Blocking Kinetics of CFTR Channel by Aromatic Carboxylic Acid Positional Isomers Characterised using a Novel Amplitude Distribution Analysis Method

To investigate the pore structure of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, we performed a systematic pore probing on CFTR channel pore with a series of small aromatic carboxylic acids, including their positional isomers, e.g., 9-anthracene carboxylic acid (9-AC) and 1-anthracene carboxylic acid (1-AC).Small compounds presumably interacting the channel protein with a few points are sensitive to structural changes of the binding site. However such low affinity blockers show fast - intermediate blocking kinetics which give us the overall affinity, but not on- and off- rates separately. To overcome this problem, we developed an iterative simulation method to estimate the on- and off- rate constants in the 9-AC or 1-AC block from the single channel amplitude distribution.The newly developed Amplitude Distribution Analysis (ADA) program first generated a single-channel current according to the given kinetic scheme and added a Gaussian noise to the currents for mimicking the background noise. The simulated currents were low-pass filtered and digitized at the same frequencies as those in the experiments and binned into an amplitude histogram. Then the program repeats a direct likelihood comparison between the simulated and experimental current amplitude distributions to find the best fitted values for the blocking kinetic parameters.The ADA program showed that the off-rate of 1-AC block is 3-fold slower than that of 9-AC and the on-rate of 1-AC is ∼3-fold faster than that of 9-AC. The voltage-dependences of on- and off- rates of 1-AC are similar to those of 9-AC, respectively. These suggest that 1-AC and 9-AC block CFTR channel by binding to a common binding site which should be modeled by a combination of a positive charge tightly surrounded by hydrophobic residues.