CFTR Background Information

CFTR Bottleneck Background Papers (with notes)

CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel and a member of ATP binding cassette (ABC) transporter superfamily. The protein is composed of 2 transmembrane domains (that form the anion conducting pore across the cell membrane), 2 nucleotide binding domains (that form two ATP binding sites in their interface) and a regulatory domain (R domain). The open state of the transmembrane domains (TMDs) is coupled to the dimerized state of the nucleotide binding domains (NBDs).

ATP binding pocket 1 (ABP1) forms between the head (Walker A and B motifs) of NBD1 and the tail (signature sequence, LSHGH) of NBD2 while ABP2 is composed of the head of NBD2 and the tail (LSGGQ) of NBD1. Only ABP2 is catalytically competent for ATP hydrolysis. When the NBDs are kept dimerized with ATP molecules bound in ABP1 and 2, the open state of TMDs is thermodynamically favored. The NBDs partially separate when ATP is hydrolyzed at ABP2. This causes the closed state of TMDs to be thermodynamically favored.

The regulatory domain prevents the NBDs from dimerizing when dephosphorylated. The cAMP-dependent protein kinase, PKA phosphorylates the R domain phosphorylation sites when there is an increase in the intracellular concentration of cAMP. When Seibert et al. (1999) mutated 15 phosphorylation sites to alanine, the response of CFTR to PKA was abolished. Ten of these sites (S422 in the N-terminus of NBD1, S660, S686, S700, S712, S737, S768, T788, S795, S813 in the R domain) are dibasic consensus sites for phosphorylation by PKA. Four sites (S670, T690, S753, T787) are monobasic sites while S790 is surrounded by positive residues. McClure et al (2012) expressed CFTR in HEK293S cells and observed phosphorylation at T717, S1444 and S1456 as well as S660, S686, S700, S712, S737, S795 using mass spectrometry.

ABPs. PNG

CFTR Bottleneck

Negoda, A., El Hiani, Y., Cowley, E. A., & Linsdell, P. (2017). Contribution of a leucine residue in the first transmembrane segment to the selectivity filter region in the CFTR chloride channel. Biochimica et Biophysica Acta - Biomembranes, 1859(5), 1049–1058. https://doi.org/10.1016/j.bbamem.2017.02.014

Gao, X., Bai, Y., & Hwang, T. C. (2013). Cysteine scanning of CFTR’s first transmembrane segment reveals its plausible roles in gating and permeation. Biophysical Journal, 104(4), 786–797. https://doi.org/10.1016/j.bpj.2012.12.048

Dawson, D. C., Liu, X., Zhang, Z.-R., & McCarty, N. A. (2003). Anion Conduction by {CFTR}: Mechanisms and Models. The Cystic Fibrosis Transmembrane Conductance Regulator, 1–34.

Mansoura, M. K., Smith, S. S., Choi, A. D., Richards, N. W., Strong, T. V, Drumm, M. L., Collins, F. S., & Dawson, D. C. (1998). Anion Binding as a Probe of the Pore. Biophysical Journal, 74(March), 1320–1332.

Lynch, J. W. (2004). Molecular structure and function of the glycine receptor chloride channel. Physiological Reviews, 84(4), 1051–1095. https://doi.org/10.1152/physrev.00042.2003

Jun, I., Cheng, M. H., Sim, E., Jung, J., Suh, B. L., Kim, Y., Son, H., Park, K., Kim, C. H., Yoon, J. H., Whitcomb, D. C., Bahar, I., & Lee, M. G. (2016). Pore dilatation increases the bicarbonate permeability of CFTR, ANO1 and glycine receptor anion channels. Journal of Physiology, 594(11), 2929–2955. https://doi.org/10.1113/JP271311

CFTR True Open

As of 7/9/2022, the "true open" position of CFTR has yet to be determined. Since the "true open" position can be used to aid in drug development and the treatment of cystic fibrosis, it is important that it is determined. CFTR has at least 2 known methods of opening, one is after phosphorylation , and there is a way for CFTR to open in its inactive conformation. This second opening is most likely has a reduced flow of ions across the membrane compared to that of the "true open" conformation. Some CFTR agonists such as ivacaftor and GLPC1837 can artificially open CFTR. CFTR has also been artificially opened by substituting cysteines in at positions _____________. The current theory in the lab is that a disulfide bridge is formed somewhere within the CFTR which stabilizes the open conformation.

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