Structure–function studies of the pore mutations. (a) Pore diameter of the WT (PDB ID 7QKO: closed and 7QL6: open) and the 9′S mutant structures of AChRs using the HOLE program. The pore is the narrowest at the level of 16′, followed by the 9′ positions (z = 0 Å). In 7QL6, the pore is ∼4 Å wider at 9′ position than in C (highlighted by a brown arrow). With the increasing number of L9′S mutants, the pore diameter progressively increases. (b) Comparison of pore diameters of 7QKO (closed), 7QL6 (open), 6UWZ (α-bungarotoxin bound non-conductive state), 4AQ5 (apparently closed), and 4AQ9 (apparently open) AChR structures. (c) Comparison of constitutive opening probability (Po) with increase in the number of L9′S mutations in the AChR pore. Representative single channel current traces (right) from AChRs with aL9′S (upper) and abL9′S mutations (lower) and the corresponding dwell-time distribution histograms (left) fitted with exponential pdfs (spline curves) are shown. Note the shift in the mean shut and open times (marked by arrows) and the increase in frequency of channel openings. (d) Effect of ACh on the gate mutants which show constitutive channel activities in the absence of any agonists. The conditions and [ACh] are displayed in the figures. (e) Simulation of single channel and macroscopic synaptic currents from WT and αL251A mutant AChRs at saturating [ACh] (1 mM). Synaptic currents simulations were performed in the QuB software suite using 1,000 molecules of AChRs, 1 mM ACh (cyan rectangular pulse), and a simple C↔AC↔AO kinetic model. (f) Upper: Scatter plots showing correlation of DDG₀ versus DDG^transfer in kcal.mol⁻¹ for L9′/V13′ (each symbol is a different mutation). DG^transfer values from Radzicka et al. (1988). Lower: The above DDG₀ versus DDG^transfer values replotted by correcting the DG^transfer values with amino acid head group volumes (Sharp et al, 1991). The R² values for the linear regression lines = 0.75 and 0.87, respectively; dotted lines, 95% confidence limit.