Yimao Zhang Susan Michaelis and Jeffrey L Brodsky 1 Introduction

The most common cause of cystic fibrosis is the deletion of a phenylalanine at position 508 (AF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). Although the majority of wild-type CFTR is degraded in the endoplasmic reticulum (ER), suggesting that its folding efficiency is low, almost all of the AF508 variant is destroyed (1-4). The process in which the ER quality control system ensures that misfolded or aberrant proteins, such as AF508 CFTR, are proteolyzed and thus...

Sample Results

As a test of the impedance hardware and software we prepared an analog circuit with resistors and capacitors with known measured values. This circuit was connected to the equipment and an impedance spectrum was obtained. The impedance values were then fitted using BLIMP to obtain estimates of the resistances and capacitances. As shown in Fig. 1, the equipment and software gave excellent estimates of the known values and an excellent fit (solid line) of the impedance spectrum. From this Nyquist...

Limited Proteolysis of Isolated Microsomes Expressing Radioactively Labeled CFTR

To assess the conformation of transiently appearing folding intermediates in the cell (e.g., the core-glycosylated wt CFTR) by limited proteolysis, the intermediates have to be labeled radioactively before isolation of microsomes. Radioactive peptide fragments, generated by proteolytic cleavage of microsomes, are immunoprecipitated with anti-CFTR antibodies, separated by SDS-PAGE, and visualized by fluorography. This approach was successful in revealing the conformational transition between...

Immunolocalization Techniques

Most immunolocalization experiments are performed using the indirect method. That is, the primary antibody reacting with the antigen does not generate the immunolocalization signal. The signal is indirectly generated by conjugates linked to a secondary antibody directed against the primary antibody (Fig. 2). This approach provides greater flexibility since it avoids the need to conjugate the primary antibody and allows the secondary antibodies (containing the conjugates) to be used with a...

Assessing Recording Quality for Quantitative Analysis of CFTR Gating

Before performing analyses on recorded data, some care must be taken to assure uniform conditions and behavior of channel activity throughout the recording interval to be analyzed. In excised inside-out patch experiments, one critical quality control factor is controlling channel rundown. Rundown takes two forms in recordings of CFTR channel activity phosphorylation-dependent and phosphorylation-independent. Here, phosphorylation-dependent rundown is defined as reductions in channel activity...

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...