Adaptation and inactivation of MscS under strong depolarizing voltages is obliterated by negatively charged substitutions for G168. Responses of a 3-s step-pulse pressure protocol activate the entire population of channels in an excised membrane patch. This protocol was repeated at varying voltages between ±20 mV and ±120 mV to demonstrate the voltage-dependent adaptation and inactivation of WT MscS at −80 and −100 mV. A substitution of alanine at residue 168 does not cause a phenotypic divergence from WT MscS in terms of voltage-dependent adaptation. However, this voltage sensitivity is completely lost in G168D, whereas G168E shows little adaptation.