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Bottleneck Project
The goal of this project is to understand how ions go through the CFTR channel, especially the narrowest part of the pore (bottleneck) and design mutations that might increase the single channel conductance. Engineered CFTR with such mutations will be useful in the future mRNA therapy for cystic fibrosis (CF) patients. We investigate ion flow throught the bottleneck by measuring the relative permeabilities and conductances of chloride, bromide, iodide, nitrate and thiocyanate ions.
Currently, we are trying to finish the Bottleneck Project experiments with L102C and R104C CFTR. Anion substitution experiments tell us how easily different anions can get into the pore of CFTR (relative permeabilities estimated by reversal potentials, see the protocol and video) and how easily they get through the bottleneck, the narrowest part of the pore. The latter is expressed as relative conductances (see the protocol). In these aspects, do we see any difference between WT, L102C and R104C CFTR? Do L102 and R104C play any role in the conduction of chloride ions through the CFTR channel? GlyH-101 experiments tell us the pharmacodynamics of this drug candidate for secretory diarrhea. GlyH-101 blocks the pore of CFTR from the extracellular side. It does not get through the CFTR channel. Does either mutagenesis (L102C or R104C) affect the binding of GlyH-101 to CFTR? The voltage-dependence of GlyH-101 block needs to be analyzed using SigmaPlot (see the protocol)
Current information for CFTR Bottleneck. This includes experiment overview of Anion Substitution and NaSCN Blockade, paper outline, references and template, as well as experiments.
L102C CFTR
L102C CFTR
Wang et al (2011) used an inside-out patch from BHK cells to show that L102C CFTR in V510A/cysless background reacts with MTSES and MTSET. They provided evidence that MTSES reacts with L102C in the open state but not in the closed state. Interestingly, L102C CFTR conductance increased with MTSET but decreased dramatically with MTSES. These charge-dependent and state-dependent effects were also observed by Gao et al (2013). They used single-channel recordings to demonstrate that the covalent modification of L102C with MTSET locks the CFTR channel into the open state with the observed open probability being close to one even in the absence of ATP. They also showed that MTSET modification decreases the single-channel current by about 46%. This suggests that MTSET modification partially blocks the pore of CFTR but locks the channel into the open state. The observations from these studies should make sense to you once you look at cryo-EM structures of the activated (6MSM) and dephosphorylated (5UAK) human CFTR. What do you think about the relative energy states of the charged chemical group attached to L102C by MTSET in the open and closed state of CFTR
R104C CFTR
R104C CFTR
R104C CFTR
R104C CFTR
Please list and summarize relevant papers.
R104C CFTR
Cui et al (2014) made single channel recordings with oocytes expressing CFTR with two engineered cysteines (R104C/E116C) and observed that the channel was locked in the open state. When DTT was applied from the extracellular side, the R104C/E116C CFTR channel closed, suggesting that C104 and C116 spontaneously form a disulfide bond and lock the channel in the open state. Cui et al (2014) observed true channel closures in single channel recordings of R104C/E116C CFTR in the absence of DTT, and ATP and PKA were required for activation of R104C/E116C CFTR, suggesting the disulfide bond between 104 and 116 is broken when the NBDs separate and TMDs form the closed state. It can also be locked into the closed state with the M2M molecular linker.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4502298/
https://www.pnas.org/content/115/50/12757
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