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.The ability of K⁺ channels to selectively conduct K⁺ ions is accomplished by a structural unit called the selectivity filter (1). The selectivity filter consists of four K⁺ binding sites built using the main chain carbonyl oxygens and the threonine side chain from the protein sequence, which is typically T-V-G-Y-G (Fig. 1A) (2, 3). This sequence, referred to as the signature sequence, is highly conserved among K⁺ channels (2). The high degree of conservation of the signature sequence indicates a similar structure for the selectivity filter of all K⁺ channels, which is in fact observed in the K⁺ channel structures presently available (4).Open in a separate windowFig. 1.The conductive and constricted conformations of the K⁺ selectivity filter. (A) Close-up view of the selectivity filter of wild-type KcsA channel at high K⁺ concentration K⁺] (PDB ID code: 1k4c). Two diagonally opposite subunits are shown in stick representation. K⁺ ions are shown as purple spheres. (B) Macroscopic currents of the wild-type KcsA channel elicited by a pH jump show inactivation. Currents were elicited at +100 mV by a rapid change of solution pH, at the arrow, from pH 7.5 (10 mM Hepes-KOH, 200 mM KCl) to pH 3.0 (10 mM succinate, 200 mM KCl). The selectivity filter of the KcsA channels at low K⁺] (C, PDB ID code: 1k4d) and in the 32-Ǻ open structure (D, PDB ID code: 3f5w) show the constricted conformation. A rotation of the Val76–Gly77 bond causes constriction of the pore. The Gly77 Cα–Cα distance in the opposite subunits is 8.1 Å for the conductive conformation and 5.4–5.5 Å for the constricted conformation at low K⁺] or in the 32-Å open state. (E) Structure of the selectivity filter of KcsA^G77dA at high K⁺] (PDB ID code: 2ih3). (F) A hypothetical structure of the KcsA^G77dA selectivity filter in the constricted conformation. Two adjacent subunits are shown. The methyl side chain of d-Ala77 of one subunit and the carbonyl oxygen atoms of the Val76 and d-Ala77 in the adjacent subunit that clash are shown in van der Waals (VDW) representation. (G) Structure of the selectivity filter of KcsA^G77dA at low K⁺] (PDB ID code: 2ih1). (H) Superposition of the selectivity filter of the KcsA^G77dA in high K⁺] (blue) and low K⁺] (red) shows that the d-Ala substitution in the selectivity filter blocks the constricted conformation.In addition to ion discrimination, the selectivity filter participates in a gating process referred to as C-type inactivation, during which the channel transitions from the conductive state to a nonconductive state (5). C-type inactivation has been extensively investigated in voltage-gated K⁺ (K_v) channels and is observed on prolonged opening of K_v channels by a sustained membrane depolarization (4, 6). C-type inactivation is an effective mechanism to control K_v channel activity and to regulate action-potential frequency in an excitable cell (7). An inactivation process, which is similar to C-type inactivation, is also observed in K⁺ channels that do not belong to the K_v family, such as the bacterial K⁺ channel KcsA. The KcsA channel is gated by pH (8). A decrease in the intracellular pH causes channel opening by conformational changes at the bundle crossing of the pore lining helices. In the closed state, the bundle crossing of the pore lining helices acts as a barrier for the movement of ions across the membrane (9). Activation of the KcsA channel is followed by inactivation during which the current decreases (Fig. 1B) (10, 11). Inactivation in the KcsA channel is proposed to be C-type as it shares a number of functional similarities with C-type inactivation in K_v channels (12–14). This similarity, coupled with the amenability of KcsA to structural studies, has made it an attractive system for elucidating the structure of the selectivity filter in the C-type inactivated state.Models for the selectivity filter in the C-type inactivated state have been proposed based on structures of the KcsA channel at low K⁺ or in the open state. The selectivity filter of the KcsA channel undergoes a conformational change from the conductive state at high K⁺ to a nonconductive state at low K⁺ (Fig. 1C) (3, 15). In the low K⁺ conformation, there is a rotation around the Gly77–Val76 peptide bond that causes the α-carbon of Gly77 to twist inwards and constrict the pore. This rotation disrupts the second and third ion binding sites in the selectivity filter and renders the channel nonconductive (Fig. 1 A, C, and D). As the rate of C-type inactivation increases at low K⁺, the conformation of the selectivity filter at low K⁺ was proposed to represent the C-type inactivated state (16). Recently, a series of structures with varying degrees of opening at the bundle crossing of the pore lining helices were obtained by using a constitutively open mutant of the KcsA channel (17). Higher degrees of opening at the bundle crossing (25–32 Å) were accompanied by a conformational change in the selectivity filter that was presumed to be nonconductive (Fig. 1D). This nonconductive conformation of the selectivity filter was proposed to represent the C-type inactivated state. The conformations of the selectivity filter in low K⁺ or in the open-channel structure are quite similar except for slight differences toward the lower half of the selectivity filter and the orientation of the Thr75 side chain. Due to their similarity, we jointly refer to these conformations as the “constricted” conformation of the selectivity filter. Changes in the conformation of the KcsA selectivity filter at low K⁺ or low pH have also been detected by solution and solid-state NMR and are consistent with the constricted conformation of the selectivity filter (18–20).However, does the constricted conformation represent the selectivity filter in the C-type inactivated state? An important caveat of the structural studies is that the C-type inactivated state must be accurately captured by the conditions used for structure determination. Experimental validation is therefore necessary before the constricted conformation can be conclusively assigned as the C-type inactivated state. Here, we used unnatural amino acid mutagenesis to test whether the constricted conformation of the selectivity filter of the KcsA channel corresponds to the C-type inactivated state. We also used unnatural amino acid mutagenesis on the archaebacterial K_v channel K_vAP, to test whether the constricted conformation is relevant during C-type inactivation in a K_v channel. We show that inactivation in the K_vAP channel is functionally similar to C-type inactivation in a eukaryotic K_v channel. To carry out unnatural amino acid mutagenesis, we developed a modular semisynthesis of the K_vAP channel that allowed us to use chemical synthesis to modify the selectivity filter. Our results on the KcsA and the K_vAP channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state.