Potassium channels are responsible for the selective permeation of K+ ions across cell membranes. K+ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose 4 K+ binding sites. Here, we report 2D IR spectra of a semisynthetic KcsA channel with site-specific 13C18O isotope labels in the selectivity filter. The ultrafast time-resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by MD simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites.

Cyclic nucleotide-gated (CNG) ion channels were first discovered in rod photoreceptors, where they are responsible for the primary electrical signal of the photoreceptor in response to light. CNG channels are highly specialized membrane proteins that open an ion-permeable pore across the membrane in response to the direct binding of intracellular cyclic nucleotides. CNG channels have been identified in a number of other tissues, including the brain, where their roles are only beginning to be appreciated. Recently, significant progress has been made in understanding the molecular mechanisms underlying their functional specializations. From these studies, a picture is beginning to emerge for how the binding of cyclic nucleotide is transduced into the opening of the pore and how this allosteric transition is modulated by various physiological effectors.

Cyclic nucleotide-gated (CNG) channels are activated in response to the direct binding of cyclic nucleotides to an intracellular domain. This domain is thought to contain a beta roll and two alpha helices, designated the B and C helices. To probe the conformational changes occurring in the ligand-binding domain during channel activation, we used the substituted cysteine accessibility method (SCAM). We found that a residue in the beta roll, C505, is more accessible in unliganded channels than in liganded channels, whereas a residue in the C helix, G597C, is more accessible in closed channels than in open channels. These results support a molecular mechanism for channel activation in which the ligand initially binds to the beta roll, followed by an opening allosteric transition involving the relative movement of the C helix toward the beta roll.

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 v AP. For semisynthesis of the K v AP 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 v AP 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.

The selectivity filter of K+ channels contains four ion binding sites (S1–S4) and serves dual functions of discriminating K+ from Na+ and of acting as a gate during C-type inactivation. C-type inactivation is modulated by ion binding to the selectivity filter sites but the underlying mechanism is not known. Here we evaluate how the ion binding sites in the selectivity filter of the KcsA channel participate in C-type inactivation and in recovery from inactivation. We use unnatural amide-to-ester substitutions in the protein backbone to manipulate the S1–S3 sites and a side chain substitution to perturb the S4 site. We develop an improved semisynthetic approach for generating these amide-to-ester substitutions in the selectivity filter. Our combined electrophysiological and X-ray crystallographic analysis of the selectivity filter mutants show that the ion binding sites play specific roles during inactivation and provide insights into the structural changes at the selectivity filter during C-type inactivation.