Interpretation of Banding Patterns

Membrane lipids can be separated by high-performance thin-layer chromatography (HP-TLC) as phospholipids, free fatty acids, monoglycerides, diglycerides, triglycerides, and cholesterol esters (4). Analysis of the total lipid extract by HP-TLC allows the determination of whether cell lipids are intact or hydrolyzed. The presence of hydrolyzed lipids in a sample, e.g., phospholipids, does not allow the analysis of phospholipid-bound fatty acids. Fatty acid methyl esters prepared following...

Solubilization and Purification of CFTR Protein Using Pentadecafluorooctanoic Acid PFO

Purification of membrane proteins requires their dissociation from interacting cellular proteins and membrane phospholipids using compatible detergents. The membrane protein of interest can then be purified from the mixed protein-detergent micelles by virtue of distinctive physicochemical properties or specific affinity with an immobilized ligand. CFTR is a very hydrophobic membrane protein and is poorly soluble in most detergents except the strong ionic detergent sodium dodecyl sulfate (SDS)...

Marybeth Howard and William J Welch 1 Introduction

Defects in protein folding constitute the basis of many genetic diseases cystic fibrosis, alpha-1 antitrypsin deficiency, familial hypercholesterolemia, and congenital nephrogenic diabetes insipidus, to name but a few (see Table 1 for a complete list). In each of these, point mutations or deletions result in a protein product that fails to achieve its properly folded state. For example, in the case of the cystic fibrosis transmembrane conductance regular protein (CFTR), the most common mutation...

CFTR Mutation Detection by Multiplex Heteroduplex mHET Analysis on MDE

Julian Zielenski, Isabel Aznarez, Tuncer Onay, John Tzounzouris, Danuta Markiewicz, and Lap-Chee Tsui Mutation detection in an integral part of disease diagnosis and patient study. For most Mendelian diseases, multiple mutations may be found in a single gene among a patient population. The type of mutations may vary from large deletions to single-base-pair (bp) substitutions, and different diseases may have different predominant types. For example, large deletions are often found in Duchenne...

Using Channel Mutants Drugs and Other Probes to Dissect CFTR Gating

Another powerful tool for dissecting CFTR gating is the use of nucle-otide and phosphate analogs. As mentioned above, the use of the nonhydrolyzable ATP analog, AMPPNP, and the NBD2 mutant K1250A, implicated the involvement of ATP hydrolysis in channel opening and closing and established the presence of two functional sites for ATP (see above). These results from few channel patches were corroborated by macroscopic relaxation experiments using rapid solution changes. Switching from AMPPNP to...

Future Directions Conclusion

Based on growing experience from in vitro gene transfer experiments, as well as in vivo studies in animal models and in Phase I and II human clinical trials, gene therapy may someday become a viable method for treating some of the clinical manifestations of cystic fibrosis. However, before this option will be feasible, substantial improvements and modifications in the technology may be necessary. In the case of adenoviral vectors, strategies that increase the efficiency and duration of viral...

Garnering Gating Information from Pre SteadyState Current Relaxations

As discussed above, single-channel studies are one way to obtain kinetic constants for channel gating. These derived rate constants can then be used to construct different models of CFTR's gating mechanism. One major drawback of the single-channel approach is that long recordings of channel activity are required to collect enough events for accurate kinetic analyses of single channels, especially if the opening and closing rates (i.e., long bursts or interburst intervals) are extremely slow....

Cftr

That oocytes and insects cells are incubated at lower temperatures, Denning et al. (6) examined the effect of incubating mammalian cells expressing AF508 CFTR at 26 C instead of 37 C. Now correct folding of the AF508 CFTR mutant was observed. Moreover, and like the situation with the insects cells and oocytes, cAMP-regulated chloride transport was observed in those cells expressing the mutant protein. Thus, we now know that the loss of a single phenylalanine residue at position 508 within the...

Generation of Mice of the Appropriate Genotype

The objective of the breeding program is to generate second generation pups that carry at least one copy of the SV40 transgene and are either non-CF (CFTR + +, or + -) or CF (CFTR - -). A male ImmortoMouse is placed with three female mice heterzygous for the S489X CFTR mutation in a microisolator cage. Multiple breeding cages are established to increase the number of first-generation offspring. When the female mice become pregnant, they are transferred to individual cages and are replaced with...

Cell Culture and Expansion

Ca2+- and Mg2+-free Hanks balanced salt solution that contains 0.05 trypsin and 0.53 mM EDTA (Gibco-BRL). 3. Collecting tubule culture media. 1 1 mix of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium (GIBCO-BRL) supplemented with 1.3 yg L sodium selenite, 1.3 yg L 3,3',5-triiodo-L-thyronine, 5 mg L insulin, 5 mg L transferrin, 25 yg L prostaglandin E1, 2.5 mM glutamine, 5 nM dexamethasone, 50000 U L nystatin, 50 mg L streptomycin, 30 mg L...

Mmzzm npe atp

Comparison of CFTR activation by rapid solution exchange and by caged ATP photolysis. (A) Trace shows activation of CFTR channels by rapid introduction of ATP, then by specific decaging of ATP-P3- 1-(2-nitrophenyl)ethyl ester (NPE-ATP). NPE-ATP does not induce activity until photolyzed by UV irradiation. Upward deflections indicate channel openings. Thin bars indicate rapid perfusion of ATP, filled bars perfusion of NPE-ATP, and stippled bars interruption of continuous perfusion....

Reconstitution of CFTR Protein into Liposomes from PFO

To study the structure and function of a purified membrane protein, it is essential that the detergent in which the protein is purified be exchanged for phospholipids. This exchange is a time- and concentration-dependent process. Typically, phospholipids are added to detergent-solubilized proteins and then the detergent is removed by dialysis or detergent adsorption resins 24,25 . Only when the detergent concentration decreases below a critical value critical micelle concentration or CMC can...