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.     

Transmembrane allosteric coupling of the gates in a potassium channel

It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98–103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.Potassium channel activation and inactivation is fundamental to many physiological functions including muscle contraction and the generation of synaptic action potentials (1). KcsA is a 160-residue pH-activated homotetrameric K⁺ channel isolated from the soil bacterium Streptomyces lividans (2, 3) with high sequence homology and functional similarity to mammalian potassium channels (4). It has provided an excellent model for studies of ion-conduction by X-ray crystallography (3, 5, 6), electrophysiology (7, 8), and NMR (9–21). Like many potassium channels, it exhibits (4, 6, 22, 23) slow, spontaneous inactivation involving the residues near the extracellular selectivity filter subsequent to channel activation. Recent results from X-ray crystallography and molecular dynamics suggest that the gates are coupled and that inactivation is prompted by channel opening, mediated via a series of intrasubunit steric contacts involving F103 with T74, T75, and M96 and an intersubunit contact with the neighboring I100 side chain (4–6, 24, 25). In separate experiments, the extracellular gate has been observed to respond directly to ambient [K⁺]: at high [K⁺] it exists in a conductive form, and at low K⁺ it collapses into a nonconductive state (3). Our NMR studies suggest that the low [K⁺] state and the low pH inactivated state may be similar; this conclusion is supported by the effect of the mutation E71A and the pattern of chemical shift perturbations in the selectivity filter when the ion is depleted (9, 19). Meanwhile, X-ray crystallography studies suggest that mutants (E71A) unable to undergo inactivation are also unable to expel ions (26).An established similarity of the low pH and the low [K⁺] states would clarify the importance of allosteric coupling and have the practical consequence that the well-behaved low K⁺ state could serve as a useful structural proxy for the otherwise fleeting inactivated state. For these reasons we tested this correspondence using NMR experiments. If the low K⁺ state is similar to the inactivated state of KcsA achieved by lowering the pH, it is expected that structural changes indicative of channel opening observed at low [K⁺] would occur not only in the selectivity filter but also in the pH gate and the hinge region. However, some studies imply that these two gates might be uncoupled or weakly coupled. For example, X-ray crystallographic studies of KcsA, where K⁺ sensitivity was largely isolated to the selectivity filter (3). In this work, we asked whether, by contrast, full-length wild-type KcsA (160 aa) reconstituted into hydrated lipid bilayers exhibits global structural changes upon ion expulsion suggestive of channel opening. To accomplish this, nearly complete ¹³C and ¹⁵N chemical shift assignments were obtained for the transmembrane and loop regions from four-dimensional (4D) solid-state nuclear magnetic resonance (SSNMR) (27), providing numerous reporters for conformational change during ion binding. In low [K⁺] ion conditions at neutral pH, not only does KcsA expel the K⁺ ions from the inner selectivity filter sites, but the channel also exhibits chemical shift perturbations at the pH gate and the hinge of the inner transmembrane helix, suggesting features akin to the inhibited state that is present at low pH and high [K⁺]. That these two distinct conditions result in a nearly identical state of the channel offers strong evidence for transmembrane allostery in the inactivation process.     

Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating

In K⁺ channels, the selectivity filter, pore helix, and outer vestibule play a crucial role in gating mechanisms. The outer vestibule is an important structurally extended region of KcsA in which toxins, blockers, and metal ions bind and modulate the gating behavior of K⁺ channels. Despite its functional significance, the gating-related structural dynamics at the outer vestibule are not well understood. Under steady-state conditions, inactivating WT and noninactivating E71A KcsA stabilize the nonconductive and conductive filter conformations upon opening the activation gate. Site-directed fluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule of open/conductive conformation is highly dynamic compared with the motional restriction experienced by the outer vestibule during inactivation gating. A wavelength-selective fluorescence approach shows a change in hydration dynamics in inactivated and noninactivated conformations, and supports a possible role of restricted/bound water molecules in C-type inactivation gating. Using a unique restrained ensemble simulation method, along with distance measurements by EPR, we show that, on average, the outer vestibule undergoes a modest backbone conformational change during its transition to various functional states, although the structural dynamics of the outer vestibule are significantly altered during activation and inactivation gating. Taken together, our results support the role of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gating mechanisms of K⁺ channels.The functional behavior of K⁺ channels is defined by a series of structural rearrangements associated with the processes of activation and inactivation gating (1–6). In response to a prolonged stimulus and in the absence of an N-terminal inactivating particle, most K⁺ channels become nonconductive through a process known as C-type inactivation (7). This C-type inactivation is crucial in controlling the firing patterns in excitable cells and is fundamental in determining the length and frequency of the cardiac action potential (8). C-type inactivation is inhibited by high extracellular K⁺ (9, 10), and the blocker tetraethylammonium (TEA) (11) can also be slowed down in the presence of permeant ions with a long residence time in the selectivity filter (Rb⁺, Cs⁺, and NH⁴⁺) (10).The prokaryotic pH-gated K⁺ channel KcsA shares most of the mechanistic properties of C-type inactivation in voltage-dependent K⁺ channels (5, 6, 12–16). Recent crystal structures of open/inactivated KcsA reveal that there is a remarkable correlation between the degree of opening at the activation gate and the conformation and ion occupancy of the selectivity filter (5). In KcsA, the selectivity filter is stabilized by a hydrogen bond network, with key interactions between residues Glu71, Asp80, and Trp67 and a bound water molecule (17). Disrupting this hydrogen bond network favors the conductive conformation of the selectivity filter (12, 13, 15).Early electrophysiological experiments have suggested that the outer vestibule (around T449 residue in Shaker and Y82 residue in KcsA) undergoes significant conformational rearrangement during C-type inactivation gating (16, 18, 19). However, comparison of the WT KcsA crystal structure, where the filter is in its conductive conformation, with either the structure obtained with low K⁺ (collapsed filter) (17) or the crystal structure of open-inactivated KcsA with maximum opening (inactivated filter) (5) does not show major conformational changes in the outer vestibule that would explain these results (Fig. 1A). We have suggested that this apparent discrepancy can be understood if we take into consideration the potential differences in the dynamic behavior of the outer vestibule changes as the K⁺ channel undergoes its gating cycle (16).Open in a separate windowFig. 1.Comparison of outer vestibule conformation in KcsA structures with conductive and collapsed/inactivated filters. (A) High-K⁺ KcsA structure [Protein Data Bank (PDB) ID code 1K4C; yellow] is compared with a low-K⁺ KcsA structure (PDB ID code 1K4D; blue) in the closed state (Left) and open/inactivated conformation (PDB ID code 3F5W; green) (Right). The outer vestibule residues are depicted as red spheres, and relevant residues are labeled. (B) Schematic representation of typical macroscopic currents elicited by pH-jump experiments in WT (inactivating) and E71A (noninactivating) KcsA channels at a depolarizing membrane potential is shown. Conditions that stabilize the closed, open/inactivated, and open/conductive conformations at the steady state are indicated with a black circle. (C) Effect of opening the lower gate on the mobility of spin-labeled outer vestibule residues in palmitoyloleoylphosphatidyl choline/palmitoyloleoylphosphatidyl glycerol (POPC/POPG) (3:1, moles/moles) reconstituted WT (Left) and noninactivating mutant E71A (Right) backgrounds for the closed (pH 7, red) and open (pH 4, black) states of KcsA, as determined by continuous wave (CW) EPR. The spectra shown are amplitude-normalized. Details are provided in SI Materials and Methods.We have probed the gating-induced structural dynamics at the outer vestibule of KcsA using site-directed fluorescence and site-directed spin labeling and pulsed EPR approaches in combination with a recently developed computational method, restrained ensemble (RE) simulations. RE simulation was used to constrain the outer vestibule using experimentally derived distance histograms in different functional states (closed, open/inactivated, and open/conductive) and to monitor the extent of backbone conformational changes during gating. To this end, we took advantage of our ability to stabilize both the open/conductive (E71A mutant) and the open/inactivated (WT) conformations of KcsA upon opening the activation gate under steady-state conditions (Fig. 1B).Our data show that the outer vestibule in the open/conductive conformation is highly dynamic. In addition, the red edge excitation shift (REES) points to a change in hydration dynamics between conductive and nonconductive outer vestibule conformations, suggesting a role of restricted water molecules in C-type inactivation gating. We suggest that, on average, the backbone conformation of the outer vestibule does not change significantly between different functional states but that local dynamics change significantly, underlining the importance of the hydrogen bond network behind the selectivity filter and the microscopic observables (e.g., dynamics of hydration) in K⁺ channel gating and C-type inactivation.     

Filter gate closure inhibits ion but not water transport through potassium channels

The selectivity filter of K⁺ channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K⁺ hydration. Ion conductivity is abrogated if some of these carbonyl groups flip out of the lumen, which happens (i) in the process of C-type inactivation or (ii) during filter collapse in the absence of K⁺. Here, we show that K⁺ channels remain permeable to water, even after entering such an electrically silent conformation. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K⁺ channel KcsA into lipid vesicles that were either C-type inactivating or noninactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. Quantification of the per-channel increment in proteoliposome water permeability with the aid of stopped-flow experiments yielded a unitary water permeability p_f of (6.9 ± 0.6) × 10⁻¹³ cm³⋅s⁻¹ for both mutants. “Collapse” of the selectivity filter upon K⁺ removal did not alter p_f and was fully reversible, as demonstrated by current measurements through planar bilayers in a K⁺-containing medium to which K⁺-free proteoliposomes were fused. Water flow through KcsA is halved by 200 mM K⁺ in the aqueous solution, which indicates an effective K⁺ dissociation constant in that range for a singly occupied channel. This questions the widely accepted hypothesis that multiple K⁺ ions in the selectivity filter act to mutually destabilize binding.     

Importance of lipid–pore loop interface for potassium channel structure and function

Potassium (i.e., K⁺) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K⁺ channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K⁺ channel inactivation and exhibits a remarkable structural plasticity that correlates to K⁺ channel inactivation. The transmembrane helix 1 unwinds when the K⁺ channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K⁺ ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K⁺ channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K⁺ channel pore.     

Selectivity filter ion binding affinity determines inactivation in a potassium channel

Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca²⁺-activated K⁺ channel from Methanobacterium thermoautotrophicum, the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K⁺ concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF’s four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K⁺ affinity (∼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K⁺, similar to KcsA, but that even a single K⁺ binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K⁺ concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK’s S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes.

Ion permeation gating within the selectivity filter (SF) of potassium (K⁺) channels has been proposed to control channel activity in different ways for different family members. In voltage-dependent K⁺ (K_V) channels, the SF has been proposed to underlie C-type inactivation (1–3), resulting in the progressive loss of current following the activation of a channel gate located near the intracellular side of the pore (4). C-type inactivation in K_V channels has been shown to be strongly dependent on the affinity of a particular binding site for permeant ions in the pore, and the affinity of these pore sites has been proposed to depend not only on the SF chemical composition, but also on regions outside of the SF (5–8). A structure-function model for this mechanism has been provided most specifically by studies of the proton-gated KcsA channel (9–11) where opening of the activation gate is correlated with a conformational constriction and a decrease in ion occupancy within the four K⁺ binding sites of the SF (named S1 to S4) (9, 12–15). Experimental and structural studies of KcsA in low K⁺ showed that the SF constriction consists of an outward flip of the carbonyl groups of the Gly77, in the middle of the signature sequence (TVGYG) of K⁺ channels, associated with a loss of K⁺ binding at site S2 of the SF (9, 14, 16–20). These conformational changes were accompanied by the binding of several water molecules behind the SF, stabilizing this constricted (also called flipped) state by sterically preventing the SF from switching back into its conductive state (16, 18–20). While some reports challenge this view (21, 22), this activation gate-coupled collapse of the SF is now generally accepted as the mechanism underlying C-type inactivation in K⁺ channels.Several types of ligand-dependent K⁺ channels, including those opened by binding Ca²⁺, such as the BK and MthK channels (23–27), do not exhibit traditional C-type inactivation, despite possessing an identical SF with K_V and KcsA channels. Furthermore, these channels have been proposed to actually gate at the SF (28–32) although a recent cryogenic electron microscopy (cryo-EM) structure of MthK in the absence of calcium (33) revealed a steric closure at the bundle crossing inner gate region, suggesting that there may be two gates involved in calcium gating. Nevertheless, at this time, the structural correlates of SF gating and the difference from inactivation are unknown.In the present study, we set out to first investigate whether we can capture different gating states of MthK by obtaining X-ray structures of its pore (Fig. 1A) in wide-ranging concentrations of K⁺. MthK channels have been previously shown to display a decrease in activity with depolarization, which is further augmented when external K⁺ concentration is lowered, a signature of SF gating and a hallmark of C-type inactivation (34). We reasoned that K⁺ titration of MthK pore structures may provide insights into the molecular causes for K⁺-dependent SF gating and will indicate whether it shares features with the C-type inactivation observed in KcsA (such as a collapsed SF). Unlike KcsA, MthK SF did not collapse in similarly low K⁺ concentrations, suggesting that the clue to why MthK does not display traditional C-type inactivation may lie in understanding the molecular underpinnings that contribute to SF conformational change. Thus, we next investigated the dependence of SF conformation on ion occupancy and used molecular dynamics (MD) simulations to reveal a uniquely low affinity central S2 site in MthK, which may play a lead role in the SF-based channel closure. Overall, our results illustrate how the exact same sequence and structure of the SF in a K⁺ channel can lead to slight variations in K⁺ binding site chemistry, which in turn can lead to distinct functional phenotypes.Open in a separate windowFig. 1.Structure of the MthK pore in different K⁺ concentrations. (A) Overall architecture of wild-type MthK pore structure (three subunits of the tetrameric pore shown for clarity) crystallized with 150 mM K⁺. The SF is highlighted by dashed lines. Alignment of this structure with that crystallized in 6 mM K⁺ yields an all-atom root-mean-square deviation (RMSD) value of 0.25 Å. (B) K⁺-omit electron density maps (2F_o – F_c contoured at 2.0 σ) for SF atoms from two opposing subunits. Structures were solved in 150, 50, 11, and 6 mM [K⁺], as indicated. Crystallographic statistics are in SI Appendix, Table S1. (C) MD system with the MthK (ribbons) embedded in a lipid bilayer (gray sticks) bathed in 200 mM KCl (K⁺ as green spheres, Cl⁻ as blue spheres, and water as red and white sticks).     

Mechanism for selectivity-inactivation coupling in KcsA potassium channels

Structures of the prokaryotic K(+) channel, KcsA, highlight the role of the selectivity filter carbonyls from the GYG signature sequence in determining a highly selective pore, but channels displaying this sequence vary widely in their cation selectivity. Furthermore, variable selectivity can be found within the same channel during a process called C-type inactivation. We investigated the mechanism for changes in selectivity associated with inactivation in a model K(+) channel, KcsA. We found that E71A, a noninactivating KcsA mutant in which a hydrogen-bond behind the selectivity filter is disrupted, also displays decreased K(+) selectivity. In E71A channels, Na(+) permeates at higher rates as seen with and flux measurements and analysis of intracellular Na(+) block. Crystal structures of E71A reveal that the selectivity filter no longer assumes the "collapsed," presumed inactivated, conformation in low K(+), but a "flipped" conformation, that is also observed in high K(+), high Na(+), and even Na(+) only conditions. The data reveal the importance of the E71-D80 interaction in both favoring inactivation and maintaining high K(+) selectivity. We propose a molecular mechanism by which inactivation and K(+) selectivity are linked, a mechanism that may also be at work in other channels containing the canonical GYG signature sequence.     

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

The sodium-potassium pump (Na⁺,K⁺-ATPase) is responsible for establishing Na⁺ and K⁺ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na⁺,K⁺-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K⁺·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K⁺, in marked contrast to previous models. Due to antagonism between ouabain and K⁺, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.     

Ion-binding properties of a K+ channel selectivity filter in different conformations

K⁺ channels are membrane proteins that selectively conduct K⁺ ions across lipid bilayers. Many voltage-gated K⁺ (K_V) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K⁺ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to K_V channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na⁺ or low [K⁺]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K⁺ ion binding to channels with an inactivated or conductive selectivity filter is different from K⁺ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.K⁺ channels are found in all three domains of life, where they selectively conduct K⁺ ions across cell membranes. Specific stimuli trigger the activation of K⁺ channels, which results in a hinged movement of the inner helix bundle (1–7). This opening on the intracellular side of the membrane initiates ion conduction across the membrane by allowing ions to enter into the channel. After a period, many channels spontaneously inactivate to attenuate the response (8–17). The inactivation process is a timer that terminates the flow of ions in the presence of an activator to help shape the response of the system. Two dominant types of inactivation have been characterized in voltage-dependent channels: N-type and C-type (18). N-type inactivation is fast and involves an N-terminal positively charged “ball” physically plugging the pore of the channel when the membrane is depolarized. C-type inactivation, on the other hand, is a slower process involving a conformational change in the selectivity filter that is initiated by a functional link between the intracellular gate and the selectivity filter (10, 19).Several experimental observations indicate a role for the selectivity filter in C-type inactivation. First, mutations in and around the selectivity filter can alter the kinetics of inactivation (20–23). Second, increasing concentrations of extracellular K⁺ ions decrease the rate of inactivation, as if the ions are stabilizing the conductive conformation of the channel to prevent a conformational change in the selectivity filter (14, 16, 17, 22). Finally, a loss of selectivity of K⁺ over Na⁺ has been observed during the inactivation process in Shaker channels, suggesting a role for the selectivity filter (24, 25). Together, these data indicate that channels in their inactivated and conductive conformations interact with K⁺ ions differently, and suggest that C-type inactivation involves a conformational change in the selectivity filter. Although several structures of K⁺ channels in their conductive state have been solved using X-ray crystallography, there is at present no universally accepted model for the C-type inactivated channel (1, 3–5, 9, 19, 26–28) (Fig. 1B).Open in a separate windowFig. 1.Macroscopic recordings and structural models of KcsA K⁺ channel. (A) Macroscopic currents of WT KcsA obtained by a pH jump from pH 8 to pH 4 reveal channel inactivation. Two models representing the conformation of the channel are shown below. (B) Conductive [Left, Protein Data Bank (PDB) ID code 1K4C] and constricted (Right, PDB ID code 1K4D) conformations of the selectivity filter are shown as sticks, and the ion-binding sites are indicated with green spheres. The thermodynamic properties of the conductive, constricted, and inactivated (Middle) conformations are the subject of this study.Inactivation in the K⁺ channel from Streptomyces lividians (KcsA) has many of the same functional properties of C-type inactivation, which has made it a model to understand its structural features (20). KcsA channels transition from their closed to open gate upon changing the intracellular pH from high to low (Fig. 1A). The rapid flux of ions through the channel is then attenuated by channel inactivation, where most open WT channels are not conducting, suggesting that crystal structures of open KcsA channels would reveal the inactivated channel. In some crystal structures of truncated WT KcsA solved with an open gate, the selectivity filter appears in the constricted conformation, similar to the conformation observed in structures of the KcsA channel determined in the presence of only Na⁺ ions or low concentrations of K⁺ ions (3, 10, 29, 30) (Fig. 1B). Solid-state and solution NMR also indicate that the selectivity filter of the KcsA channel is in the constricted conformation when the cytoplasmic gate is open (31–33).However, a recently published study shows that even when the constricted conformation of KcsA’s selectivity filter is prevented by a nonnatural amino acid substitution, the channel inactivates like WT channels, suggesting the constricted filter does not correspond to the functionally observed inactivation in KcsA (28). In this study, we use isothermal titration calorimetry (ITC) to quantify the ion-binding properties of WT and mutant KcsA K⁺ channels with their selectivity filters in different conformations and EPR spectroscopy to determine the conformation of the channels’ intracellular gates. A comparison of these ion-binding properties leads us to conclude that the conductive and inactivated filters are energetically more similar to each other than the constricted and inactivated filters.     

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K+ channels

K⁺ channels distinguish K⁺ from Na⁺ in the selectivity filter, which consists of four ion-binding sites (S1–S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of C-type inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K⁺ binding at the S2 site. We also show that an ester substitution at the S2 site in the K_vAP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K⁺ channels in K⁺ compared with Rb⁺ are due to different ion occupancies at the S2 site.Potassium channels are a ubiquitous family of integral membrane proteins that facilitate the selective conduction of K⁺ ions across cellular membranes (1). K⁺ selectivity is achieved by a structural element in the K⁺ channel pore called the selectivity filter (2). The selectivity filter consists of four sequential ion-binding sites (labeled S1–S4, from the outside to inside) that are built using protein backbone carbonyl oxygen atoms and the threonine side chain from the protein sequence T-V-G-Y-G (Fig. 1A) (4, 5).Open in a separate windowFig. 1.Ester substitutions in the selectivity filter of the KcsA channel. (A) Close-up view of the selectivity filter of the wild-type KcsA channel [Protein Data Bank (PDB): 1K4C]. Two opposite subunits are shown in stick representation, and the K⁺ ions bound are shown as purple spheres. The amide bonds (1′–4′) and the ion-binding sites in the selectivity filter (S1–S4) are labeled. (B) Macroscopic currents for the KcsA channels were elicited at +100 mV by a rapid change in pH from 7.5 to 3.0. (C) Single-channel currents recorded at steady-state conditions at pH 3.0. The pH 3.0 solution is 10 mM succinate, 200 mM KCl, and the pH 7.5 solution is 10 mM Hepes–KOH, 200 mM KCl. The data for KcsA^WT are from ref. 3.In addition to selective conduction of K⁺, the selectivity filter acts as a gate to regulate the flow of ions through the pore (6–8). During this gating process, conformational changes at the selectivity filter convert it from a conductive to a nonconductive state. In voltage-gated K⁺ (K_v) channels, this gating process is referred to as C-type inactivation (9). C-type inactivation is a physiologically important process as it plays a direct role in regulating neuronal firing and in pacing cardiac action potentials (8). The KcsA K⁺ channel from Streptomyces lividans undergoes an inactivation process that is functionally similar to C-type inactivation in a eukaryotic K_v channel (10–13). As the KcsA channel is easily amenable to structural studies, it has become an important model system for understanding the structure of the selectivity filter in the C-type–inactivated state and the forces that drive inactivation (14, 15).One of the hallmarks of C-type inactivation is a dependence on the permeant ion (6, 7). The rate of C-type inactivation decreases when the K⁺ concentration is increased or when the permeant ion is changed from K⁺ to Rb⁺ (16, 17). Crystallographic studies on K⁺ channels have shown that a change in the permeant ion or its concentration results in changes in the ion occupancy at the binding sites in the selectivity filter (18, 19). For example, K⁺ and Rb⁺ at similar concentrations show different occupancies at the ion-binding sites, and the channel exhibits different rates of inactivation in K⁺ compared with Rb⁺ (3, 16, 20), which suggests a link between ion occupancy at the selectivity filter and inactivation (21, 22). The influence of permeant ions on inactivation has been proposed to arise from a “foot in the door”-like effect in which ion binding at a specific site prevents inactivation, similar to the presence of a foot in the doorway that prevents a door from closing (16, 23). The binding site responsible for the foot in the door effect is suspected to be at the extracellular side of the channel, but the exact location of the binding site, whether in the selectivity filter or at the extracellular mouth of the filter, is not known (6).In this study, we investigate this link between ion binding at the selectivity filter and inactivation. The approach that we use is to alter ion binding at the selectivity filter sites and to determine the effect on inactivation. The S1–S3 ion-binding sites in the selectivity filter are constructed by backbone carbonyl oxygens. Therefore, conventional site-directed mutagenesis does not allow us to alter these sites. Instead, we use chemical synthesis and nonsense suppression approaches to introduce amide-to-ester substitutions in the protein backbone to perturb ion binding to specific sites in the selectivity filter (24, 25).Amide-to-ester substitutions have previously been used to engineer the protein backbone for studies on protein stability and folding (26). Ester bonds are isosteric to amide bonds but have altered hydrogen-bonding properties and reduced electronegativity at the carbonyl oxygen (27). This reduction in the electronegativity of the carbonyl oxygen, by roughly one-half compared with an amide bond, perturbs ion binding to the selectivity filter. Amide-to-ester substitutions have previously been reported in the selectivity filters of the Kir2.1 and the KcsA K⁺ channels (28, 29). In the Kir2.1 channel, an ester substitution for the 3′ amide bond (see Fig. 1A for nomenclature) was found to reduce channel conductance and to produce distinct subconductance levels. In the KcsA channel, an ester substitution for the 1′ amide bond was found to reduce channel conductance, and a crystal structure of the ester mutant showed that the ester substitution decreased ion occupancy at the S1 site. Neither of these studies examined the effect of the ester substitutions on inactivation.Here we substitute the 1′, 2′, and 3′ amide bonds in the selectivity filter of the KcsA K⁺ channel with esters and investigate the effect on inactivation. We determine the crystal structure of the 2′ ester mutant of the KcsA channel to examine the effect of the ester substitution on the structure and ion occupancy of the selectivity filter. We also investigate the effect of an ester substitution at the 2′ amide bond in the selectivity filter on inactivation in the voltage-gated K⁺ channel, K_vAP. Our results show that the S1 and S2 sites in the selectivity filter do not act as the foot in the door sites to prevent inactivation. Unexpectedly, we find that a lack of ion binding at the S2 site reduces inactivation.     

Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex

Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na⁺,K⁺-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na⁺,K⁺-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P–ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg²⁺ is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K⁺ in the E2P–bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1–2.Cardiotonic steroids (CTSs) induce diverse physiological effects on, for example, heart muscle and blood pressure regulation, but the underlying mechanisms remain unknown, despite a long history of therapeutic applications and model studies. It is widely recognized that they target Na⁺,K⁺-ATPase, and a direct consequence of their binding is an inhibition of the enzyme. Their positive inotropic effect in cardiomyocytes has been related to coupling between Na⁺,K⁺-ATPase and Na⁺/Ca²⁺-exchanger through the intracellular Na⁺ concentration, whereas numerous other outcomes observed on the cellular level have led to hypotheses of the existence of signaling cascade mechanisms with Na⁺,K⁺-ATPase acting as a receptor. The minimal functional unit of the enzyme is an αβ-complex, and because there exist four α- and three β-isoforms of the Na⁺,K⁺-ATPase, the variations in the heterodimer composition and a vast number of CTSs differing in apparent isoform specificities (1) add to the complexity and multiplicity of reported physiological responses.The conserved structural core shared by all CTSs includes a cis-trans-cis ring-fused steroid core with two methyl substituents at steroid positions C10_β and C13_β, two hydroxyl groups (OH3_β and OH14_β), and an unsaturated lactone ring at the C17_β position, among which the lactone and OH14_β are critical for binding to Na⁺,K⁺-ATPase (2, 3). The type of lactone at the C17_β position divides natural CTSs into cardenolides (five-membered lactone rings) and bufadienolides (six-membered lactone rings). Finally, many CTSs are glycosylated by one to four carbohydrate residues at OH3_β (Fig. S1). It has been shown that glycosylation improves CTS affinity toward the Na⁺,K⁺-ATPase and contributes (at least in the case of digoxin and digitoxin) to their Na⁺,K⁺-ATPase isoform selectivity, with up to fourfold preference for α2/α3 over α1 (1).The recently published crystal structure of the Na⁺,K⁺-ATPase phosphoenzyme (E2P) in complex with the widely studied CTS ouabain (4, 5) showed that the high-affinity CTS-binding site is constituted by the transmembrane helices αM1–6 of the catalytic α-subunit, forming a pocket exposed to the extracellular side and overlapping with the extracellular ion exchange pathway (6). The E2P–ouabain structure also revealed details on protein–ligand interactions facilitating high-affinity CTS binding compared with a low-affinity ouabain complex (7). Among the important features brought to view by the high-affinity complex structure were (i) a Mg²⁺ ion occupying cation-binding site II, (ii) the rearrangement of αM4, forming the structural basis for the well-known antagonistic effect of K⁺ on ouabain binding, and (iii) an E2P-specific configuration of αM1–2 on the cytoplasmic side, whereas the extracellular end of this helix pair closes in on the CTS-binding site (4). Biochemical experiments showing competitive interactions between K⁺ and Mg²⁺ suggested that the nature of the cation in site II is a determinant for ouabain affinity. In addition, long-range interactions between the unsaturated, polarized five-membered lactone ring of ouabain and the Mg²⁺ ion were suggested as a factor for CTS recognition and differentiation. Despite previous reports showing that glycosylated CTSs have higher Na⁺,K⁺-ATPase affinity than their aglycones, no specific interactions were observed between the sugar moiety of ouabain and the protein to explain that effect.To gain a better understanding of the structure–activity relationship of the CTSs, we have crystallized the E2P form of the pig kidney Na⁺,K⁺-ATPase (α₁β₁γ) in complex with two CTSs: bufalin (a nonglycosylated bufadienolide) and digoxin (a trisaccharide-conjugated cardenolide) (Fig. 1A), which also are pharmacological agents. We further performed experiments on CTS binding to Na⁺,K⁺-ATPase, including the aglycones digitoxigenin and ouabagenin (Fig. S1). The data revealed notable qualitative differences in kinetics of the enzyme interactions with the glycosylated vs. nonglycosylated CTSs as well as a remarkable insensitivity of bufalin binding to K⁺. The time course of Na⁺,K⁺-ATPase inhibition under steady-state conditions, mimicking the interactions with CTSs in vivo, revealed that binding occurs in two steps. The impact of separate structural components, such as sugar and lactone moieties, on the individual steps of CTS binding is discussed on the basis of our structural and biochemical data.Open in a separate windowFig. 1.Structural comparison of the crystal structures of the high-affinity Na⁺,K⁺-ATPase α₁β₁γ E2P–CTS complexes. The phosphoenzyme stabilized by bufalin, digoxin, and ouabain (5) is depicted in blue, green, and gray cartoons, respectively, and the bufalin, digoxin, and ouabain molecules are represented by magenta, orange, and dark gray sticks, respectively. The K⁺ and Mg²⁺ ions are represented by purple and yellow spheres, respectively. (A) Structural representation of the CTSs digoxin, bufalin, and ouabain. (B and C) The final 2F_o-F_c electron density maps of the E2P–bufalin and E2P–digoxin, respectively, complexes (contoured at 1.0σ level). The maps are represented by gray mesh. (D) Structural alignment of the E2P–bufalin, E2P–digoxin, and E2P–ouabain complexes performed on the segments αM7–10 showing a high degree of overall structural similarity. (E) The CTS-binding site visualized from the extracellular site based on the same alignment as above. The alignment reveals similar hydrophobic interactions between the α-surface of the CTS core and αM4–6. In contrast, different interactions are formed between the substituents at the β-surface of the CTS core and αM1–2, leading to minor CTS-induced rearrangements. (F) The CTS-binding site visualized from αM1–2. αM4 overlays well for Mg²⁺-bound complexes of E2P–digoxin and E2P–ouabain as well as the E2P–bufalin complex, despite potassium bound in the cation-binding sites.     

Hypokalemic Paralysis Complicated by Concurrent Hyperthyroidism and Chronic Alcoholism: A Case Report

Thyrotoxic periodic paralysis (TPP) is characterized by the presence of muscle paralysis, hypokalemia, and hyperthyroidism. We report the case of a young man with paralysis of the lower extremities, severe hypokalemia, and concurrent hyperthyroidism. TPP was suspected; therefore, treatment consisting of judicious potassium (K⁺) repletion and β-blocker administration was initiated. However, urinary K⁺ excretion rate, as well as refractoriness to treatment, was inconsistent with TPP. Chronic alcoholism was considered as an alternative cause of hypokalemia, and serum K⁺ was restored through vigorous K⁺ repletion and the addition of K⁺-sparing diuretics.The presence of thyrotoxicosis and hypokalemia does not always indicate a diagnosis of TPP. Exclusion of TPP can be accomplished by immediate evaluation of urinary K⁺ excretion, acid-base status, and the amount of potassium chloride required to correct hypokalemia at presentation.     

Crystal structure of the high-affinity Na+,K+-ATPase–ouabain complex with Mg2+ bound in the cation binding site

The Na⁺,K⁺-ATPase maintains electrochemical gradients for Na⁺ and K⁺ that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na⁺,K⁺-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na⁺,K⁺-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg²⁺, and crystallographic studies indicate that Rb⁺ and Mn²⁺, but not Na⁺, bind to this site. Comparison with the low-affinity [K₂]E2–MgF_x–ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742–13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg²⁺. K⁺ binding at the same site unwinds a turn of αM4, dragging residues Ile318–Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K⁺–Mg²⁺ competition and explain the well-known antagonistic effect of K⁺ on CTS binding.     

Binding of cardiotonic steroids to Na+,K+-ATPase in the E2P state

The sodium pump (Na⁺, K⁺-ATPase, NKA) is vital for animal cells, as it actively maintains Na⁺ and K⁺ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF₃⁻ complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K⁺-antagonism and suggest a route to isoform specific CTSs.

Under physiological conditions, Na⁺,K⁺-ATPase (NKA) actively extrudes three cytoplasmic Na⁺ ions in exchange for two extracellular K⁺ ions per ATP hydrolyzed (see Fig. 1 for a simplified reaction diagram). The established gradients for Na⁺ and K⁺ are pivotal for generating a membrane potential, regulation of cell volume, and providing chemical energy for various secondary active transporters. They are expressed in all animal cells and are finely tuned. In humans, the catalytic α-subunit exists in four isoforms. α1 is ubiquitous and best studied. α2 is most abundant in skeletal and heart muscle, whereas α3 is found in brain cells and α4 in cells of the testis. The α-subunit complexes with a β-subunit (isoforms β1–4 in humans) and a tissue specific regulatory protein FXYD (1–7 in humans) (for a recent general review, see, e.g., refs. 1, 2).Open in a separate windowFig. 1.A reaction diagram of Na⁺,K⁺-ATPase with special emphasis on the backward phosphorylation with P_i and inhibition by CTSs. CTSs can bind to at least three E2P species with different affinities. The states demonstrated to allow high-affinity binding of CTSs appear in purple letters, and those supposed to allow high-affinity binding but not demonstrated are in red letters; the state that allows low-affinity binding is in orange letters. E2P^ATP is the physiological E2P ground state (path A); E2P^Pi can be formed in the backward reaction starting from E2 with P_i (path B). This reaction places Mg²⁺ at the phosphorylation site of NKA but likely to incorporate another Mg²⁺ at site II (E2P^Pi·Mg²⁺). If the backward phosphorylation reaction starts from E2·2K⁺, E2P·2K⁺ will be (transiently) formed in which two K⁺ bind to NKA in E2P with high affinity (E2P·2K⁺_high). CTSs can stabilize a different state, in which two K⁺ are bound with (presumably) low affinity (E2P^Pi·2K⁺_low; path C). Crystal structures available are boxed and phosphate analogs used are shown below the boxes. The crystal structures obtained in this study are highlighted (yellow boxes). PDB ID codes for published crystal structures are: E1∼P·ADP·3Na⁺, 3WGU; E2P^Pi·Mg²⁺(OBN), 4HYT; E2P^Pi·Mg²⁺(DGX), 4RET; E2P^Pi·2K⁺ (BUF), 4RES; E2·P_i·2K⁺, 2ZXE; E2·P_i·2K⁺(OBN), 3A3Y.Cardiotonic glycosides, such as digoxin (DGX) and digitoxin (DTX), are specific inhibitors of NKA and have been prescribed for patients with heart failure for centuries. Canonical cardiotonic glycosides, including ouabain (OBN), the best studied member, have a tripartite structure: a central steroid core, a five-membered or six-membered lactone ring, and a carbohydrate moiety of one to four residues. Each part appears to have a different role in binding. As summarized by Glynn (3), critical features of high affinity cardiac glycosides are: 1) The unsaturated lactone ring attached in the correct configuration at C17; 2) the cis configuration of the AB and CD ring junctions in the steroid nucleus; 3) the presence of a hydroxyl group at C14; and 4) the presence of an appropriate sugar at C3. A wide range of cardiotonic steroids (CTSs), including aglycones, as the sugar at C3 does not necessarily improve the affinity, showing vastly different inhibitory properties, have been developed in order to improve their usability in the clinical setting.Indeed, several new members, such as rostafuroxin (ROS) (4) and istaroxime (IST) (5), now under clinical trials, have distinct chemical structures. ROS is proposed as a potent antihypertensive compound in ouabain-dependent models of hypertension (4). It is reported to be capable of displacing OBN from NKA at a concentration 10 times lower than that expected from its K_I, which is 1,000 times greater than that of OBN (6). IST has only a carbonyl group instead of the unsaturated lactone, and an aminoalkyloxime group instead of the sugar, but shows an inhibitory potency similar to that of digoxin (5). It is reported to have a significant inotropic effect but with a lower risk of causing cardiac arrhythmia compared to digoxin (5). Why these compounds can replace OBN at a much lower concentration than that expected from their binding affinities is paradoxical and addressed in this study.Reflecting their very long history, the accumulated literature on CTSs is huge. Numerous studies report on their inhibitory activities, but they appear rather inconsistent, partly due to differences in experimental conditions (7). It is well established that CTSs preferentially bind to NKA in the E2P ground state from the extracellular face (8). However, the E2P states formed in different routes show distinct properties. In the physiological route, in the presence of Mg²⁺ and Na⁺, the E2P state is reached through phosphorylation by ATP (path A in Fig. 1) and denoted here as E2P^ATP. The E2P state can be reached by backward phosphorylation by P_i in the presence of Mg²⁺ (path B in Fig. 1) and denoted as E2P^Pi [denoted previously as E′2P (9)]. These states show different kinetic properties. In particular, dephosphorylation of E2P^ATP is fast if K⁺ is present, whereas that of E2P^Pi is slow and hardly accelerated by K⁺ (9, 10). As this insensitivity is due to the binding of a second Mg²⁺ to the ATPase in E2P^Pi (10), it would be more appropriate to denote this state as E2P^Pi·Mg²⁺ (Fig. 1). As the affinity of Mg²⁺ in E2P^Pi is ∼0.5 mM (10), the majority of the ATPase molecules phosphorylated by P_i will be in this state. E2P^ATP has a low affinity for Mg²⁺ (not saturated at 6 mM) (10). Therefore, the transmembrane cation binding sites and, accordingly, the CTS-binding cavity will be different in the two E2P states. Indeed, the signal from RH421, a voltage-sensitive styryl dye, is clearly different (9). Then, the inhibitory properties of CTSs will also be different in these two E2P states (type I and II complexes in refs. 11, 12). Furthermore, if phosphorylation by P_i + Mg²⁺ is performed in the presence of K⁺, another type of E2P form with loosely occluded K⁺, termed E2P^Pi·2K⁺, is generated (path C in Fig. 1). This form has a high rate of dephosphorylation (9, 10). OBN is well known to have a much-reduced affinity in the presence of K⁺ (K⁺ antagonism) (e.g., ref. 13), but other CTSs have not been well characterized in this regard. Indeed, Laursen et al., reported that bufalin (BUF) requires K⁺ for high-affinity binding (14). In a recent report (15), the difference in K⁺ antagonism is attributed to the lactone ring. Therefore, systematic measurements on the inhibitory potency in the three E2P states are clearly required, in addition to the one under turnover conditions.Confusion in the literature is apparent even in structural studies. There are several crystal structures published for NKA with bound CTSs: those in E2·P_i·2K⁺ with ouabain at low affinity (2.8-Å resolution) (16), BUF in E2P^Pi·2K⁺ (3.4-Å resolution) (14), and those in E2P^Pi·Mg²⁺ with ouabain at high affinity (3.4 Å) (17) or digoxin (3.9 Å) (14). All of the crystals of the high-affinity complexes are generated in the presence of a high concentration (>100 mM) of Mg²⁺, and indeed, Mg²⁺ is observed to occupy site II for K⁺. Therefore, the E2P state stabilized by CTSs should be denoted as E2P^Pi·Mg²⁺ (Fig. 1). These crystal structures have established that the high affinity of CTSs primarily arises from complementarity between the M5 helix and the α-face of the steroid core, consistent with mutagenesis studies (18–22). However, other than this, there seems to be serious discrepancies between biochemical and structural data. For instance, ouabagenin (OBG), which lacks rhamnose attached to C3, has a 300-fold reduced affinity in binding to NKA in E2P^Pi·Mg²⁺, but Laursen et al. (17) describes that the sugar moiety in ouabain does not interact with the ATPase. Mutagenesis studies have identified residues responsible for isoform dependence (23, 24), but the crystal structure failed to explain why (24). We really do not know if any structural changes are caused by CTS binding to NKA, because no structure is available for the E2P ground state without CTS.We answer this question in this report, as we now have crystal structures of the BeF₃⁻ complex of NKA, an E2P ground state analog, free of CTS. Systematic measurements of the inhibitory properties of various CTSs, including ROS and IST, under four different conditions provide a basis for addressing their structure-activity relationships. One striking finding is that ROS shows a much higher affinity under turnover conditions than in E2P^Pi·Mg²⁺, in marked contrast to OBN. Such differences, as well as the K⁺ antagonism, are nicely explained by the crystal structures of NKA with various CTSs. The crystal structures also explain the isoform dependence unambiguously and suggest ways to confer α2 specificity on CTSs.     

Cryo-EM structure of gastric H+,K+-ATPase with a single occupied cation-binding site

Gastric H⁺,K⁺-ATPase is responsible for gastric acid secretion. ATP-driven H⁺ uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K⁺, resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H⁺/2K⁺ to 1H⁺/1K⁺ per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K⁺ congener (Rb⁺), the 8-Å resolution structure of H⁺,K⁺-ATPase in the transition state of dephosphorylation, (Rb⁺)E2∼AlF, which is distinct from the preceding Rb⁺-free E2P state. A strong density located in the transmembrane cation-binding site of (Rb⁺)E2∼AlF highly likely represents a single bound Rb⁺ ion, which is clearly different from the Rb⁺-free E2AlF or K⁺-bound (K⁺)E2∼AlF structures. Measurement of radioactive ⁸⁶Rb⁺ binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb⁺ is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H⁺/1K⁺/1ATP transport mode of H⁺,K⁺-ATPase, which is a prerequisite for generation of the 10⁶-fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the β subunit’s N terminus and the P domain observed in the (Rb⁺)E2∼AlF structure, we propose a refined vectorial transport model of H⁺,K⁺-ATPase, which must prevail against the highly acidic state of the gastric lumen.     

Association between Serum Potassium and Outcomes in Patients with Reduced Kidney Function

Background and objectives

Patients with CKD are more likely than others to have abnormalities in serum potassium (K⁺). Aside from severe hyperkalemia, the clinical significance of K⁺ abnormalities is not known. We sought to examine the association of serum K⁺ with mortality and hospitalization rates within narrow eGFR strata to understand how the burden of hyperkalemia varies by CKD severity. Associations were examined between serum K⁺ and discontinuation of medications that block the renin-angiotensin-aldosterone system (RAAS), which are known to increase serum K⁺.

Design, setting, participants, & measurements

A cohort of patients with CKD (eGFR<60 ml/min per 1.73 m²) with serum K⁺ data were studied (n=55,266) between January 1, 2009, and June 30, 2013 (study end). Serum K⁺, eGFR, and covariates were considered on a time-updated basis. Mortality, major adverse cardiovascular events (MACE), hospitalization, and discontinuation of RAAS blockers were considered per time at risk.

Results

During the study, serum K⁺ levels of 5.5–5.9 and ≥6.0 mEq/L were most prevalent at lower eGFR: they were present, respectively, in 1.7% and 0.2% of patient-time for eGFR of 50–59 ml/min per 1.73 m² versus 7.6% and 1.8% of patient-time for eGFR<30 ml/min per 1.73 m². Serum K⁺ level <3.5 mEq/L was present in 1.2%–1.4% of patient-time across eGFR strata. The median follow-up time was 2.76 years. There was a U-shaped association between serum K⁺ and mortality; pooled adjusted incidence rate ratios were 3.05 (95% confidence interval, 2.53 to 3.68) and 3.31 (95% confidence interval, 2.52 to 4.34) for K⁺ levels <3.5 mEq/L and ≥6.0 mEq/L, respectively. Within eGFR strata, there were U-shaped associations of serum K⁺ with rates of MACE, hospitalization, and discontinuation of RAAS blockers.

Conclusions

Both hyperkalemia and hypokalemia were independently associated with higher rates of death, MACE, hospitalization, and discontinuation of RAAS blockers in patients with CKD who were not undergoing dialysis. Future studies are needed to determine whether interventions targeted at maintaining normal serum K⁺ improve outcomes in this population.     

K regulates bacteroid-associated functions of Bradyrhizobium

Cowpea Bradyrhizobium 32H1 cells, when grown under 0.2% O₂, synthesize nitrogenase, as well as a methylammonium (ammonium) transport system and an electrogenic K⁺/H⁺ antiporter. This effect was seen in growth medium containing 8-12 mM K⁺ but not with 50 μM K⁺. Addition of K⁺ to cells growing under low O₂ tensions in low-K⁺ medium led to various phenotypic properties associated with bacteroids, including the ability to reduce acetylene, induction of an ammonium transport carrier and the K⁺/H⁺ antiporter, and increased synthesis of two heme-biosynthetic enzymes, δ-aminolevulinate synthase and δ-aminolevulinate dehydratase. K⁺ addition caused the repression of glutamine synthetase and of capsular polysaccharide synthesis, functions related to the free-living state. A similar pattern of regulation was observed in Bradyrhizobium japonicum. In addition, K⁺-mediated depression in Bradyrhizobium 32H1 was inhibited by exudate of Vigna unguiculata, its host plant. We conclude that K⁺ ions, in addition to low O₂ tension, are needed for the expression of several bacteroid-related functions in bradyrhizobia and thus are a major controlling influence in bacteroid development.     

Tissue kallikrein permits early renal adaptation to potassium load

Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K⁺ intake, we hypothesized that this enzyme might regulate plasma K⁺ concentration ([K⁺]). We showed in wild-type mice that renal K⁺ and TK excretion increase in parallel after a single meal, representing an acute K⁺ load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption (TK^−/−) were used to assess the impact of the enzyme on plasma [K⁺]. A single large feeding did not lead to any significant change in plasma [K⁺] in TK^+/+, whereas TK^−/− mice became hyperkalemic. We next examined the impact of TK disruption on K⁺ transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK^−/− mice exhibit net transepithelial K⁺ absorption because of abnormal activation of the colonic H⁺,K⁺-ATPase in the intercalated cells. Finally, in CCDs isolated from TK^−/− mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H⁺,K⁺-ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K⁺ load.     

Structures,Bonding and Sensor Properties of Some Alkaline o-Phthalatocuprates

Comprehensive study of the structure and bonding of disodium, dipotassium and diammonium di-o-phthalatocuprates(II) dihydrates has been undertaken. The crystal structure of ammonium o-phthalatocuprate has been determined. The identity of structures of phthalatocuprate chains in potassium and ammonium salts has been revealed. Vibrational spectra of all three compounds have been recorded, and the assignment of vibrational bands has been made. Force field calculations have shown a minor effect of outer-sphere cations (Na⁺, K⁺, NH₄⁺) on both intraligand (C-O) and metal–ligand bond strengths. Synthesized compounds have been tested as electrochemical sensors on D-glucose, dopamine and paracetamol. Their sensitivity to analytes varied in the order of Na⁺ > K⁺ > NH₄⁺. This effect has been explained by the more pronounced steric hindrance of copper ions in potassium and ammonium salts.     

Rearrangement of a unique Kv1.3 selectivity filter conformation upon binding of a drug

We report two structures of the human voltage-gated potassium channel (Kv) Kv1.3 in immune cells alone (apo-Kv1.3) and bound to an immunomodulatory drug called dalazatide (dalazatide–Kv1.3). Both the apo-Kv1.3 and dalazatide–Kv1.3 structures are in an activated state based on their depolarized voltage sensor and open inner gate. In apo-Kv1.3, the aromatic residue in the signature sequence (Y447) adopts a position that diverges 11 Å from other K⁺ channels. The outer pore is significantly rearranged, causing widening of the selectivity filter and perturbation of ion binding within the filter. This conformation is stabilized by a network of intrasubunit hydrogen bonds. In dalazatide–Kv1.3, binding of dalazatide to the channel’s outer vestibule narrows the selectivity filter, Y447 occupies a position seen in other K⁺ channels, and this conformation is stabilized by a network of intersubunit hydrogen bonds. These remarkable rearrangements in the selectivity filter underlie Kv1.3’s transition into the drug-blocked state.

Potassium channels form K⁺-selective pores that span cell membranes in virtually all living organisms. In humans, a family of 78 genes encodes four classes of K⁺ channels (voltage-gated, calcium-activated, inward rectifier, and two-pore channels), which are involved in a multitude of physiological functions in both electrically excitable and nonexcitable cells (1). All four classes of channels conduct K⁺ ions selectively and rapidly, but they differ in how they are gated. The selectivity filter is the structural element responsible for the exquisitely K⁺-selective pore (2–5). It is the narrowest part of the ion conduction pathway and connects a water-filled cavity in the center of the protein with an outer vestibule in the extracellular solution. The filter accommodates K⁺ ions at four sites called S1, S2, S3, and S4 starting at the extracellular side. The signature sequence G(Y/F)G in the selectivity filter plays a critical role in making the pore K⁺ selective (6, 7). In all K⁺ channel structures determined, both bacterial and eukaryotic, the aromatic residue (Y or F) in the signature sequence is nearly identical in position, although these channels differ in the conformation (closed or open) of the S6 helical inner gate (8). In the hERG/Kv11.1 channel, a subtle deviation in the position of F627 in the signature sequence causes a slight widening of the selectivity filter, which has been suggested to underlie the channel’s transition into the C-type inactivated state (8).The voltage-gated potassium channel (Kv) Kv1.3–Kvβ2 in lymphocytes and microglia provides the counterbalancing cation efflux to promote calcium entry necessary for calcium signaling (9, 10). Selective blockers of Kv1.3–Kvβ2 treat diverse autoimmune and neuroinflammatory diseases in rodent models (9, 10), highlighting the channel’s physiological and pharmacological importance. Here, we determined structures of Kv1.3 complexed to its accessory subunit Kvβ2 alone (apo-Kv1.3) and bound to dalazatide (dalazatide–Kv1.3), a potent and selective peptide inhibitor of Kv1.3 in clinical trials for autoimmune and neuroinflammatory diseases (10–14). Both apo-Kv1.3 and dalazatide–Kv1.3 are in the activated state based on the depolarized voltage sensor and open S6 helical inner gate. Comparison of the two structures reveals substantial conformational changes in the selectivity filter. In apo-Kv1.3, Y447 in the signature sequence diverges more than 11 Å from the position of corresponding aromatic residues in other K⁺ channels, both in eukaryotes and bacteria. The outer pore is wider at S1 and S2 and narrowed at S0 K⁺-binding sites, resulting in loss of the K⁺ ion from site S2. A network of intrasubunit hydrogen bonds (H451–Y447, H451–D449) stabilizes this unique conformation of the selectivity filter of apo-Kv1.3, and, interestingly, the intrasubunit hydrogen bond (W436–D449) that prevents C-type inactivation (15) is absent. Apo-Kv1.3’s selectivity filter and voltage-sensing domain (VSD) differ significantly from two structures of Kv1.3 that were recently described (16). In dalazatide–Kv1.3, dalazatide’s interaction with H451 disrupts the H451–Y447 hydrogen bond, freeing Y447 to swing back into the interior of the selectivity filter and adopt a position seen in other K⁺ channels. The selectivity filter is narrower, and K⁺ ions are present at sites S2–S4 but not at S1. This conformation is stabilized by a network of intersubunit hydrogen bonds (Y447–W437, Y447–T441, and H451–D449), but the intrasubunit hydrogen bond (W436–D449) that prevents C-type inactivation (15) is likely absent. Our structures provide a basis for the design of Kv1.3 inhibitors for use as immunomodulatory therapeutics.