Dual-color quantum dot detection of a heterotetrameric potassium channel (hKCa3.1)

2011 ◽  
Vol 300 (4) ◽  
pp. C843-C849 ◽  
Author(s):  
Daniel E. J. Waschk ◽  
Anke Fabian ◽  
Thomas Budde ◽  
Albrecht Schwab
Keyword(s):  
Quantum Dots ◽  
Plasma Membrane ◽  
Membrane Potential ◽  
Cell Membrane ◽  
Quantum Dot ◽  
Fluorescence Microscopy ◽  
Potassium Channel ◽  
Binomial Distribution ◽  
Hek293 Cells ◽  
F Cells

Potassium channels play a key role in establishing the cell membrane potential and are expressed ubiquitously. Today, more than 70 mammalian K+ channel genes are known. The diversity of K+ channels is further increased by the fact that different K+ channel family members may assemble to form heterotetramers. We present a method based on fluorescence microscopy to determine the subunit composition of a tetrameric K+ channel. We generated artificial “heteromers” of the K+ channel hKCa3.1 by coexpressing two differently tagged hKCa3.1 constructs containing either an extracellular hemagglutinin (HA) or an intracellular V5 epitope. hKCa3.1 channel subunits were detected in the plasma membrane of MDCK-F cells or HEK293 cells by labeling the extra- and intracellular epitopes with differently colored quantum dots (QDs). As previously shown for the extracellular part of hKCa3.1 channels, its intracellular domain can also bind only one QD label at a time. When both channel subunits were coexpressed, 27.5 ± 1.8% and 24.9 ± 2.1% were homotetramers consisting of HA- and V5-tagged subunits, respectively. 47.6 ± 3.2% of the channels were heteromeric and composed of both subunits. The frequency distribution of HA- and V5-tagged homo- and heteromeric hKCa3.1 channels is reminiscent of the binomial distribution ( a + b)2 = a2 + 2 ab + b2. Along these lines, our findings are consistent with the notion that hKCa3.1 channels are assembled from two homomeric dimers and not randomly from four independent subunits. We anticipate that our technique will be applicable to other heteromeric membrane proteins, too.

scholarly journals Plasma Membrane Potential of Candida albicans Measured by Di-4-ANEPPS Fluorescence Depends on Growth Phase and Regulatory Factors

2019 ◽  
Vol 7 (4) ◽  
pp. 110 ◽  
Author(s):  
Suchodolski ◽  
Krasowska
Keyword(s):  
Candida Albicans ◽  
Plasma Membrane ◽  
Membrane Potential ◽  
Cell Membrane ◽  
Growth Phase ◽  
Fungal Pathogen ◽  
Physiological State ◽  
External Factors ◽  
Good Tool ◽  
Opportunistic Fungal Pathogen

The potential of the plasma membrane (Δѱ) regulates the electrochemical potential between the outer and inner sides of cell membranes. The opportunistic fungal pathogen, Candida albicans, regulates the membrane potential in response to environmental conditions, as well as the physiological state of the cell. Here we demonstrate a new method for detection of cell membrane depolarization/permeabilization in C. albicans using the potentiometric zwitterionic dye di-4-ANEPPS. Di-4-ANEPPS measures the changes in the cell Δѱ depending on the phases of growth and external factors regulating Δѱ, such as potassium or calcium chlorides, amiodarone or DM-11 (inhibitor of H+-ATPase). We also demonstrated that di-4-ANEPPS is a good tool for fast measurement of the influence of amphipathic compounds on Δѱ.

A heteromeric potassium channel involved in the modulation of the plasma membrane potential is essential for the survival of African trypanosomes

2015 ◽  
Vol 29 (8) ◽  
pp. 3228-3237 ◽  
Author(s):  
Michael E. Steinmann ◽  
Amaia González-Salgado ◽  
Peter Bütikofer ◽  
Pascal Mäser ◽  
Erwin Sigel
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Potassium Channel ◽  
Plasma Membrane Potential ◽  
African Trypanosomes

scholarly journals nanoPaint: Dynamic Imaging of Nanoscopic Structural Plasticity of the Plasma Membrane

2018 ◽  
Author(s):  
Mariana Tasso ◽  
Thomas Pons ◽  
Nicolas Lequeux ◽  
Julie Nguyen ◽  
Zsolt Lenkei ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Plasma Membrane ◽  
Cell Membrane ◽  
Time Scale ◽  
Membrane Surface ◽  
Structural Plasticity ◽  
Cb1 Receptor ◽  
Single Particle Tracking ◽  
Dynamic Imaging ◽  
Surface Topology

Single-particle tracking with quantum dots (QDs) constitutes a powerful tool to track the nanoscopic dynamics of individual cell membrane components unveiling their membrane diffusion characteristics. Here we tested the possibility of extracting from the nano-resolved (16 ms and 30 nm) population dynamics of several quantum dots, time-binned at the second time-scale, the rapid structural changes of the cell membrane surface. We used for this proof-of-concept study bright, small and stable biofunctional QD nanoconstructs recognizing the neuronal cannabinoid type 1 (CB1) receptor and a commercial point-localization microscope to reconstruct in 3D the dynamics of the plasma membrane surface of cultured cells with a spatial resolution of tens of nanometers. CB1 receptor was chosen because it’s a highly expressed and fast diffusing membrane protein. Therefore, rapid QD diffusion on the axonal plasma membrane of cultured hippocampal neurons allowed highly precise reconstruction of the membrane surface in less than one minute. QD nanoconstructs diffused into the membrane of synaptic clefts allowing the entire topological reconstruction of the presynaptic component. In addition, we demonstrated successful reconstruction of the remarkably high dynamics of membrane surface topology at the second time-scale both in HEK-293 cell filopodia and axons. Our results show that this novel technique, which we named nanoPaint, is a powerful precision tool for the study of the structural plasticity of cell membrane surfaces.

scholarly journals Caveolin-1 directly interacts with UT-A1 urea transporter: the role of caveolae/lipid rafts in UT-A1 regulation at the cell membrane

2009 ◽  
Vol 296 (6) ◽  
pp. F1514-F1520 ◽  
Author(s):  
Xiuyan Feng ◽  
Haidong Huang ◽  
Yuan Yang ◽  
Otto Fröhlich ◽  
Janet D. Klein ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Lipid Rafts ◽  
Hek293 Cells ◽  
Cell Fractionation ◽  
Urea Transporter ◽  
Cell Plasma Membrane ◽  
Caveolin 1 ◽  
Cell Plasma

The cell plasma membrane contains specialized microdomains called lipid rafts which contain high amounts of sphingolipids and cholesterol. Lipid rafts are involved in a number of membrane protein functions. The urea transporter UT-A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. In this study, we investigated the possible role of lipid rafts in UT-A1 membrane regulation. Using sucrose gradient cell fractionation, we demonstrated that UT-A1 is concentrated in the caveolae-rich fraction both in stably expressing UT-A1 HEK293 cells and in freshly isolated kidney IMCD suspensions. In these gradients, UT-A1 at the cell plasma membrane is codistributed with caveolin-1, a major component of caveolae. The colocalization of UT-A1 in lipid rafts/caveolae was further confirmed in isolated caveolae from UT-A1-HEK293 cells. The direct association of UT-A1 and caveolin-1 was identified by immunoprecipitation and GST pull-down assay. Examination of internalized UT-A1 in pEGFP-UT-A1 transfected HEK293 cells fluorescent overlap with labeled cholera toxin subunit B, a marker of the caveolae-mediated endocytosis pathway. Disruption of lipid rafts by methyl-β-cyclodextrin or knocking down caveolin-1 by small-interference RNA resulted in UT-A1 cell membrane accumulation. Functionally, overexpression of caveolin-1 in oocytes decreased UT-A1 urea transport activity and UT-A1 cell surface expression. Our results indicate that lipid rafts/caveolae participate in UT-A1 membrane regulation and this effect is mediated via a direct interaction of caveolin-1 with UT-A1.

Extracellularly applied ruthenium red and cADP ribose elevate cytosolic Ca2+ in isolated rat osteoclasts

1996 ◽  
Vol 270 (3) ◽  
pp. F469-F475 ◽  
Author(s):  
O. A. Adebanjo ◽  
V. S. Shankar ◽  
M. Pazianas ◽  
B. J. Simon ◽  
F. A. Lai ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Cell Membrane ◽  
Cell Surface ◽  
Divalent Cation ◽  
Ruthenium Red ◽  
The Face ◽  
A Cell ◽  
Cation Sensing ◽  
Isolated Rat

We demonstrated recently that the divalent cation-sensing receptor on the osteoclast, the Ca2+ receptor (CaR), is a functional component of a cell surface-expressed ryanodine receptor-like molecule (RyR). The objective of the present study was to further characterize this putative RyR by use of the two well-known cell-impermeant RyR modulators, ruthenium red and adenosine 3',5'-cyclic diphosphate ribose (cADPr). We found that, when applied extracellularly, ruthenium red (5 x 10(-8)-10(-4) M) and cADPr (5 x 10(-6) M) triggered an elevation of cytosolic [Ca2+]. Depolarization of the cell membrane by the application of 0.1 M K+ in the presence of 5 x 10(-6) M. valinomycin resulted in a concentration-dependent increase in the magnitude of the cytosolic Ca2+ response to extracellular ruthenium red (5 x 10(-9) and 5 x 10(-5) M), a phenomenon that was not seen when osteoclasts were hyperpolarized using 5 x 10(-3) M K+ with 5 x 10(-6) M valinomycin. In the presence of an intact nonleaky cell membrane, these results would favor a plasma membrane locus of action for the two modulators. Furthermore, pretreatment of osteoclasts with either modulator resulted in a markedly attenuated cytosolic Ca2+ transient elicited in response to the CaR agonist Ni2+, thus confirming an interaction between the cADPr- and ruthenium red-sensitive sites and the osteoclast CaR. The inhibition of the cytosolic Ca2+ response to Ni2+ induced by ruthenium red remained unchanged in the face of membrane potential changes. Finally, the cytosolic Ca2+ response to caffeine (5 x 10(-4) M), another RyR modulator, was also strongly attenuated by pretreatment with 5 x 10(-9) M ruthenium red. We conclude that ruthenium red and cADPr act on plasma membrane-resident sites and that both these sites interact with the process of divalent cation sensing.

Glutathione-capped quantum dots for plasma membrane labeling and membrane potential imaging

Nano Research ◽  
2019 ◽  
Vol 12 (6) ◽  
pp. 1321-1326 ◽  
Author(s):  
Guangcun Chen ◽  
Yejun Zhang ◽  
Zhao Peng ◽  
Dehua Huang ◽  
Chunyan Li ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Plasma Membrane ◽  
Membrane Potential ◽  
Membrane Labeling

scholarly journals Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry

Zygote ◽  
2015 ◽  
Vol 24 (4) ◽  
pp. 529-536 ◽  
Author(s):  
Chihiro Kanno ◽  
Sung-Sik Kang ◽  
Yasuyuki Kitade ◽  
Yojiro Yanagawa ◽  
Yoshiyuki Takahashi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Membrane Potential ◽  
Fluorescence Microscopy ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Plasma Membrane Integrity ◽  
Bovine Spermatozoa ◽  
Acrosomal Integrity

SummaryThe present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen–thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE–PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE–PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE–PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE–PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously.

Enhanced cell membrane enrichment and subsequent cellular internalization of quantum dots via cell surface engineering: illuminating plasma membranes with quantum dots

2016 ◽  
Vol 4 (5) ◽  
pp. 834-843 ◽  
Author(s):  
Hong-Yin Wang ◽  
Xian-Wu Hua ◽  
Hao-Ran Jia ◽  
Peidang Liu ◽  
Ning Gu ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Plasma Membrane ◽  
Cell Membrane ◽  
Cell Surface ◽  
Hydrophobic Interaction ◽  
Plasma Membranes ◽  
Surface Engineering ◽  
Cellular Internalization ◽  
Cell Surface Engineering

Through hydrophobic interaction-based cell surface engineering, enhanced plasma membrane enrichment and subsequent cellular internalization of quantum dots were achieved.

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