years ago.

Agriculture↗

PubMed2004

Conformational defects slow Golgi exit, block oligomerization, and reduce raft affinity of caveolin-1 mutant proteins.

Caveolin-1, a structural protein of caveolae, is cleared unusually slowly from the Golgi apparatus during biosynthetic transport. Furthermore, several caveolin-1 mutant proteins accumulate in the Golgi apparatus. We examined this behavior further in this mutant study. Golgi accumulation probably resulted from loss of Golgi exit information, not exposure of cryptic retention signals, because several deletion mutants accumulated in the Golgi apparatus. Alterations throughout the protein caused Golgi accumulation. Thus, most probably acted indirectly, by affecting overall conformation, rather than by disrupting specific Golgi exit motifs. Consistent with this idea, almost all the Golgi-localized mutant proteins failed to oligomerize normally (even with an intact oligomerization domain), and they showed reduced raft affinity in an in vitro detergent-insolubility assay. A few mutant proteins formed unstable oligomers that migrated unusually slowly on blue native gels. Only one mutant protein, which lacked the first half of the N-terminal hydrophilic domain, accumulated in the Golgi apparatus despite normal oligomerization and raft association. These results suggested that transport of caveolin-1 through the Golgi apparatus is unusually difficult. The conformation of caveolin-1 may be optimized to overcome this difficulty, but remain very sensitive to mutation. Disrupting conformation can coordinately affect oligomerization, raft affinity, and Golgi exit of caveolin-1.

Amino Acid Sequence↗

PubMed2002

Placental alkaline phosphatase is efficiently targeted to rafts in supported lipid bilayers.

Evidence is growing that biological membranes contain lipid microdomains or "rafts" that may be involved in processes such as cellular signaling and protein trafficking. In this study, we have used atomic force microscopy to examine the behavior of rafts in supported lipid bilayers. We show that bilayers composed of equimolar dioleoylphosphatidylcholine and sphingomyelin spontaneously form rafts, which are detectable as raised features. A comparison of the extents of protrusion of the rafts in monolayers and bilayers indicates that the rafts in the two leaflets of the bilayer coincide. The rafts were observed both in the absence and presence of cholesterol (33 mol %). Cholesterol reduced raft protrusion presumably by increasing the thickness of the non-raft bilayer. PLAP (glycosylphosphatidylinositol-anchored protein placental alkaline phosphatase) was purified and shown to exist as a dimer. Following its incorporation into supported lipid bilayers, PLAP was found to be targeted efficiently to rafts, both in the absence and presence of cholesterol. We suggest that atomic force microscopy provides a powerful tool for the study of raft structure and properties.

Alkaline Phosphatase↗

PubMed2002

NCX1 surface expression: a tool to identify structural elements of functional importance.

The rat Na(+)/Ca(2+) exchanger isoforms of the NCX1 gene have 14 cysteine residues. Each of these cysteines can be mutated individually to alanine or serine without loss of functional expression in transfected HEK293 cells. Yet sequential exchange starting from the amino terminal end of three or more cysteines results in reduced transport activity and surface expression. As more and more cysteines are replaced, transport activity and surface expression decrease in parallel, and the cysteineless mutant exhibits only traces of Na(+)/Ca(2+) exchange activity and surface expression. No significant differences are detected in the amount of total cell exchanger protein between the wild-type exchanger and its functional or nonfunctional cysteine mutants. Reduced surface expression of the Na(+)/Ca(2+) exchanger NCX1 is also observed when HEK293 cells expressing the transporter are treated with cyclosporin A (CsA) or with PSC833. The reductions in transport activity and surface expression are concentration dependent and parallel. No reduction is obtained in the total amount of exchanger protein by CsA or PSC833 treatment, suggesting that the effects of these drugs on NCX1 expression are posttranslational. FK506 and rapamycin treatment of HEK293 cells expressing rat NCX1 isoforms has no effect on transport activity, surface expression, or the total amount of exchanger protein in the transfected cells.

Amino Acid Sequence↗