A single amino acid gates the KcsA channel

•pH-dependent gating of the KcsA channel is regulated by the CPD.•E146 is the most essential amino acid for pH sensing by the KcsA.•The protonated-mimicking mutant, E146Q, is constitutively open independent of pH.•Minimal rearrangement of the CPD is sufficient for opening of the KcsA. The KcsA channel is a proton-activated potassium channel. We have previously shown that the cytoplasmic domain (CPD) acts as a pH-sensor, and the charged states of certain negatively charged amino acids in the CPD play an important role in regulating the pH-dependent gating. Here, we demonstrate the KcsA channel is constitutively open independent of pH upon mutating E146 to a neutrally charged amino acid. In addition, we found that rearrangement of the CPD following this mutation was not large. Our results indicate that minimal rearrangement of the CPD, particularly around E146, is sufficient for opening of the KcsA channel.