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.     

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.     

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.Ion conduction through the pore domain of cation channels is regulated by two gates: an inner gate at the bundle crossing of the pore-lining transmembrane helices and an outer gate located at the selectivity filter (Fig. 1 B and C). These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1–4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4–10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5–14). However, flux-dependent inactivation occurs in Na⁺ and Ca²⁺ channels as well and would likely require a structurally different mechanism to explain coupling between the selectivity filter and inner gate (7, 13–18).Open in a separate windowFig. 1.Crystal structures of the nonselective cation channel NaK and the potassium-selective NaK2K mutant show structural changes restricted to the area of the selectivity filter. Alignment of the WT NaK (gray; PDB 3E8H) and NaK2K (light blue; PDB 3OUF) selectivity filters shows a KcsA-like four-ion-binding-site selectivity filter is created by the NaK2K mutations (D66Y and N68D) (A), but no structural changes occur outside the vicinity of the selectivity filter (B). (C) Full-length NaK (green; PDB 2AHZ) represents a closed conformation. Alignment of this structure with NaK (gray) highlights the changes in the M2 hinge (arrow), hydrophobic cluster (residues F24, F28, and F94 shown as sticks), and constriction point (arrow; residue Q103 shown as sticks) upon channel opening. Two (A) or three monomers (B and C) from the tetramer are shown for clarity.This study provides experimental evidence of structural and dynamic coupling between the inner gate and selectivity filter in the NaK channel, a nonselective cation channel from Bacillus cereus (19). These results were entirely unexpected given the available high-resolution crystal structures (20, 21). The NaK channel has the same basic pore architecture as K⁺ channels (Fig. 1 B and C) and has become a second model system for investigating ion selectivity and gating due to its distinct selectivity filter sequence (₆₃TVGDGN₆₈) and structure (19–23). Most strikingly, there are only two ion binding sites in the selectivity filter of the nonselective NaK channel (Fig. 1A) (21, 24). However, mutation of two residues in the selectivity filter sequence converts the NaK selectivity filter to the canonical KcsA sequence (₆₃TVGYGD₆₈; Fig. 1 A and B), leading to K⁺ selectivity and a KcsA-like selectivity filter structure with four ion binding sites (21, 23). This K⁺-selective mutant of NaK is called NaK2K. Outside of the immediate vicinity of the two mutations in the selectivity filter, high-resolution crystal structures of NaK and NaK2K are essentially identical (Fig. 1B) with an all-atom rmsd of only 0.24 Å.NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating because there is no evidence for any collapse or structural change in the selectivity filter. The NaK selectivity filter structure is identical in Na⁺ or K⁺ (22) and even in low-ion conditions (25), consistent with its nonselective behavior. Even the selective NaK2K filter appears structurally stable in all available crystal structures (25). Here we use NMR spectroscopy to study bicelle-solubilized NaK. Surprisingly, we find significant differences in the NMR spectra of NaK and NaK2K that extend throughout the protein and are not localized to the selectivity filter region. This, combined with NMR dynamics studies of NaK, suggests a dynamic pathway for transmembrane coupling between the inner gate and selectivity filter of NaK.     

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.     

Initial steps of inactivation at the K+ channel selectivity filter

K⁺ efflux through K⁺ channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel’s selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K⁺ channel. Either Mg²⁺ or Ca²⁺ can reduce K⁺ efflux through MthK channels. However, Ca²⁺, but not Mg²⁺, can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca²⁺ ions can partially dehydrate, enabling selective accessibility of Ca²⁺ to a site at the entry to the selectivity filter. Ca²⁺ binding at the site interacts with K⁺ ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.Potassium (K⁺) channels are activated and opened by a variety of stimuli, including ligand binding and transmembrane voltage, to enable K⁺ efflux and thus, modulate physiological processes related to electrical excitability, such as regulation of action potential firing, smooth muscle contraction, and hormone secretion (1). In addition, many K⁺ channels are further controlled by a gating phenomenon known as C-type inactivation, in which K⁺ conduction is stopped, despite the continued presence of an activating stimulus (2). The mechanisms underlying C-type inactivation in voltage-gated K⁺ channels (Kv channels) are linked to both intracellular and extracellular permeant ion concentrations, and several lines of evidence have suggested that C-type inactivation is associated with a conformational change near the external mouth of the K⁺ channel pore (i.e., at the canonical K⁺ channel selectivity filter) (3–11).In Shaker Kv channels, C-type inactivation is known to be enhanced and recovery from inactivation is slowed by impermeant cations accessing the cytoplasmic side of the channel (5, 6, 10). Enhancement of inactivation by these cations suggests a working hypothesis, in which the impermeant ion prevents refilling of the selectivity filter with K⁺ (6). Thus, K⁺ presumably dissociates from the filter to the external solution, and this vacancy leaves the filter susceptible to a conformational change that underlies the nonconducting, inactivated state. However, the physical basis for the relation between ion movements and C-type inactivation as well as the structural underpinnings of the mechanism remain unclear.Here, we use divalent metal cations (Mg²⁺, Ca²⁺, and Sr²⁺) as probes of inactivation mechanisms in MthK, a model K⁺ channel of known structure (Fig. 1) (12–14). Specifically, we analyze conduction and gating of single MthK channels by electrophysiology combined with analysis of ion and protein movements by molecular simulation. Our electrophysiological experiments indicate that, although each of these divalent metal ions can reduce the size of single channel currents, only Ca²⁺ and Sr²⁺ can enhance inactivation, whereas Mg²⁺ does not. Using molecular simulation and potential of mean force (PMF) calculations, we find that Ca²⁺, but not Mg²⁺, can shed its hydration shell waters to access a site, termed S5, at the entry to the channel’s selectivity filter (Fig. 1C) after displacement of K⁺ ions to the extracellular side of the channel. Subsequent dissociation of a K⁺ ion from the filter, in turn, favors a conformational change within the selectivity filter, contributing to enhanced inactivation. These results support a working hypothesis that directly relates dissociation of K⁺ with a structural change in the selectivity filter to initiate inactivation of K⁺ channels.Open in a separate windowFig. 1.Structure and activation properties of MthK. (A) Presumed biological structure of MthK shown as a Cα-trace [Protein Data Bank (PDB) ID code 3RBZ]. The channel consists of a transmembrane pore domain tethered to a ring of RCK domains, which mediate channel activation by cytoplasmic Ca²⁺ (green spheres). The gray-shaded region represents the presumed plasma membrane; dashed lines represent the linker region between the pore and RCK gating ring that is unresolved in the crystal structure. (B) High-resolution structure of the MthK pore domain, with the selectivity filter shown in ball and stick representation (PDB ID code 3LDC). Subunits in the front and back have been removed for clear visualization of the conduction pathway (inside dashed rectangle), with K⁺ ions shown as purple spheres and ordered water molecules shown as red spheres. (C) –Magnified view of the MthK conduction pathway (boxed region in B) with potential ion binding sites (S0–S_cav) indicated. (D) Po vs. [Ca²⁺] (black symbols) and [Cd²⁺] (red symbols) from currents recorded at −100 mV. MthK activation requires ∼20-fold lower [Cd²⁺] compared with [Ca²⁺]. Curves represent fits with a Hill equation with the following parameters: EC₅₀ = 1.0 mM and n_H = 9.5 for Ca²⁺; EC₅₀ = 49 μM and n_H = 8.4 for Cd²⁺. (E) Representative single channel currents from reconstituted MthK at depolarized voltages with 200 mM KCl at both sides of the membrane and Ca²⁺ or Cd²⁺ at the cytoplasmic side of the channel as indicated. Cd²⁺ can fully activate MthK at concentrations that produce much less fast blockade than Ca²⁺. O and C indicate open and closed current levels, respectively. (F) Unitary current vs. voltage for MthK channels activated with 30 and 100 μM Cd²⁺ (green and red, respectively) and 2 mM Ca²⁺ (black). Smooth curves are drawn for display only; 100 μM Cd²⁺ results in nominal levels of fast blockade, yielding large outward current.     

BK channel opening involves side-chain reorientation of multiple deep-pore residues

Three deep-pore locations, L312, A313, and A316, were identified in a scanning mutagenesis study of the BK (Ca²⁺-activated, large-conductance K⁺) channel S6 pore, where single aspartate substitutions led to constitutively open mutant channels (L312D, A313D, and A316D). To understand the mechanisms of the constitutive openness of these mutant channels, we individually mutated these three sites into the other 18 amino acids. We found that charged or polar side-chain substitutions at each of the sites resulted in constitutively open mutant BK channels, with high open probability at negative voltages, as well as a loss of voltage and Ca²⁺ dependence. Given the fact that multiple pore residues in BK displayed side-chain hydrophilicity-dependent constitutive openness, we propose that BK channel opening involves structural rearrangement of the deep-pore region, where multiple residues undergo conformational changes that may increase the exposure of their side chains to the polar environment of the pore.Large-conductance, Ca²⁺-activated K⁺ (BK) channels regulate physiological processes such as neurotransmitter release, smooth muscle contraction, and hair cell frequency tuning (1–12). BK channel proteins are homotetramers formed by BK α-subunits, which then associate with different β- or γ-subunits in a tissue-specific manner (13–22). The α-subunit of the BK (K_Ca1.1) channel is encoded by the KCNMA1 gene, first discovered in Drosophila as the slowpoke mutation (dSlo) (23, 24), and later identified in mouse (mSlo1) and human (hSlo1) (25, 26). Each α-subunit has seven transmembrane segments (S0–S6), with the S6 segments lining the pore. A similar structural arrangement is found in other members of the K⁺ channel protein family.From a functional point of view, the gating behavior of BK channels can be described as a central C⇔O (i.e., closed⇔open) transition, influenced by voltage sensor movement and/or Ca²⁺ binding (27–31). The structural basis for this C⇔O transition is believed to be conformational changes of the S6 segment and/or the selectivity filter (32, 33), controlling the passage of K⁺ ions across the membrane.Molecular details of gating-related conformational changes have been probed with mutagenesis-based methods for BK and related channels. Although it has been demonstrated that voltage-gated (Kv) K⁺ channels open by a cytoplasmic S6 “bundle crossing” gate (34–37), evidence has accumulated that the opening conformational change of BK, like cyclic nucleotide-gated (CNG) channels, occurs deeper in the pore, closer to the selectivity filter (38–46). With the goal of further understanding the opening conformational change in BK channels, we used a histidine substitution/protonation strategy and identified a residue in BK S6 (M314 in hSlo1) whose side chain turns more toward the pore when the channel is open. The open conformation can be stabilized by the presence of side-chain charges at this location, with the aspartate mutant being the most effective in keeping the channel open in neutral pH (47).To uncover more dynamic details at other pore residues during BK channel gating, we scanned the S6 segment cytoplasmic to the selectivity filter with single aspartate substitutions (I308D→N328D). Because the S6 residues of K⁺ channels reside on the interface between a polar (the water and ion filled pore) and a nonpolar (the rest of the protein in the membrane) environment, we expect the charged side chains of substituted aspartates to prefer the more aqueous environment of the pore, making the conformations with such side-chain orientation energetically favorable. If these conformations correspond to any functional states of the channel, such states may be stabilized, and will be functionally measurable. Our previous studies identified the M314D mutant channels as favoring the open state, consistent with greater exposure of this side chain to the pore upon opening.In addition, the S6-formed pore is believed to host a “gate” that, when closed, prevents the passage of ions. The nature of such a gate, in its closed conformation, is proposed to be an occlusion structure formed by the side chains of amino acid residues at the gate location. In the structural model of the closed KcsA channel, at three residue locations (T107, A111, V115) to the intracellular end of its TM2 (equivalent of S6 in mammalian K⁺ channels) (Fig. 1), one could see the side chains at equivalent locations from the four subunits come very close to each other in the pore, occluding passage of K⁺ (48, 49). In the structural model of an open KcsA channel, pore diameters at the same locations are significantly larger (50, 51).Open in a separate windowFig. 1.Sequence alignment and structural models. (A) Sequence alignment of KcsA (accession no. {"type":"entrez-protein","attrs":{"text":"NP_631700","term_id":"21225921","term_text":"NP_631700"}}NP_631700), Shaker (accession no. {"type":"entrez-protein","attrs":{"text":"CAA29917","term_id":"288442","term_text":"CAA29917"}}CAA29917), and hSlo1 (accession no. {"type":"entrez-protein","attrs":{"text":"AAB65837","term_id":"507922","term_text":"AAB65837"}}AAB65837), starting from the threonine in the “TVGYGD” signature sequence of the K⁺ channel selectivity filter. The numbers by the channel name indicate the sequence number for the threonine in the corresponding channel. Highlighted in yellow are T107, A111, and V115 of KcsA, and P475, V478, and F481 of Shaker. Three residues of hSlo1 are highlighted as follows: L312, blue; A313, red; A316, green. The brown bar below the sequences indicates the selectivity filter, and the gray bar indicates residues scanned by aspartate substitution. (B) Side, top, and bottom views of two KcsA structure models: 1R3J, the closed conformation; and 3F5W, the open conformation. Only the P and S6 helices are shown. In the side views, one of the four subunits was taken away to better view the pore. The brown vertical lines indicate ion permeation through the selectivity filter. The equivalent residues to L312, A313, and A316 are colored the same way as in A.One way to functionally locate such a tightly packed occlusion structure is to try to disrupt it with small, charged, or large side-chain amino acid substitutions. Application of this method on the Shaker K⁺ channels, together with prior evidence from cysteine accessibility experiments, identified V478 or F481 as candidate locations for the gate (52). With location P475, the aspartate mutant is constitutively open, the result of a shifted energetic balance between the closed and the open states (52, 53) (Fig. 1). If the BK channel gate is also formed by specific, intersubunit hydrophobic interactions between four equivalent residues in the tetramer, such as suggested by the KcsA structure, the charges on the substituted aspartate side chains may also prevent such interactions and reveal the location of the gate.     

Catalysis of Na+ permeation in the bacterial sodium channel NaVAb

Determination of a high-resolution 3D structure of voltage-gated sodium channel Na_VAb opens the way to elucidating the mechanism of ion conductance and selectivity. To examine permeation of Na⁺ through the selectivity filter of the channel, we performed large-scale molecular dynamics simulations of Na_VAb in an explicit, hydrated lipid bilayer at 0 mV in 150 mM NaCl, for a total simulation time of 21.6 μs. Although the cytoplasmic end of the pore is closed, reversible influx and efflux of Na⁺ through the selectivity filter occurred spontaneously during simulations, leading to equilibrium movement of Na⁺ between the extracellular medium and the central cavity of the channel. Analysis of Na⁺ dynamics reveals a knock-on mechanism of ion permeation characterized by alternating occupancy of the channel by 2 and 3 Na⁺ ions, with a computed rate of translocation of (6 ± 1) × 10⁶ ions⋅s⁻¹ that is consistent with expectations from electrophysiological studies. The binding of Na⁺ is intimately coupled to conformational isomerization of the four E177 side chains lining the extracellular end of the selectivity filter. The reciprocal coordination of variable numbers of Na⁺ ions and carboxylate groups leads to their condensation into ionic clusters of variable charge and spatial arrangement. Structural fluctuations of these ionic clusters result in a myriad of ion binding modes and foster a highly degenerate, liquid-like energy landscape propitious to Na⁺ diffusion. By stabilizing multiple ionic occupancy states while helping Na⁺ ions diffuse within the selectivity filter, the conformational flexibility of E177 side chains underpins the knock-on mechanism of Na⁺ permeation.The rapid passage of cations in and out of excitable cells through selective pathways underlies the generation and regulation of electrical signals in all living organisms (1–4). The metazoan cell membrane is exposed to a high-Na⁺, low-K⁺ concentration on the extracellular (EC) side, and to a low-Na⁺, high-K⁺ concentration on the intracellular (IC) side. Selective voltage-gated Na⁺ and K⁺ channels control the response of the cell to changes in the membrane potential. In particular, voltage-gated Na⁺ channels (Na_V) are responsible for the initiation and propagation of action potentials in cardiac and skeletal myocytes, neurons, and endocrine cells (1–4). Mutations in Na_V channel genes are responsible for a wide range of debilitating channelopathies, including congenital epilepsy, paramyotonia, erythromelalgia, familial hemiplegic migraine, paroxysmal extreme pain disorder, and periodic paralyses (5, 6), underlining the importance of deciphering the relationship between the structure and function of Na_V channels. Here, we use molecular simulations to study the binding and permeation of Na⁺ in bacterial sodium channel Na_VAb.Although several atomic structures of K⁺-selective channels have been solved over the past decade (7–12), the atomic structure of an Na⁺-selective channel from the bacterium Arcobacter butzleri, Na_VAb, was reported only recently (13). In the preopen state of Na_VAb (13), the pore is closed at the IC gate, but the selectivity filter (SF) appears to be in its open, functional state. The molecular structure of the SF of Na_VAb (TLESW) differs significantly from that of potassium channels such as KcsA (TVGYG), in that it is both wider and shorter. In KcsA, channel coordination of permeating cations consists almost entirely of direct interactions with backbone carbonyl oxygen atoms. In contrast, in Na_VAb, the SF is lined with amino acid side chains from S178 and E177 in addition to backbone carbonyl groups from T175 and L176 (7, 8, 10, 13). Due to the tetrameric domain arrangement of Na_VAb, the E177 site forms a ring of four glutamate side chains (EEEE) in the same sequence positions as the characteristic DEKA ring of eukaryotic sodium channels (14, 15). The presence of charged and titratable carboxylate groups in the SF of Na_v channels raises major questions about the catalytic mechanism for ionic permeation and the structural basis for ion selectivity.As a first step toward elucidating the structural basis of ionic permeation and selectivity, we examine the movement of Na⁺ ions in and out of the pore from equilibrium molecular dynamics (MD) simulations of Na_VAb in a hydrated lipid bilayer (Fig. S1). Forty-seven time trajectories totaling 21.6 μs were generated at 300 K in the presence of 150 mM NaCl to mimic the physiological environment of the periplasm. We analyzed Na⁺ diffusion at a potential of 0 mV, similar to the peak of macroscopic Na⁺ current during an action potential or a voltage clamp experiment in nerve or muscle cells. The analysis of hundreds of spontaneous events of Na⁺ diffusion through the SF provides detailed insight into a knock-on mechanism of Na⁺ permeation involving alternating ion-occupancy states and resulting in an estimated translocation rate of (6 ± 1) × 10⁶ ions⋅s⁻¹.     

Family of prokaryote cyclic nucleotide-modulated ion channels

Cyclic nucleotide-modulated ion channels are molecular pores that mediate the passage of ions across the cell membrane in response to cAMP or GMP. Structural insight into this class of ion channels currently comes from a related homolog, MloK1, that contains six transmembrane domains and a cytoplasmic cyclic nucleotide binding domain. However, unlike eukaryote hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, MloK1 lacks a C-linker region, which critically contributes to the molecular coupling between ligand binding and channel opening. In this study, we report the identification and characterization of five previously unidentified prokaryote homologs with high sequence similarity (24–32%) to eukaryote HCN and CNG channels and that contain a C-linker region. Biochemical characterization shows that two homologs, termed AmaK and SthK, can be expressed and purified as detergent-solubilized protein from Escherichia coli membranes. Expression of SthK channels in Xenopus laevis oocytes and functional characterization using the patch-clamp technique revealed that the channels are gated by cAMP, but not cGMP, are highly selective for K⁺ ions over Na⁺ ions, generate a large unitary conductance, and are only weakly voltage dependent. These properties resemble essential properties of various eukaryote HCN or CNG channels. Our results contribute to an understanding of the evolutionary origin of cyclic nucleotide-modulated ion channels and pave the way for future structural and functional studies.Hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels belong to the superfamily of voltage-gated K⁺ channels. Both types of channels share a similar domain topology with six transmembrane domains, a C-linker region, and a cyclic nucleotide binding domain (CNBD). The S5–S6 segment forms the channel pore, including the selectivity filter for cations. The S4 segment contains several positively charged amino acids, suggesting that it acts as voltage sensor. Despite these similarities in sequence, the function of HCN and CNG channels is noticeably different: HCN channels activate upon membrane hyperpolarization and can be modulated by cyclic nucleotides. They are weakly selective for K⁺ over Na⁺ ions (for reviews, see refs. 1–3). In contrast, CNG channels are activated by the binding of cyclic nucleotides solely and their activity depends only weakly on voltage. The ionic current is carried by both monovalent and divalent cations (for reviews, see refs. 4 and 5).Insight into the structure of HCN channels has been gained only from crystal structures of the isolated intracellular C-linker and CNBD of mammalian HCN1, HCN2, HCN4, and invertebrate spHCN1. These parts of the channel assemble into tetramers (6–9). Further structural information comes from prokaryote ion channels that are homologous to HCN and CNG channels, such as the bacterial cyclic nucleotide-regulated K⁺ channel MloK1 (10–13). MloK1 lacks a C-linker region, but has a CNBD with an overall structure that is remarkably similar to the CNBD of eukaryote HCN channels (10). Based on the dimer assembly of the MloK1 CNBD in the crystal structure, a gating mechanism has been proposed in which the pore opening in the tetrameric channel arises from the action of the four CNBDs as a dimer of dimers (10). The crystal structure of the MloK1 transmembrane domain (11) reveals a domain topology that resembles that of the voltage-gated K⁺ channel Kv1.2 (14), but with important differences. The MloK1 structure suggests that the S1–S4 domain and its associated linker in MloK1 can serve as a clamp to constrain the gate and possibly function in concert with the CNBD to regulate channel opening (11). Additionally, crystal structures have also been determined for the C-linker and cyclic nucleotide binding homology domain (CNBHD) of related ion channels, including the zebrafish EAG-like (ELK) K⁺ channel (15), the mosquito ERG K⁺ channel (16), and the mouse EAG1 K⁺ channel (17). Structural insight into the mechanism of ion permeation has been derived from a prokaryote ion channel NaK (18), which was mutated to mimic the CNG channel pore region (19). Collectively, these structural data have brought valuable information about the determinants of ion permeation, domain assembly, ligand recognition, channel gating and regulation, as well as effects of disease-causing mutations (20).Despite this tremendous progress, crystal structures for whole-eukaryote HCN and CNG channels are still not available at present, and structural insight into fundamental aspects of ion channel function is still lacking, such as the inverse voltage sensitivity in HCN channels and the coupling between cyclic nucleotide binding and channel opening by the C-linker domain, which is, as mentioned, absent in the MloK1 channel (10). In contrast, a putative voltage-gated K⁺ channel containing a C-linker region and CNBD similar to eukaryote channels was identified in the genome of the cyanobacterium Trichodesmium erythraeum (21), here termed TerK, and it was suggested to possibly represent an ancestral HCN or CNG channel (21). However, neither structure nor function of this prokaryote homolog is known. In this study, we report the characterization of TerK and four additional prokaryote ion channels, which all contain six putative transmembrane domains, a C-linker region, and a CNBD, and apparently form a family of prokaryote ion channels with close similarity to eukaryote HCN and CNG channels. We describe the expression in Escherichia coli, detergent screening and biochemical purification of these different homologs. Moreover, we identified two homologs, SthK (from Spirochaeta thermophila) and AmaK (from Arthrospira maxima), which can be stably extracted with detergents and purified in sufficiently high amounts for biochemical and structural studies. Using confocal fluorescence microscopy and electrophysiological recordings, we describe essential functional properties of one homolog, SthK. We find that SthK has electrophysiological properties that closely resemble those of eukaryote CNG channels as it is gated by intracellular cAMP and produces large unitary currents, whereas its activity is relatively insensitive to voltage. However, unlike CNG channels, SthK contains the selectivity filter sequence -TIGYGD-, which is more similar to HCN channels and other K⁺ selective channels. We could experimentally demonstrate that SthK channels are highly selective for K⁺ over Na⁺ ions. Importantly, SthK has several sequence features that closely resemble eukaryote cyclic nucleotide-modulated channels, including a C-linker region, which is missing in previously studied prokaryote homologs, such as MloK1 (10, 12, 13) and MmaK (22). Together, these data make the SthK channel a promising candidate for future structural analysis to learn more about how mammalian CNG and HCN channels work.     

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

Over the past two decades, enormous progress has been made in designing fluorescent sensors or probes for divalent metal ions. In contrast, the development of fluorescent sensors for monovalent metal ions, such as sodium (Na⁺), has remained underdeveloped, even though Na⁺ is one the most abundant metal ions in biological systems and plays a critical role in many biological processes. Here, we report the in vitro selection of the first (to our knowledge) Na⁺-specific, RNA-cleaving deoxyribozyme (DNAzyme) with a fast catalytic rate [observed rate constant (k_obs) ∼0.1 min⁻¹], and the transformation of this DNAzyme into a fluorescent sensor for Na⁺ by labeling the enzyme strand with a quencher at the 3′ end, and the DNA substrate strand with a fluorophore and a quencher at the 5′ and 3′ ends, respectively. The presence of Na⁺ catalyzed cleavage of the substrate strand at an internal ribonucleotide adenosine (rA) site, resulting in release of the fluorophore from its quenchers and thus a significant increase in fluorescence signal. The sensor displays a remarkable selectivity (>10,000-fold) for Na⁺ over competing metal ions and has a detection limit of 135 µM (3.1 ppm). Furthermore, we demonstrate that this DNAzyme-based sensor can readily enter cells with the aid of α-helical cationic polypeptides. Finally, by protecting the cleavage site of the Na⁺-specific DNAzyme with a photolabile o-nitrobenzyl group, we achieved controlled activation of the sensor after DNAzyme delivery into cells. Together, these results demonstrate that such a DNAzyme-based sensor provides a promising platform for detection and quantification of Na⁺ in living cells.Metal ions play crucial roles in a variety of biochemical processes. As a result, the concentrations of cellular metal ions have to be highly regulated in different parts of cells, as both deficiency and surplus of metal ions can disrupt normal functions (1–4). To better understand the functions of metal ions in biology, it is important to detect metal ions selectively in living cells; such an endeavor will not only result in better understanding of cellular processes but also novel ways to reprogram these processes to achieve novel functions for biotechnological applications.Among the metal ions in cells, sodium (Na⁺) serves particularly important functions, as changes in its concentrations influence the cellular processes of numerous living organisms and cells (5–8), such as epithelial and other excitable cells (9). As one of the most abundant metal ions in intracellular fluid (10), Na⁺ affects cellular processes by triggering the activation of many signal transduction pathways, as well as influencing the actions of hormones (11). Therefore, it is important to carefully monitor the concentrations of Na⁺ in cells. Toward this goal, instrumental analyses by atomic absorption spectroscopy (12), X-ray fluorescence microscopy (13), and ²³Na NMR (14) have been used to detect the concentration of intracellular Na⁺. However, it is difficult to use these methods to obtain real-time dynamics of Na⁺ distribution in living cells. Fluorescent sensors provide an excellent choice to overcome this difficulty, as they can provide sensitive detection with high spatial and temporal resolution. However, despite significant efforts in developing fluorescent metal ion sensors, such as those based on either genetically encoded probes or small molecular sensors, most fluorescent sensors reported so far can detect divalent metal ions such as Ca²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ (15–21). Among the limited number of Na⁺ sensors, such as sodium-binding benzofuran isophthalate (22), Sodium Green (23), CoroNa Green/Red (24, 25), and Asante NaTRIUM Green-1/2 (26), most of them are not selective for Na⁺ over K⁺ (22–25, 27, 28) or have a low binding affinity for Na⁺ (with a K_d higher than 100 mM) (25, 27–31). Furthermore, the presence of organic solvents is frequently required to achieve the desired sensitivity and selectivity for many of the Na⁺ probes (32–34), making it difficult to study Na⁺ under physiological conditions. Therefore, it is still a major challenge to design fluorescent sensors with strong affinity for Na⁺ and high selectivity over other monovalent and multivalent metal ions that work under physiological conditions.To meet this challenge, our group and others have taken advantage of an emerging class of metalloenzymes called DNAzymes (deoxyribozymes or catalytic DNA) and turned them into metal ion probes. DNAzymes were first discovered in 1994 through a combinatorial process called in vitro selection (35). Since then, many DNAzymes have been isolated via this selection process. Among them, RNA-cleaving DNAzymes are of particular interest for metal ion sensing, due to their fast reaction rate and because the cleavage, which is catalyzed by a metal ion cofactor, can easily be converted into a detectable signal (36–38). Unlike the rational design of either small-molecule or genetically encoded protein sensors, DNAzymes with desired sensitivity and specificity for a metal ion of interest can be selected from a large library of DNA molecules, containing up to 10¹⁵ different sequences (35, 39). A major advantage of DNAzymes as metal ion sensors is that metal-selective DNAzymes can be obtained without prior knowledge of necessary metal ion binding sites or specific metal–DNA interaction (40, 41). In addition, through the in vitro selection process, metal ion binding affinity and selectivity can be improved by tuning the stringency of selection pressure and introducing negative selection against competing metal ions (39, 40). Finally, DNA is easily synthesized with a variety of useful modifications and its biocompatibility makes DNAzyme-based sensors excellent tools for live-cell imaging of metal ions. As a result, several metal-specific DNAzymes have been isolated and converted into sensors for their respective metal ion cofactors, including Pb²⁺ (35, 42, 43), Cu²⁺ (44, 45), Zn²⁺ (46), UO₂²⁺ (47), and Hg²⁺ (48). They have recently been delivered into cells for monitoring UO₂²⁺ (41, 49), Pb²⁺ (50), Zn²⁺ (51), and histidine (52) in living cells.However, in contrast to the previously reported DNAzymes with divalent metal ion selectivity, no DNAzymes have been reported to have high selectivity toward a specific monovalent metal ion. Although DNAzymes that are independent of divalent metal ions have been obtained (53–55), including those using modified nucleosides with protein-like functionalities (i.e., guanidinium and imidazole) (56–58), no DNAzyme has been found to be selective for a specific monovalent metal ion over other monovalent metal ions. For example, the DNAzyme with the highest reported selectivity for Na⁺ still binds Na⁺ over K⁺ with only 1.3-fold selectivity (54). More importantly, those DNAzymes require very high concentrations of monovalent ions (molar ranges) to function and display very slow catalytic rates (e.g., 10⁻³ min⁻¹) (53–55). The poor selectivity, sensitivity, and slow catalytic rate render these DNAzymes unsuitable for cellular detection of Na⁺, due to interference from other monovalent ions such as K⁺ (which is present in concentrations about 10-fold higher than Na⁺), and the need to image the Na⁺ rapidly.In this study, we report the in vitro selection and characterization of an RNA-cleaving DNAzyme with exceptionally high selectivity (>10,000-fold) for Na⁺ over other competing metal ions, with a dynamic range covering the physiological Na⁺ concentration range (0.135–50 mM) and a fast catalytic rate (k_obs, ∼0.1 min⁻¹). This Na⁺-specific DNAzyme was transformed into a DNAzyme-based fluorescent sensor for imaging intracellular Na⁺ in living cells, by adopting an efficient DNAzyme delivery method using a cationic polypeptide, together with a photocaging strategy to allow controllable activation of the probe inside cells.     

Plastidial transporters KEA1, -2, and -3 are essential for chloroplast osmoregulation,integrity, and pH regulation in Arabidopsis

Multiple K⁺ transporters and channels and the corresponding mutants have been described and studied in the plasma membrane and organelle membranes of plant cells. However, knowledge about the molecular identity of chloroplast K⁺ transporters is limited. Potassium transport and a well-balanced K⁺ homeostasis were suggested to play important roles in chloroplast function. Because no loss-of-function mutants have been identified, the importance of K⁺ transporters for chloroplast function and photosynthesis remains to be determined. Here, we report single and higher-order loss-of-function mutants in members of the cation/proton antiporters-2 antiporter superfamily KEA1, KEA2, and KEA3. KEA1 and KEA2 proteins are targeted to the inner envelope membrane of chloroplasts, whereas KEA3 is targeted to the thylakoid membrane. Higher-order but not single mutants showed increasingly impaired photosynthesis along with pale green leaves and severely stunted growth. The pH component of the proton motive force across the thylakoid membrane was significantly decreased in the kea1kea2 mutants, but increased in the kea3 mutant, indicating an altered chloroplast pH homeostasis. Electron microscopy of kea1kea2 leaf cells revealed dramatically swollen chloroplasts with disrupted envelope membranes and reduced thylakoid membrane density. Unexpectedly, exogenous NaCl application reversed the observed phenotypes. Furthermore, the kea1kea2 background enables genetic analyses of the functional significance of other chloroplast transporters as exemplified here in kea1kea2Na⁺/H⁺ antiporter1 (nhd1) triple mutants. Taken together, the presented data demonstrate a fundamental role of inner envelope KEA1 and KEA2 and thylakoid KEA3 transporters in chloroplast osmoregulation, integrity, and ion and pH homeostasis.The regulation of ion and pH homeostasis is a vitally important feature of all living organisms. The existence of organelles in eukaryotic cells has added complexity to this circumstance. Proper function of chloroplasts in plant cells is not only crucial for the organism’s survival but affects all life forms as chloroplasts convert light into chemical energy and fix carbon from the atmosphere. With up to 10% of the dry weight, K⁺ is the most abundant cation found in plants; it fulfills numerous essential roles: for example, in osmoregulation, as a pH regulator, in motor cell movements, and in membrane polarization (1).Early studies on isolated chloroplasts suggested that K⁺ transport occurs in exchange for H⁺ (2, 3). Later studies on reconstituted envelope membranes supported the notion that K⁺ and H⁺ transport are functionally connected (4, 5). A K⁺ transport system across envelope membranes has been proposed to be crucial for chloroplast function because even small changes in the osmotic pressure or electrochemical potential led to a dramatic decrease in photosynthesis (6, 7). However, the molecular mechanisms that mediate K⁺ transport across chloroplast membranes and that regulate the osmotic pressure and pH and ion homeostasis of the chloroplast are poorly understood. Two possible mechanisms have been proposed for K⁺ transport across the inner envelope membrane: K⁺ channels (4, 5) and K⁺/H⁺ antiporters (7, 8). Furthermore, it remains unknown whether K⁺ transport across chloroplast membranes is rate-limiting for chloroplast function. These gaps in knowledge could be closed by genetic analyses to assess the proposed roles of K⁺ in chloroplast function and could also lead to a mechanistic understanding for transport models. In this study we sought to characterize K⁺ transporters of the chloroplast and to identify their biological functions in plants.A variety of different ion/H⁺ transporters, K⁺ channels, and H⁺ pumps are located in the plasma and endomembranes of plants. These transporters maintain organelle-specific ion contents and pH, which create gradients over membranes that not only energize secondary transport processes but also result in unique biochemical reaction compartments. The electro neutral cation/H⁺ antiporters in Arabidopsis thaliana build the superfamily of monovalent cation/proton antiporters (CPA) (44 predicted genes), which further subdivides into the CPA1 and CPA2 families (9, 10). CPA1 consists of the Na⁺(K⁺)(Li⁺)/H⁺ exchangers NHX1–8. Although NHX1–6 were identified in endomembranes (11–13), NHX7 and -8 localize to the plasma membrane (14, 15) and are more distantly related to the first six members, thus forming a subfamily (10, 16). SOS1/NHX7 has been studied in detail because loss-of-function of the gene leads to salt sensitivity (14).The second family CPA2 covers two larger subfamilies, which include Cation/H⁺ exchangers (CHX) and putative K⁺-efflux antiporters (KEA) (10). Twenty-eight different CHXs exist in A. thaliana, with members targeted to the plasma membrane, prevacuolar membrane, or the endoplasmic reticulum, where they exchange K⁺ against H⁺ (17–19). The significance of this transporter class was shown in chx20 loss-of-function mutants, in which endomembrane dynamics and osmoregulation needed for stomatal opening is affected (20). Therefore, K⁺/H⁺ antiporters represent major osmo- and pH-regulators for organelles (9). A CPA2 family member, CHX23, long thought to be a chloroplast K⁺/H⁺ antiporter, was recently found not to target to chloroplasts but to the endoplasmic reticulum (19, 21). In addition, CHX23 was found to be preferentially expressed in pollen (9), and the described phenotypes in A. thaliana CHX23 RNAi and chx23-1 tilling mutants (22) could not be confirmed in T-DNA mutants (19, 21); thus, the molecular nature and biological function of chloroplast K⁺ transporters remain unknown.Recently, strong evidence was presented that the KEA2 protein could fulfill the role of plastidial K⁺/H⁺ antiport. A half-sized C-terminal transmembrane domain containing an AtKEA2 fragment was shown to complement a yeast mutant deficient in the endosomal Na⁺(K⁺)/H⁺ exchanger NHX1p (23). In addition, in vitro measurements showed K⁺/H⁺ transport capacity for the half-sized protein fragment. A 100-aa N-terminal protein fragment of AtKEA2 suggested that the full-length AtKEA2 protein may be targeted to chloroplasts (23). However, no mutant phenotypes or chloroplast functions have yet been ascribed for KEA2 and investigation of the full-length gene was unsuccessful because of gene toxicity in Escherichia coli (23). Here, we have identified three members of the CPA2 family, KEA1, KEA2, and KEA3, as chloroplast K⁺/H⁺ antiporters that have critical function in the inner envelope (KEA1, KEA2) and in the thylakoid membrane (KEA3). Our findings reveal their essential role in plant chloroplast function, osmoregulation, and pH regulation.     

Heme impairs the ball-and-chain inactivation of potassium channels

Fine-tuned regulation of K⁺ channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K⁺ channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K⁺ channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K⁺ current with an apparent EC₅₀ value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.A-type K⁺ channels, a family of voltage-gated K⁺ (Kv) channels, play a vital role in the control of neuronal excitability, regulation of presynaptic spike duration, Ca²⁺ entry, and neurotransmitter release (1). One of the prominent features of A-type K⁺ channels is their inactivation, which is mediated by two structurally distinct processes (2, 3). The fast inactivation is initiated by the N-terminal protein structure, thereby termed N-type inactivation, whereas the slow C-type inactivation is related to the pore structure (2, 3). N-type inactivation proceeds according to a “ball-and-chain” mechanism; the positive charges of the N-terminal ball domains bring the structures to the pore domain of the channel and the distal segment of one of the four intrinsically disordered N-terminal ball domains enters the hydrophobic central cavity/vestibule of the inner pore of the channel thus obstructing the flow of K⁺ (2–5).Acute enzymatic or mutational removal of the distal N terminus eliminates N-type inactivation, and in such inactivation-removed channels, intracellular application of peptides corresponding to the N-terminal sequence restores inactivation (4, 6, 7). Structural analysis suggests that the N-terminal inactivation structure needs to be flexible or even intrinsically disordered to reach the receptor in the channel’s cavity (8, 9).“Tuning” of rapid N-type inactivation is an effective way of adapting cells to specific needs. For example, molecular processes affecting the speed and degree of N-type inactivation in Kv1.4 (KCNA4) channels include redox regulation of a cysteine residue in the N-terminal ball structure (C13) (10), protonation of histidine at position 16 (11), interaction with membrane lipids (12), and Ca²⁺-dependent phosphorylation (13). Furthermore, low-molecular-weight compounds affecting N-type inactivation (N-type disinactivators) have been discussed as potential drugs regulating cellular excitability (14).Heme [Fe(II) protoporphyrin-IX] is well known as a protein cofactor, often conferring gas sensitivity as exemplified in hemoglobin, cytochromes, myoglobin, and soluble guanylyl cyclase. In many heme proteins including soluble guanylyl cyclase, heme is bound or coordinated in part by an amino acid sequence typically containing a histidine or cysteine residue, which acts as an axial fifth ligand (in addition to the four bonds provided by the nitrogen atoms of the protoporphyrin-IX ring to the iron center) to the redox-sensitive iron center, and water or a bound gas molecule acts as the sixth ligand (15). However, recent advances revealed a novel role of heme as a nongenomic modulator of ion channel functions, first exemplified for the large-conductance voltage- and Ca²⁺-dependent K⁺ channel (Slo1 BK) (16) and later for the epithelial Na⁺ channel (17). Detailed analysis of the biophysical action of heme [ferrous iron (Fe²⁺)] or hemin [ferric iron (Fe³⁺)] on the Slo1 BK channel demonstrated that hemin is a potent modulator of the allosteric gating mechanism of the channel (18), and mutagenesis studies have indicated the sequence CKACH located in the cytoplasmic C terminus of the channel plays a critical role (16, 19). However, neither for Slo1 BK channels nor for epithelial Na⁺ channels, the interaction of heme with the ion channel protein is structurally resolved. In this study, we found that the fast N-type inactivation of Kv1.4 A-type K⁺ channels is potently modulated by heme/hemin. Furthermore, we provide structural insight into heme interaction with a channel explaining how heme prevents A-type channels from entering an inactivated state.     

Angiotensin II signaling via protein kinase C phosphorylates Kelch-like 3, preventing WNK4 degradation

Hypertension contributes to the global burden of cardiovascular disease. Increased dietary K⁺ reduces blood pressure; however, the mechanism has been obscure. Human genetic studies have suggested that the mechanism is an obligatory inverse relationship between renal salt reabsorption and K⁺ secretion. Mutations in the kinases with-no-lysine 4 (WNK4) or WNK1, or in either Cullin 3 (CUL3) or Kelch-like 3 (KLHL3)—components of an E3 ubiquitin ligase complex that targets WNKs for degradation—cause constitutively increased renal salt reabsorption and impaired K⁺ secretion, resulting in hypertension and hyperkalemia. The normal mechanisms that regulate the activity of this ubiquitin ligase and levels of WNKs have been unknown. We posited that missense mutations in KLHL3 that impair binding of WNK4 might represent a phenocopy of the normal physiologic response to volume depletion in which salt reabsorption is maximized. We show that KLHL3 is phosphorylated at serine 433 in the Kelch domain (a site frequently mutated in hypertension with hyperkalemia) by protein kinase C in cultured cells and that this phosphorylation prevents WNK4 binding and degradation. This phosphorylation can be induced by angiotensin II (AII) signaling. Consistent with these in vitro observations, AII administration to mice, even in the absence of volume depletion, induces renal KLHL3^S433 phosphorylation and increased levels of both WNK4 and the NaCl cotransporter. Thus, AII, which is selectively induced in volume depletion, provides the signal that prevents CUL3/KLHL3-mediated degradation of WNK4, directing the kidney to maximize renal salt reabsorption while inhibiting K⁺ secretion in the setting of volume depletion.Hypertension affects 1 billion people worldwide and is a major risk factor for death from stroke, myocardial infarction, and congestive heart failure. The study of Mendelian forms of hypertension has demonstrated the key role of increased renal salt reabsorption in disease pathogenesis (1–4). Observational and intervention trials (5, 6) also indicate that increased dietary K⁺ lowers blood pressure; however, the mechanism of this effect has been unclear.Pseudohypoaldosteronism type II (PHAII; Online Mendelian Inheritance in Man no. 145260), featuring hypertension and hyperkalemia, has revealed a previously unrecognized mechanism that regulates the balance between renal salt reabsorption and K⁺ secretion in response to aldosterone (7). Aldosterone is produced by the adrenal glomerulosa in volume depletion, in response to angiotensin II (AII), and in hyperkalemia via membrane depolarization (8). In volume depletion, aldosterone maximizes renal salt reabsorption, whereas in hyperkalemia, aldosterone promotes maximal renal K⁺ secretion. Volume depletion increases both the NaCl cotransporter (NCC) (9) and electrogenic Na⁺ reabsorption via the epithelial Na⁺ channel (ENaC) (10). The lumen-negative potential produced by ENaC activity provides the electrical driving force for paracellular Cl⁻ reabsorption (11). In hyperkalemia, the lumen-negative potential promotes K⁺ secretion via the K⁺ channel Kir1.1 (renal outer medullary K⁺ channel ROMK), reducing plasma K⁺ level (12, 13). Additionally, recent studies have implicated aldosterone signaling in intercalated cell transcellular Cl⁻ flux (14). In these cells, hyperkalemia induces phosphorylation of the mineralocorticoid receptor (MR) ligand-binding domain, making it incapable of ligand binding and activation. AII signaling induces dephosphorylation, and activation of the MR by aldosterone then induces transcellular Cl⁻ flux, which is required for defense of intravascular volume (14, 15). Because electrogenic Cl⁻ reabsorption and K⁺ secretion both dissipate the lumen-negative potential produced by ENaC, maximal Cl⁻ reabsorption inhibits K⁺ secretion and vice versa.Patients with PHAII have constitutive reabsorption of NaCl with concomitant inhibition of K⁺ secretion, resulting in hypertension and hyperkalemia, despite normal levels of aldosterone (7). Dominant mutations in the serine–threonine kinases with-no-lysine 4 (WNK4) or WNK1, or in CUL3 or KLHL3, elements of a ubiquitin ligase complex, cause this disease (2, 4). WNK4 modulates the activities of NCC, ENaC, Kir1.1, and MR (14, 16–21), and WNK4 function can be modulated by phosphorylation (21). CUL3/KLHL3 has been shown to target WNK4 and WNK1 for ubiquitination and degradation, and disease-causing mutations impair this binding and degradation (22–24). In particular, dominant mutations in the Kelch domain of KLHL3 prevent binding to WNKs; reciprocally, disease-causing point mutations in WNK4 also prevent WNK4–KLHL3 binding.These findings suggest that regulation of WNK degradation by CUL3/KLHL3 is highly regulated and that disease-causing mutations might phenocopy a state in which WNKs are normally turned off, producing constitutive salt reabsorption and inhibited K⁺ secretion. We now demonstrate that this inference is correct and implicate AII signaling in this process.     

On the principle of ion selectivity in Na+/H+-coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na⁺ or H⁺, thereby harnessing the energy stored in the form of transmembrane electrochemical potential gradients to sustain their activities. The molecular and environmental factors that control and modulate the ion specificity of most of these systems are, however, poorly understood. Here, we use isothermal titration calorimetry to determine the Na⁺/H⁺ selectivity of the ion-driven membrane rotor of an F-type ATP synthase. Consistent with earlier theoretical predictions, we find that this rotor is significantly H⁺ selective, although not sufficiently to be functionally coupled to H⁺, owing to the large excess of Na⁺ in physiological settings. The functional Na⁺ specificity of this ATP synthase thus results from two opposing factors, namely its inherent chemical selectivity and the relative availability of the coupling ion. Further theoretical studies of this membrane rotor, and of two others with a much stronger and a slightly weaker H⁺ selectivity, indicate that, although the inherent selectivity of their ion-binding sites is largely set by the balance of polar and hydrophobic groups flanking a conserved carboxylic side chain, subtle variations in their structure and conformational dynamics, for a similar chemical makeup, can also have a significant contribution. We propose that the principle of ion selectivity outlined here may provide a rationale for the differentiation of Na⁺- and H⁺-coupled systems in other families of membrane transporters and enzymes.Gradients in the electrochemical potential of H⁺ or Na⁺ across biological membranes sustain a wide range of essential cellular process. The resulting proton or sodium motive forces (pmf, smf) are the predominant energy source for secondary-active membrane transporters, which mediate the uptake of many substances required by the cell (1–3), and also enable pathogenic bacteria to protect themselves from human-made antibiotics and other toxic compounds (4–6). Downhill membrane permeation of Na⁺ and H⁺ across the membrane also powers the ATP synthase (7), which produces most of cellular ATP, and energizes the rotation of bacterial flagella (8). Thus, the importance of this mode of energy transduction in cells cannot be overstated. Nevertheless, little is known about the factors that control and modulate the specificity for Na⁺ or H⁺ in most of these processes.It seems clear, although, that there is no correlation between function type and ion specificity; that is, the same process in different species can be coupled to either Na⁺ or H⁺ (2, 5–7, 9–11). Organism-specific environmental factors, such as temperature or pH, also do not provide a consistent rationale; for example, ATP synthases from thermoalkaliphilic bacteria use a H⁺ gradient despite the scarcity of H⁺ and the potentially greater degree of H⁺ leakage across the membrane at high temperatures, compared with Na⁺ (12, 13). Indeed, pmf- and smf-driven systems are often found within the same organism, and sometimes with the same or similar function; for example, malate uptake in Bacillus subtilis is mediated by the H⁺-coupled symporter CimH (14) and by the Na⁺-coupled MaeN (15). Moreover, specific membrane transporters and enzymes are sometimes coupled to both Na⁺ and H⁺, either concurrently (using multiple binding sites), such as the multidrug efflux pump NorM of Vibrio cholera (16) and the Methanosarcina acetivorans ATP synthase (17), or alternately (using a single binding site), such as the Escherichia coli melibiose permease (18) and some membrane-integral pyrophosphatases (19).At the molecular level, the architecture of pmf- and smf-driven membrane proteins within the same family has consistently been found to be largely conserved (20–28). Thus, it appears as if the Na⁺ or H⁺ specificity of these systems is dictated by localized variations in their amino acid sequence, rather than by major structural or mechanistic adaptations. This conclusion leads to an intriguing dilemma. Under most physiological conditions, the concentration of Na⁺ exceeds that of protons by many orders of magnitude (for example, a millionfold in mitochondria, or a billionfold in alkaline environments). Thus, in membrane-protein families with members that are driven by the smf or the pmf, the latter must have evolved amino acid adaptions that result in an extreme H⁺ selectivity, so as to counter the large Na⁺ concentration excess. Conversely, specific coupling to the smf would not actually require a strong Na⁺ selectivity; weak H⁺ selectivity or nonselectivity would be sufficient for Na⁺-coupling, physiologically. How can variation changes at the amino acid level result in such extreme variations in the H⁺ selectivity of a protein structure, spanning 10 or more orders of magnitude?In previous theoretical studies, we have addressed this question for the family of ion-motive ATPases (17, 29, 30), which comprises eukaryotic and prokaryotic ATP synthases, as well as vacuolar ion pumps. In these multicomponent enzymes, a membrane-embedded substructure known as the rotor ring can revolve around its axis, relative to the rest of the protein’s membrane domain. This rotational motion enables the ring to capture Na⁺ or H⁺ ions as they enter the protein via an access channel, and to shuttle them to a separate exit channel, in a sequential manner. Because the entry and exit channels are not colinear, there is a strict correspondence between the direction of ion permeation and the sense of rotation of the ring (which in turn determines the type of activity of the catalytic sector of the enzyme, i.e., ATP synthesis or hydrolysis).The principle of ion selectivity emerging from the abovementioned computational studies posits that rotor rings are universally H⁺ selective, owing to the fact that ion binding is consistently mediated by a conserved carboxylic side chain (the intrinsic H⁺/Na⁺ selectivity of a carboxylic group in solution is 10,000-fold), and that this inherent H⁺ selectivity is enhanced or suppressed by multiple orders of magnitude, depending on the balance between hydrophobic and polar groups lining the ion-binding sites (aside from the key carboxyl side chain); geometric factors may additionally fine-tune the selectivity of these sites, for a given chemical composition.In this study, we challenge the validity of this theory through experimental measurements and further computational analyses. Specifically, we set out to experimentally determine the thermodynamic ion selectivity of a rotor ring from a Na⁺-driven ATP synthase, and to compare this ring with others physiologically driven by either Na⁺ or H⁺. We find that the results of this analysis are qualitatively and quantitatively consistent with the theory of ion selectivity proposed previously, and provide novel insights into the influence of conformational factors.     

Convergent Ca2+ and Zn2+ signaling regulates apoptotic Kv2.1 K+ currents

A simultaneous increase in cytosolic Zn²⁺ and Ca²⁺ accompanies the initiation of neuronal cell death signaling cascades. However, the molecular convergence points of cellular processes activated by these cations are poorly understood. Here, we show that Ca²⁺-dependent activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is required for a cell death-enabling process previously shown to also depend on Zn²⁺. We have reported that oxidant-induced intraneuronal Zn²⁺ liberation triggers a syntaxin-dependent incorporation of Kv2.1 voltage-gated potassium channels into the plasma membrane. This channel insertion can be detected as a marked enhancement of delayed rectifier K⁺ currents in voltage clamp measurements observed at least 3 h following a short exposure to an apoptogenic stimulus. This current increase is the process responsible for the cytoplasmic loss of K⁺ that enables protease and nuclease activation during apoptosis. In the present study, we demonstrate that an oxidative stimulus also promotes intracellular Ca²⁺ release and activation of CaMKII, which, in turn, modulates the ability of syntaxin to interact with Kv2.1. Pharmacological or molecular inhibition of CaMKII prevents the K⁺ current enhancement observed following oxidative injury and, importantly, significantly increases neuronal viability. These findings reveal a previously unrecognized cooperative convergence of Ca²⁺- and Zn²⁺-mediated injurious signaling pathways, providing a potentially unique target for therapeutic intervention in neurodegenerative conditions associated with oxidative stress.Calcium has long been recognized as a critical component of neuronal cell death pathways triggered by oxidative, ischemic, and other forms of injury (1). Indeed, Ca²⁺ deregulation has been associated with a variety of detrimental processes in neurons, including mitochondrial dysfunction (2), generation of reactive oxygen species (3), and activation of apoptotic signaling cascades (4). More recently, zinc, a metal crucial for proper cellular functioning (5), has been found to be closely linked to many of the injurious conditions in which Ca²⁺ had been thought to play a prominent role (6–10). In fact, it has been suggested that a number of deleterious properties initially attributed to Ca²⁺ may have significant Zn²⁺-mediated components (11, 12). Although it is virtually impossible to chelate, or remove, Ca²⁺ without disrupting Zn²⁺ levels (13), the introduction of techniques to monitor Ca²⁺ and Zn²⁺ simultaneously in cells (14) has made it increasingly apparent that both cations have important yet possibly distinct roles in neuronal cell death (12, 15–18). However, the relationship between the cell death signaling pathways activated by the cations is unclear, and possible molecular points of convergence between these signaling cascades have yet to be identified.Injurious oxidative and nitrosative stimuli lead to the liberation of intracellular Zn²⁺ from metal binding proteins (19). The released Zn²⁺, in turn, triggers p38 MAPK- and Src-dependent Kv2.1 channel insertion into the plasma membrane, resulting in a prominent increase in delayed rectifier K⁺ currents in dying neurons, with no change in activation voltage, ∼3 h following a brief exposure to the stimulus (20–26). The increase in Kv2.1 channels present in the membrane mediates a pronounced loss of intracellular K⁺, likely accompanied by Cl⁻ (27, 28), that facilitates apoptosome assembly and caspase activation (20, 29–34). Indeed, K⁺ efflux appears to be a requisite event for the completion of many apoptotic programs, including oxidant-induced, Zn²⁺-mediated neuronal death (21).Ca²⁺ has been suggested to regulate the p38 MAPK signaling cascade via Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)-mediated activation of the MAP3K apoptosis signaling kinase-1 (ASK-1) (35). Because ASK-1 is also required for p38-dependent manifestation of the Zn²⁺-triggered, Kv2.1-mediated enhancement of K⁺ currents (36), we hypothesized that the p38 activation cascade may provide a point of convergence between Ca²⁺ and Zn²⁺ signals following oxidative injury. Here, we report that Ca²⁺ and Zn²⁺ signals do, in fact, converge on a cellular event critical for the K⁺ current enhancement, and that CaMKII is required for this process. However, CaMKII does not act upstream of p38 activation as originally hypothesized, but instead interacts with the N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein syntaxin, which we showed to be necessary for the insertion of Kv2.1-encoded K⁺ channels following an apoptotic stimulus (23).     

From the Cover: Water access points and hydration pathways in CLC H+/Cl? transporters

CLC transporters catalyze transmembrane exchange of chloride for protons. Although a putative pathway for Cl⁻ has been established, the pathway of H⁺ translocation remains obscure. Through a highly concerted computational and experimental approach, we characterize microscopic details essential to understanding H⁺-translocation. An extended (0.4 µs) equilibrium molecular dynamics simulation of membrane-embedded, dimeric ClC-ec1, a CLC from Escherichia coli, reveals transient but frequent hydration of the central hydrophobic region by water molecules from the intracellular bulk phase via the interface between the two subunits. We characterize a portal region lined by E202, E203, and A404 as the main gateway for hydration. Supporting this mechanism, site-specific mutagenesis experiments show that ClC-ec1 ion transport rates decrease as the size of the portal residue at position 404 is increased. Beyond the portal, water wires form spontaneously and repeatedly to span the 15-Å hydrophobic region between the two known H⁺ transport sites [E148 (Glu_ex) and E203 (Glu_in)]. Our finding that the formation of these water wires requires the presence of Cl⁻ explains the previously mystifying fact that Cl⁻ occupancy correlates with the ability to transport protons. To further validate the idea that these water wires are central to the H⁺ transport mechanism, we identified I109 as the residue that exhibits the greatest conformational coupling to water wire formation and experimentally tested the effects of mutating this residue. The results, by providing a detailed microscopic view of the dynamics of water wire formation and confirming the involvement of specific protein residues, offer a mechanism for the coupled transport of H⁺ and Cl⁻ ions in CLC transporters.The chloride channel (CLC) family (1, 2) includes both passive Cl⁻ channels and secondary active H⁺-coupled Cl⁻ transporters (3–8). The latter, also known as H⁺/Cl⁻ exchangers, drive uphill movement of H⁺ by coupling the process to downhill movement of Cl⁻ or vice versa, thereby exchanging the two types of ions across the membrane at fixed stoichiometry (9). ClC-ec1, a CLC from Escherichia coli, has served as the prototype CLC for biophysical studies because of its known crystal structures (10, 11), its tractable biochemical behavior, and its structural and mechanistic similarities to mammalian CLC transporters (3–8, 12–17). Detailed structural and functional studies of ClC-ec1 (9, 11, 18–27) have shed light on some of its key mechanistic aspects. Most prominently, these studies have characterized the Cl⁻ permeation pathway and its lining residues (10, 18, 25) and established the role of E148, also known as Glu_ex, as the extracellular gate for the Cl⁻ pathway (9, 11).Although much less is known about the H⁺ translocation pathway (and mechanism), experimental studies have provided key information on the involvement of specific residues in H⁺ transport (9, 13, 14, 20, 22, 27, 28). Extensive site-directed mutagenesis studies have zeroed in on two glutamate residues essential for H⁺ transport (Fig. 1A): E148 (Glu_ex), which acts as the main extracellular H⁺ binding site (9, 11, 27), and E203 (Glu_in), which plays a similar role on the cytoplasmic side (20, 22, 28). Neutralization of either glutamate eliminates H⁺ translocation by ClC-ec1 (9, 28). However, the discovery of these H⁺ binding sites also raised a mechanistic puzzle (3, 23): How do protons translocate between the two sites, which are separated by a ∼15-Å-long, largely hydrophobic region within the lumen of the protein?Open in a separate windowFig. 1.Cl⁻ and H⁺ permeation pathways in ClC-ec1. (A) View of the ClC-ec1 structure in a lipid bilayer (the simulation system used here), with the identical subunits shown in yellow and orange. The presumed Cl⁻/H⁺ permeation pathways are indicated by green and red lines, respectively. The dashed segment of the red line denotes the pathway investigated in this study. (B) Close-up of the central hydrophobic region, with the residues forming this region shown as orange sticks and labeled. Also shown are key glutamate residues (E202, E203, and E148) as well as the Cl⁻ at the central anion binding site. (C) Hydration of the central hydrophobic region during the 0.4-µs equilibrium simulation, measured as the number of water molecules in this region for each subunit.Since the report of its first crystal structure, a large number of computational studies have aimed at investigating various molecular details related to the CLC H⁺ transport mechanism (27, 29–34). One model emerging from these studies proposes that water molecules may connect the two H⁺ sites (Glu_ex and Glu_in) and, thereby, facilitate H⁺ transport (29, 30, 34). This idea was initially proposed by Kuang and coworkers (29) on the basis of a hole-searching algorithm applied to static crystal structures of ClC-ec1. In their proposed pathway, water molecules are suggested to form two half-wires that are then connected by the hydroxyl group of Y445 to form a complete path for H⁺ transfer. However, it is known from experiments on the Y445F mutant that this hydroxyl is not required for H⁺ transport (20). Wang and Voth (30) proposed another pathway by combining an improved search algorithm for buried water with short molecular dynamics (MD) simulations, thereby taking into account the dynamic nature of the protein. Their pathway did not rely on Y445 but required reorientation of the side chain of E203 to connect the two H⁺ sites. In another study, these investigators further carried out semiempirical free energy calculations to investigate the Cl⁻/H⁺ coupling mechanism (33).Although the idea of water-mediated H⁺ transport is intriguing and could be key to understanding H⁺ transport in ClC-ec1, several questions relevant to a water wire mechanism remain unanswered: Can the hydrophobic region between the two H⁺ sites actually be hydrated under equilibrium conditions? What is the access/entry point or points for water from the bulk into the hydrophobic region, which is buried inside the protein, approximately at the midpoint of the membrane? Is it possible to observe the spontaneous formation of water wires through MD simulations? If so, how much do the simulated wire structures differ from the ones proposed by the prior studies based on search algorithms? How could the protein affect the dynamics and/or the thermodynamics of water wires?In the current study, we have addressed these questions through a combined computational and experimental approach. An extended 0.4-µs MD simulation of a membrane-embedded model of wild-type (WT) ClC-ec1 reveals that the central hydrophobic region can indeed be hydrated by water molecules mainly from the cytoplasmic bulk phase through pathways near the dimer interface via a portal lined by residues E202, E203, and A404. Water wires connecting the two H⁺ sites form spontaneously and repeatedly during the equilibrium simulation. Formation of wires requires a side-chain conformational change of I109 and the occupancy of the central Cl⁻ binding site, S_cen. These simulation results make two strong and testable predictions: that mutations at A404 and I109 will reduce ClC-ec1 activity and that the reduction in activity occurs via effects on the H⁺ branch of the transport mechanism. Our experimental tests and additional simulations performed on one mutant form of the protein fully support these predictions.