scholarly journals Characterization of the scrambling domain of the TMEM16 family

2017 ◽  
Vol 114 (24) ◽  
pp. 6274-6279 ◽  
Author(s):  
Sayuri Gyobu ◽  
Kenji Ishihara ◽  
Jun Suzuki ◽  
Katsumori Segawa ◽  
Shigekazu Nagata
Keyword(s):  
Plasma Membranes ◽  
Point Mutations ◽  
Transmembrane Segment ◽  
Stepping Stones ◽  
Transmembrane Segments ◽  
Outer Leaflet ◽  
Intracellular Ca ◽  
A Cell ◽  
Phospholipid Scrambling

The TMEM16 protein family has 10 members, each of which carries 10 transmembrane segments. TMEM16A and 16B are Ca2+-activated Cl− channels. Several other members, including TMEM16F, promote phospholipid scrambling between the inner and outer leaflets of a cell membrane in response to intracellular Ca2+. However, the mechanism by which TMEM16 proteins translocate phospholipids in plasma membranes remains elusive. Here we show that Ca2+-activated, TMEM16F-supported phospholipid scrambling proceeds at 4 °C. Similar to TMEM16F and 16E, seven TMEM16 family members were found to carry a domain (SCRD; scrambling domain) spanning the fourth and fifth transmembrane segments that conferred scrambling ability to TMEM16A. By introducing point mutations into TMEM16F, we found that a lysine in the fourth transmembrane segment of the SCRD as well as an arginine in the third and a glutamic acid in the sixth transmembrane segment were important for exposing phosphatidylserine from the inner to the outer leaflet. However, their role in internalizing phospholipids was limited. Our results suggest that TMEM16 provides a cleft containing hydrophilic “stepping stones” for the outward translocation of phospholipids.

Functional characterization of the transmembrane segment VII of the NHE1 isoform of the Na+/H+ exchangerThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

2007 ◽  
Vol 85 (3-4) ◽  
pp. 319-325 ◽  
Author(s):  
Jie Ding ◽  
Raymond W.P. Ng ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
70Th Birthday ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Transmembrane Segment ◽  
Transmembrane Segments ◽  
Pore Lining ◽  
Positively Charged ◽  
Selection Of

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH. It extrudes 1 intracellular H+ in exchange for 1 extracellular Na+. It has 2 large domains, an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the critical transmembrane segment TM VII. Residues Leu 255, Leu 258, Glu 262, Leu 265, Asn 266, Asp 267, Val 269, Val 272, and Leu 273 were all mutated to cysteine residues in the cysteineless NHE1 isoform. Mutation of amino acids E262, N266, and D267 caused severe defects in activity and targeting of the intact full length protein. The balance of the active mutants were examined for sensitivity to the sulfhydryl reactive reagents, positively charged MTSET ((2- (trimethylammonium)ethyl)methanethiosulfonate) and negatively charged MTSES ((2-sulfonatoethyl)methanethiosulfonate). Leu 255 and Leu 258 were sensitive to MTSET but not to MTSES. The results suggest that these amino acids are pore-lining residues. We present a model of TM VII that shows that residues Leu 255, Leu 258, Glu 262, Asn 266, and Asp 267 lie near the same face of TM VII, lining the ion transduction pore.

Na+/Ca2+ Exchange in Plasma Membranes and Microsomal Fraction of Brain Cortex

1985 ◽  
Vol 43 ◽  
pp. 490-491
Author(s):  
O.P. Coutinho ◽  
W.N. Norton ◽  
C.A.M. Carvalho ◽  
A.P. Carvalho
Keyword(s):  
Plasma Membranes ◽  
Brain Cortex ◽  
Microsomal Fraction ◽  
Primary Objective ◽  
Synaptic Plasma Membranes ◽  
Intracellular Ca ◽  
Sheep Brain ◽  
Gradient Separation ◽  
Density Gradient Separation

The Na+/Ca2+ exchange system is one of the mechanisms responsible for regulating intracellular Ca2+ concentrations in neurons. However, the precise mechanism of Na+/Ca2+ exchange among synaptic plasma membranes and microsomes of neurons has yet to be resolved. The primary objective of the present investigation was to determine the specific transport properties with regard to Na+/Ca2+ exchange among neural synaptic plasma membranes and the various fractions of a microsomal density gradient separation.Synaptic plasma membranes were obtained by hypotonic disruption of sheep brain cortex synaptosomes (Fig. 1). Enzymatic characterization of the synaptic plasma membranes revealed high Na+/K+ ATPase activity and low RNA content, which suggest that the material is predominantly of plasma membrane origin. The most common constituents consisted of membrane fragments and resealed vesicles (Fig. 2).

scholarly journals The Marginal Cells of the Caenorhabditis elegans Pharynx Scavenge Cholesterol and Other Hydrophobic Small Molecules

2019 ◽  
Author(s):  
Muntasir Kamal ◽  
Houtan Moshiri ◽  
Lilia Magomedova ◽  
Duhyun Han ◽  
Ken CQ Nguyen ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Small Molecules ◽  
Plasma Membranes ◽  
Bacterial Suspension ◽  
Filter Feeder ◽  
C Elegans ◽  
Sphingomyelin Synthase ◽  
Outer Leaflet ◽  
Marginal Cells

AbstractThe nematode worm Caenorhabditis elegans is a bacterivore filter feeder. Through the contraction of the worm’s pharynx, a bacterial suspension is sucked into the pharynx’s lumen. Excess liquid is then shunted out of the buccal cavity through ancillary channels that are made from specialized pharyngeal cells called marginal cells. Through the characterization of our library of worm-bioactive small molecules (a.k.a. wactives), we found that more than one third of wactives visibly accumulate inside of the marginal cells as crystals or globular spheres. Wactives that visibly accumulate are typically more hydrophobic than those that do not. To understand why wactives accumulate specifically in marginal cells, we performed a forward genetic screen for mutants that resist the lethality associated with one crystallizing wactive. We identified a presumptive sphingomyelin-synthesis pathway that is necessary for crystal and sphere accumulation. Sphingomyelin is a phospholipid that is enriched in the outer leaflet of the plasma membranes of most metazoans. We find that the predicted terminal enzyme of this pathway, sphingomyelin synthase 5 (SMS-5), is expressed in the pharynx, contributes to sphingomyelin abundance, and that its expression in marginal cells is sufficient for wactive accumulation. We also find that the expression of SMS-5 in the marginal cells is necessary for the proper absorption of exogenous cholesterol, without which C. elegans cannot develop. We conclude that the sphingomyelin-rich plasma membrane of the marginal cells acts as a sink to scavenge important hydrophobic nutrients from the filtered liquid that might otherwise be shunted back into the environment.One sentence summaryThe anterior pharynx of C. elegans is a Sink for Hydrophobic Small Molecules

scholarly journals Characterization of Transmembrane Segments 3, 4, and 5 of MalF by Mutational Analysis

2001 ◽  
Vol 183 (1) ◽  
pp. 375-381 ◽  
Author(s):  
Angelika Steinke ◽  
Sandra Grau ◽  
Amy Davidson ◽  
Eckhard Hofmann ◽  
Michael Ehrmann
Keyword(s):  
Structural Model ◽  
Cytoplasmic Membrane ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Transport Channel ◽  
Transmembrane Segments ◽  
Maltose Transporter ◽  
Membrane Spanning ◽  
Membrane Components

ABSTRACT MalF and MalG are the cytoplasmic membrane components of the binding protein-dependent ATP binding cassette maltose transporter inEscherichia coli. They are thought to form the transport channel and are thus of critical importance for the mechanism of transport. To study the contributions of individual transmembrane segments of MalF, we isolated 27 point mutations in membrane-spanning segments 3, 4, and 5. These data complement a previous study, which described the mutagenesis of membrane-spanning segments 6, 7, and 8. While most of the isolated mutations appear to cause assembly defects, L323Q in helix 5 could interfere more directly with substrate specificity. The phenotypes and locations of the mutations are consistent with a previously postulated structural model of MalF.

Mutations affecting transmembrane segment interactions impair adhesiveness of E-cadherin

1999 ◽  
Vol 112 (23) ◽  
pp. 4415-4423 ◽  
Author(s):  
O. Huber ◽  
R. Kemler ◽  
D. Langosch
Keyword(s):  
Cell Adhesion ◽  
Self Assembly ◽  
Transmembrane Domain ◽  
Point Mutations ◽  
Cell Surface Expression ◽  
Transmembrane Segment ◽  
Reporter System ◽  
Transmembrane Segments ◽  
E Cadherin ◽  
Cell Cell

Lateral clustering of E-cadherin molecules is required for the adhesive properties of this cell-cell adhesion molecule. Both the extracellular domain and the cytoplasmic region of E-cadherin were previously reported to contribute to lateral clustering, but little is known about a role of the transmembrane domain in this respect. Following our previous findings indicating self-assembly of artificial transmembrane segments based on leucine residues, we asked whether the leucine-rich transmembrane segment of E-cadherin participates in lateral clustering. Here, we demonstrate that its transmembrane domain self-assembles as analyzed using the ToxR reporter system. Certain point mutations within the transmembrane domain markedly reduced self-assembly. To study whether the same point mutations also affect E-cadherin-mediated adhesion in vivo, wild-type and mutant E-cadherin cDNAs were transfected into Ltk(-) cells. Indeed, cell aggregation assays revealed significantly reduced adhesiveness when mutations had been introduced which disrupted transmembrane segment interaction. In control experiments, cell-surface expression, interaction with catenins and the cytoskeleton as well as trypsin-resistance of the protein were unaffected. These data suggest that interactions between the transmembrane segments are important for the lateral association of E-cadherin molecules required for cell-cell adhesion.

scholarly journals Biophysical Characterization of Endotoxin Inactivation by NK-2, an Antimicrobial Peptide Derived from Mammalian NK-Lysin

2004 ◽  
Vol 48 (5) ◽  
pp. 1593-1599 ◽  
Author(s):  
Jörg Andrä ◽  
Michel H. J. Koch ◽  
Rainer Bartels ◽  
Klaus Brandenburg
Keyword(s):  
Antimicrobial Peptide ◽  
Lipid A ◽  
Model Organisms ◽  
Amino Acid Residues ◽  
Biophysical Characterization ◽  
Biological Assays ◽  
Outer Leaflet ◽  
A Cell ◽  
Sugar Chains

ABSTRACT NK-2, a membrane-acting antimicrobial peptide, was derived from the cationic core region of porcine NK-lysin and consists of 27 amino acid residues. It adopts an amphipathic, α-helical secondary structure and has been shown to interact specifically with membranes of negatively charged lipids. We therefore investigated the interaction of NK-2 with lipopolysaccharide (LPS), the main, highly anionic component of the outer leaflet of the outer membrane of gram-negative bacteria, by means of biophysical and biological assays. As model organisms and a source of LPS, we used Salmonella enterica strains with various lengths of the LPS carbohydrate moiety, including smooth LPS, rough LPS, and deep rough LPS (LPS Re) mutant strains. NK-2 binds to LPS Re with a high affinity and induces a change in the endotoxin-lipid A aggregate structure from a cubic or unilamellar structure to a multilamellar one. This structural change, in concert with a significant overcompensation of the negative charges of LPS, is thought to result in the neutralization of the endotoxic LPS activity in a cell culture system. Neutralization of LPS activity by NK-2 as well as its antibacterial activity against the various Salmonella strains strongly depends on the length of the sugar chains of LPS, with LPS Re being the most sensitive. This suggests that a hydrophobic peptide-LPS interaction is necessary for efficient neutralization of the biological activity of LPS and that the long carbohydrate chains, besides their function as a barrier for hydrophobic drugs, also serve as a trap for polycationic substances.

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