Average time constant of the OFF sensing current of different mutants of Ci-VSP. (A) The OFF sensing current recorded from the PBM mutants (open symbols) R253A-R254A-C363S (n = 5), R253K-R254K-C363S (n = 4), R245Q-R246Q-C363S (n = 4), and R245A-R246A (n = 4) were significantly faster than those recorded from the C363S mutant (filled squares; n = 5) for all potentials tested above +20 mV (HP = −60 mV). (B) The mutant R245Q-R246Q C363S displayed slower OFF sensing current than those from the mutant R253A-R254A-C363S for pulses above +100 mV.