Methods to study CFTR protein in vitro.

Dale J Benos; Bakhrom K Berdiev; Iskander I Ismailov; Lynda S Ostedgaard; Ilana Kogan; Canhui Li; Mohabir Ramjeesingh; Christine E Bear

doi:10.1016/j.jcf.2004.05.018

PubMed · 15463933

Methods to study CFTR protein in vitro.

Dale J Benos·Bakhrom K Berdiev·Iskander I Ismailov·Lynda S Ostedgaard·Ilana Kogan·Canhui Li·Mohabir Ramjeesingh·Christine E Bear

View original source ↗Export metadata ↓

Abstract

CFTR is a cyclic AMP and nucleotide-related chloride-selective channel with a low unitary conductance. Many of the physiological roles of CFTR are effectively studied in intact cells and tissues. However, there are also several clear advantages to the application of cell-free technologies to the study of the biochemical and biophysical properties of CFTR. When expressed in heterologous cells, CFTR is processed relatively poorly, depending, however, on the cell-type analysed. In some cells, only 20-25% of the protein which is initially synthesized exits the endoplasmic reticulum to insert into the cell membrane [Cell 83 (1995) 121; EMBO J. 13 (1994) 6076]. Further, many of the disease-causing mutants of CFTR result in even lower processing efficiencies. Therefore, several procedures have been developed to study regulated CFTR channel function expressed in microsomal membranes and following its purification and reconstitution. These experimental approaches and their application are discussed here.

Explore related subjects

Cell Membrane Clinical Laboratory Techniques Cystic Fibrosis Transmembrane Conductance Regulator Humans In Vitro Techniques Membrane Proteins Journal Article Review

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Dale J Benos, Bakhrom K Berdiev, Iskander I Ismailov, Lynda S Ostedgaard, Ilana Kogan, Canhui Li, Mohabir Ramjeesingh, Christine E Bear. 2004. Methods to study CFTR protein in vitro.. https://doi.org/10.1016/j.jcf.2004.05.018

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2025

Time-resolved transcriptomics of S. cerevisiae and S. pastorianus in response to plasma membrane stresses.

Yeasts are beneficial microorganisms for human society and are utilized for academic and industrial purposes. For academic purposes, S. cerevisiae is a well-investigated model for studying eukaryotic cellular processes. For industrial purposes, S. pastorianus, which has a hybrid genome of S. cerevisiae and S. eubayanus, has been served for lager beer production. During fermentation, S. pastorianus produces ~7% of EtOH, which induces plasma membrane (PM)/cell wall stress in yeast. Therefore, S. pastorianus may experience PM stress and adapt to the self-forming environment during fermentation. However, how yeast adapts to PM stress remains unclear. Here, we investigated the temporal cellular responses of S. cerevisiae and S. pastorianus during adaptation to PM stresses by time-resolved mRNA-seq analysis. Our data showed different transcriptional phenotypes between S. cerevisiae and S. pastorianus during adaptation. The results may reflect the distinct nature of the two yeasts that have evolved in different nutritional environments. The dataset presented here would provide a promising resource for studying the characteristic nature of these differentially domesticated yeasts upon PM stresses.

Cell Membrane↗

PubMed2007

Fluorinated and hemifluorinated surfactants as alternatives to detergents for membrane protein cell-free synthesis.

Hemifluorinated and fluorinated surfactants are lipophobic and, as such, non-detergent. Although they do not solubilize biological membranes, they can, after conventional solubilization, substitute for detergents to keep membrane proteins soluble, which generally improves their stability [Breyton, Chabaud, Chaudier, Pucci and Popot (2004) FEBS Lett. 564, 312-318]. In the present study, we show that (hemi)fluorinated surfactants can be used for in vitro synthesis of membrane proteins: they do not interfere with protein synthesis, and they provide a suitable environment for MscL, a pentameric mechanosensitive channel, to fold and oligomerize to its native functional state. Following synthesis, both types of surfactants can be used to deliver MscL directly to pre-formed lipid vesicles. The electrophysiological activity of MscL synthesized in vitro in the presence of either hemi- or per-fluorinated surfactant is similar to that of the protein expressed in vivo.

Cell Membrane↗

PubMed2007

Human biliverdin reductase, a previously unknown activator of protein kinase C betaII.

Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) betaII, activated by insulin and free radicals (Miralem, T., Hu, Z., Torno, M. D., Lelli, K. M., and Maines, M. D. (2005) J. Biol. Chem. 280, 17084-17092; Lerner-Marmarosh, N., Shen, J., Torno, M. D., Kravets, A., Hu, Z., and Maines, M. D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7109-7114). Here, by using 293A cells co-transfected with pcDNA3-hBVR and PKC betaII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC betaII, but not the reductase and PKC zeta, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC betaII K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC betaII for myelin basic protein; activation was independent of phospholipids and extended to the phosphorylation of S2, a PKC-specific substrate. The increase in substrate phosphorylation was blocked by specific inhibitors of conventional PKCs and attenuated by sihBVR. The effect of the latter could be rescued by subsequent overexpression of hBVR. To a large extent, the activation was a function of the hBVR N-terminal chain of valines and intact ATP-binding site and the cysteine-rich C-terminal segment. The cobalt protoporphyrin-activated hBVR phosphorylated a threonine in a peptide corresponding to the Thr500 in the human PKC betaII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC betaII nor PKC zeta activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR.PKC betaII immunocomplex. The potential biological relevance of the hBVR activation of PKC betaII was suggested by the finding that in cells transfected with the PKC betaII, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC betaII, as well as in untransfected cells, upon treatment with phorbol myristate acetate, the PKC translocated to the plasma membrane and co-localized with hBVR. hBVR activation of PKC betaII underscores its potential function in propagation of signals relayed through PKCs.

Cell Membrane↗