Semisynthetic K⁺ channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state

C-type inactivation of K ⁺ channels plays a key role in modulating cellular excitability. During C-type inactivation, the selectivity filter of a K ⁺ channel changes conformation from a conductive to a nonconductive state. Crystal structures of the KcsA channel determined at low K ⁺ or in the open state revealed a constricted conformation of the selectivity filter, which was proposed to represent the C-type inactivated state. However, structural studies on other K ⁺ channels do not support the constricted conformation as the C-type inactivated state. In this study, we address whether the constricted conformation of the selectivity filter is in fact the C-type inactivated state. The constricted conformation can be blocked by substituting the first conserved glycine in the selectivity filter with the unnatural amino acid d -Alanine. Protein semisynthesis was used to introduce d -Alanine into the selectivity filters of the KcsA channel and the voltage-gated K ⁺ channel K ᵥAP. For semisynthesis of the K ᵥAP channel, we developed a modular approach in which chemical synthesis is limited to the selectivity filter whereas the rest of the protein is obtained by recombinant means. Using the semisynthetic KcsA and K ᵥAP channels, we show that blocking the constricted conformation of the selectivity filter does not prevent inactivation, which suggests that the constricted conformation is not the C-type inactivated state.