Membrane receptors transfection

It is possible that the water-filled a-LTX channel, which is relatively wide ( 10A at its narrowest (Krasilnikov and Sabirov 1992 Orlova et al. 2000), can pass small molecules. Indeed, a-LTX channels inserted in the membranes of synaptosomes, NMJ nerve terminals, and receptor-transfected COS7 cells appear to pass fluorescein (Stokes-Einstein radius, Re = 4.5 A) and norepinephrine (Re < 4 A) (Davletov et al. 1998 Rahman et al. 1999 Volynski et al. 2000), shown in Figure 2 for comparison with 8-hydrated calcium ion (Rc = 4.2 A) and the toxin channel. Analysis of impermeant cations commonly used in channel studies reveals that a-LTX channels are poorly permeable (Hurlbut et al. 1994) to glucosamine H+(Re = 4.6 A) and not significantly permeable (Tse and Tse 1999) to N-methyl-D-glucamine (Re = 5.2 A), thus limiting the pore diameter by 10 A. 

When the efficacy of biphalin-stimulated G protein activation was examined (Table 3) in delta opioid receptor-transfected CHO cells, an efficacy ratio of 0.42 was determined as compared with deltorphin-II and DPDPE, the latter a reference delta-selective agonist. Such low efficacy values suggest that biphalin does not efficiently stimulate the G protein through the delta receptor [9]. Relative affinities of biphalin and morphine for mu, delta, and kappa binding sites in guinea pig brain membranes are shown in Table 4. 

Fig. 4. Competition studies between the antagonists pirenzepine and tripitramine and the radioligand W-[ H]methylscopolamine (filled symbols) in membranes prepared from COS-7 cells co-transfected with M, -l-Mj-tail (a), M3 -l-Mj-trunc (b), M3 -l-M2(Asn404- Ser) (c) and M 3-short-I-Mj-trunc (d). The open symbols in panel a, b and c represent curves obtained in membranes from COS-7 cells separately transfected with M2 and M3-tail , M3 and Mj-trunc and M3 and M2(Asn404 Ser) , respectively. The open symbols in panel d represents the curve obtained with the Mj-short receptor transfected alone.

KEY EXPERIMENT TRANSFECTION OF MEMBRANE RECEPTORS WHAT ABOUT LIPIDS ... 

Figure 4.16 Bright field (a) and time-resolved luminescence (b delay lOOfis) of transfected living HEK cells expressing the tagged membrane receptor GABAB2 as revealed by [Eu(L29)f -conjugated antibodies. Reproduced with permission from [158], Copyright 2007, Elsevier...

FIGURE 5.4 Microphysiometry responses of HEK 293 cells transfected with human calcitonin receptor, (a) Use of microphysiometry to detect receptor expression. Before transfection with human calcitonin receptor cDNA, HEK cells do not respond to human calcitonin. After transfection, calcitonin produces a metabolic response, thereby indicating successful membrane expression of receptors, (b) Cumulative concentration-response curve to human calcitonin shown in real time. Calcitonin added at the arrows in concentrations of 0.01, 0.1, 1.10, and lOOnM. Dose-response curve for the effects seen in panel B. 

The process by which cells take up large molecules is called endocytosis. Some of these molecules (eg, polysaccharides, proteins, and polynucleotides), when hydrolyzed inside the cell, yield nutrients. Endocytosis provides a mechanism for regulating the content of certain membrane components, hormone receptors being a case in point. Endocytosis can be used to learn more about how cells function. DNA from one cell type can be used to transfect a different cell and alter the latter s function or phenotype. A specific gene is often employed in these experiments, and this provides a unique way to smdy and analyze the regulation of that gene. DNA transfection depends upon endocytosis endocy-... 

Another potential source of iron, at least for hepatocytes, is receptor-independent uptake of iron from transferrin. This appears to involve an iron uptake pathway from transferrin which is neither suppressed in hepatocytes by antibodies to TfR (Trinder et at, 1988), nor by transfection of HuH-7 hepatoma cells with transferrin receptor anti-sense cDNA (Trinder etat, 1996). The same pathway may also be utilized for iron uptake from isolated transferrin N-lobe, which is not recognized by the receptor (Thorstensen et at, 1995). The possible role of TfR2 in this process remains to be established, as does the physiological importance of this pathway in intact liver. Human melanoma cells (Richardson and Baker, 1994) and Chinese hamster cells lacking transferrin receptors but transfected with melanotransferrin (Kennard et at, 1995) use another pathway for transferrin iron uptake, independent of the transferrin receptor, but utilizing iron transfer from transferrin or simple iron chelates to membrane-anchored melanotransferrin, and from there onwards into the cellular interior. 

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