Tryptophan substitution mutants on S5 largely retain normal force activation. (A, top) A diagram of the S5 sequence. (bottom) A helical belt diagram produced by DNASTAR showing the relative amino acid positions on S5. Residues, the replacements of which with tryptophan produced constitutive channel activities, are labeled with black dots. Those were replaced with alanine for further analyses. (B) Representative traces of wild type (WT) and 14 tryptophan mutants and 4 alanine mutants showing robust force responses to 150-mmHg pressure. Whereas the wild type was tested at 10⁻⁵ M Ca²⁺, the mutants were examined at different [Ca²⁺] to better demonstrate their responses to pressure from appropriate basal activities, as many of these mutants affect basal activities and channel kinetics like the insertion mutants in Figs. 3 and 5. (C) Quantifications of the mechanosensitivity of wild type and mutants in B as the nP_o fold increase in response to 150-mmHg pressure. No significant difference can be discerned by this measure. P > 0.05. For those mutants whose saturation level could be determined, Δα values are compared with wild type in Fig. 7. Means ± SD (n = 4 for wild type and n = 3 for each mutant).