The movement and interaction of multiple ions passing through in single file underlie various fundamental K+ channel properties, from the effective conduction of K+ ions to channel blockade by Ba2+ ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba2+ with permeant ions within the ion conduction pathway of the MthK K+ channel. We found that, as typical of K+ channels, the MthK channel was blocked by Ba2+ at the internal side, and the Ba2+-blocking effect was enhanced by external K+. We also obtained crystal structures of the MthK K+ channel pore in both Ba2+–Na+ and Ba2+–K+ environments. In the Ba2+–Na+ environment, we found that a single Ba2+ ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba2+–K+ environment, Ba2+ ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba2+ blockade of K+ channels.
K+ channels catalyze selective and rapid transport of K+ ions across the cell membrane (Hille, 2001). The combination of two apparently contradictory characteristics, strong selectivity for K+ over other cations and very high K+ conductance, arises from the presence of multiple ions moving and interacting in single file (Hodgkin and Keynes, 1955; Hille and Schwarz, 1978). The crystal structures of KcsA K+ channel in its closed state (Fig. 1, left) reveal an ion conduction pathway that consists of a selectivity filter with four dehydrated K+ ion–binding sites (S1–S4, extracellular to intracellular) formed by 20 oxygen atoms from backbone carbonyl and side-chain hydroxyl groups and an adjacent central cavity containing hydrated ions (Doyle et al., 1998; Zhou et al., 2001). Such an ion conduction pathway allows multiple K+ ions to simultaneously occupy inside and to interact with each other, warranting the very high conductance and strong K+ selectivity (Morais-Cabral et al., 2001).
Ba2+ is the only alkaline earth metal that blocks K+ channels, presumably because it uniquely shares high similarity with the alkali metal K+ in terms of the ionic radius (1.35 Å of Ba2+ vs. 1.33 Å of K+) while doubling the charge. The similar size allows Ba2+ ions to fit into the K+ channel selectivity filter. However, the charge apparently causes tight binding, preventing the rapid flow of K+. The inhibitory properties of Ba2+ ions on K+ channels have been extensively used to functionally probe the K+ channel structure (Armstrong and Taylor, 1980; Vergara and Latorre, 1983; Neyton and Miller, 1988a,b; Piasta et al., 2011). Among these studies, Neyton and Miller (1988a,b) showed that Ba2+ inhibition of Ca2+-activated K+ channels is sensitive to the K+ ions externally or internally, and they identified three K+-binding sites with different affinities and effects on Ba2+ blockades. An external lock-in site preferentially binds K+ ions with high affinity over Na+ ions by a factor of ∼1,000. K+ binding at the external lock-in site prevents Ba2+ ions from exiting to the external side. Higher external concentration (500 mM) causes K+ binding at an enhancement site, driving Ba2+ ions to dissociate rapidly to the internal side. Internal K+ ions also cause a similar lock-in effect, whereas the internal lock-in site is nonselective for both K+ and Na+. These studies suggest that the ion conduction pathway of K+ channels contains multiple distinct ion-binding sites. The determination of the first K+ channel structure (Doyle et al., 1998) invoked structural interpretations of these phenomena (Jiang and MacKinnon, 2000). Ba2+-soaked KcsA crystal structure in the absence of K+ showed that Ba2+ ions preferably bind at sites 2 and 4 (Lockless et al., 2007). However, experimental and computational studies of Ba2+ blockades in the KcsA K+ channels yielded different interpretations of Ba2+ blockades (Kim and Allen, 2011; Piasta et al., 2011; Rowley and Roux, 2013), so further studies are needed.
The structures of the isolated ion conduction pore of MthK in its open conductive state (Fig. 1, right) revealed a similar selectivity filter and a central cavity (Ye et al., 2010), whereas the high resolution allowed us to probe the Ba2+ block mechanism in a more highly accurate structural way than the earlier studies (Jiang and MacKinnon, 2000; Lockless et al., 2007). In this current study, we characterized the Ba2+ blockades of MthK K+ channel at the single-channel level and demonstrated that the MthK K+ channel displays a typical K+ channel Ba2+ blockade property. We further determined the positions of Ba2+ ions in the pore of MthK K+ channel at two different ionic environments, which allowed us to deduce ionic configurations and to analyze the interactions of Ba2+ ions and the permeation ions.
MATERIALS AND METHODS
The MthK K+ channel was expressed and purified in n-decyl-β-d-maltoside as described previously (Jiang et al., 2002). Purified MthK A88D mutant was reconstituted into lipid vesicles composed of 3:1 POPE/POPG. To increase the fusion efficiency of the channel in the synthetic lipid bilayers, protein/lipid ratios of 1:100 were used in the reconstitution. Single-channel data were recorded in a vertical lipid bilayer setup in which a planar lipid bilayer of 3:1 POPE/POPG (20 mg/ml) in decane was painted over an ∼150-µm hole in a polystyrene partition separating the internal and external solutions. To induce fusion of channel-containing vesicles, the solution on the side to which vesicles were added (external side) contained 150 mM NaCl and 10 mM Tris, pH 8.0, whereas the opposite side (internal side) contained 15 mM KCl and 10 mM Tris, pH 8.0. After the appearance of channels in the membrane, as monitored under voltage pulses, KCl concentration in the internal solution was raised to 150 mM. Ba2+ was added only to the intracellular side of the channel. Membrane voltage was controlled and current was recorded using an amplifier (Axopatch 200B; Molecular Devices) with an analogue-to-digital converter (Digidata 1322A; Axon Instruments). Currents were low-pass filtered at 1–2 kHz and sampled at 10 kHz.
Crystallization, data collection, and structure determination
The MthK K+ channel pores were purified and crystallized as described previously (Ye et al., 2010). The crystals were soaked either in a solution containing 3.5 M 1,6-hexandiol, 10 mM BaCl2, 100 mM NaCl, 5 mM LDAO, and 100 mM HEPES, pH 7.5 (Ba2+–Na+ complex), or in an otherwise identical solution containing 10 mM BaCl2 and 100 mM KCl (Ba2+–K+ complex) overnight. To avoid the interference of the Na+ ion, if any, we neutralized the HEPES buffer with KOH for the Ba2+/K+ soaking solution. The soaked crystals were directly frozen in liquid nitrogen. X-ray data were collected at the Shanghai Synchrotron Radiation Facility BL17U beamline. The data were indexed, integrated, and scaled using the program HKL-2000 (HKL Research, Inc.; Otwinowski and Minor, 1997). The structures were determined by molecular replacement using Phaser in the CCP4 suite (Collaborative Computational Project, Number 4, 1994) with the MthK pore structure (Protein Data Bank accession no. 3LDC) as a search model, followed by model building with XtalView (McRee, 1999) and refinement with REFMAC (Collaborative Computational Project, Number 4, 1994). Detail data collection and refinement statistics are listed in Table 1. The structure coordinates and reflection files are deposited in the Protein Data Bank under accession nos. 4QE7 and 4QE9.
One-dimensional electron density profiles
We obtained one-dimensional electron density profiles of bound ions in the selectivity filter as described previously (Morais-Cabral et al., 2001). All data were scaled to 2.4-Å resolution against the K+ complex crystal (100 mM K+; Protein Data Bank accession no. 3LDC) before map calculation. We obtained one-dimensional profiles by sampling the Fo–Fc difference maps or the anomalous difference maps, with ions and selectivity filter residues omitted, along the central axis of the filter using MAPMAN (Kleywegt and Jones, 1996). Occupancies were estimated for Ba2+–Na+ and Ba2+–K+ complexes by comparing one-dimensional electron density profiles with that of the K+ complex as a standard of known occupancy based on the assumption that the relative peak areas in one-dimensional electron density maps calculated from these model maps were nearly proportional to the relative number of electrons in the model ion.
Ba2+ blockades in the MthK K+ channel
A purified MthK A88D mutant channel was reconstituted into lipid vesicles, and its functional properties were studied in a vertical lipid bilayer membrane. We chose A88D mutant instead of wild-type MthK channel in this study because this mutant channel is constitutively active with extremely high open probability in the absence of Ca2+ (Shi et al., 2011), which allows us to record the single-channel outward current by avoiding the reduction of the outward conductance caused by intracellular [Ca2+] (Zadek and Nimigean, 2006; Li et al., 2007).
Fig. 2 A shows sample traces of the outward currents of this mutant recorded at 100 mV with 150 mM K+ at internal and 150 mM Na+ at external sides. The channel is fully open, with only rapid closings within a burst (intraburst closings). The dwell time distributions of the closed states can be fit with one exponential component, as shown beside the trace (Fig. 2 A). The time constant of the rapid intraburst closings (τintraburst) is 0.76 ms, similar to that of a wild-type channel from an earlier study, indicating that it is an intrinsic property of the ion conduction pore (Li et al., 2007).
200 µM Ba2+ was then added to the internal side of the channel to induce block (Fig. 2 B), whereas ∼10 mM Ba2+ was needed to see significant blockade effect at the external side (Fig. 2 D), indicating that the MthK channel shares an asymmetric Ba2+ blockade with many other K+ channels (Standen and Stanfield, 1978a,b; Armstrong and Taylor, 1980; Armstrong et al., 1982; Vergara and Latorre, 1983; Piasta et al., 2011). As shown in Fig. 2 B, the addition of an internal 200 µM Ba2+ induces interburst in addition to the intraburst closings. Analysis of the dwell time distributions of the closed states gives rise to a τintraburst of 0.83 ms and a time constant for the interburst closings (τinterburst) of 10.7 ms. The similar τintraburst before and after Ba2+ block indicates that this fast gating process is independent of added Ba2+ ions, whereas the interburst closings reflect channel-blocking events induced by Ba2+ ions. Similarly, the dwell time distribution of the closed states can be fit with two exponential components with 10 mM Ba2+ in the external side (Fig. 2 D).
The Ba2+ block kinetics is strongly affected by externally applied K+ ions. As shown in Fig. 2 (C and E), 10 mM K+ dramatically increased Ba2+ block duration. The dwell time distributions are fit by three well-separated exponentials of time constants of 0.7 ms (τintraburst), 11.2 ms (τinterburst1), and 179 ms (τinterburst2) for internal Ba2+ blockade (Fig. 2 C) or of 0.89 ms (τintraburst), 12.2 ms (τinterburst1), and 164.8 ms (τinterburst2) for external Ba2+ blockade (Fig. 2 E). This is very similar to the K+ lock-in effect reported earlier on the BK channel (Neyton and Miller, 1988b) and recently on KcsA (Piasta et al., 2011). In addition to the intraburst closings, the interburst closings contain a shorter and longer duration fraction. The shorter one has a similar time constant (τinterburst1) to that without external K+ ions (Fig. 2, B and D), whereas the external K+ produces a much slower block component.
Ion occupancy in the selectivity filter
We soaked the MthK pore crystals in a stabilization solution containing 10 mM Ba2+ and 100 mM Na+ (Ba2+–Na+ complex) or 10 mM Ba2+ and 100 mM K+ (Ba2+–K+ complex) and determined their structures at 2.4- and 2.15-Å resolution, respectively (Table 1). To study the occupancy of Ba2+ ions in the selectivity filter at the Ba2+–Na+ environment, we first calculated the Fo–Fc electron density map of the Ba2+–K+ complex after omitting the selectivity filter and ions (Fig. 3 C) and compared it with those at Na+ and K+ environments (Fig. 3, A and B). The electron density in the filter of the Ba2+–Na+ complex is stronger than that of the Na+ complex (in 100 mM Na+) and K+ complex (in 100 mM K+; Fig. 3 E), indicating that the selectivity filter is occupied by a heavier Ba2+ ion (56 electrons). By assuming that the area under the peaks of the one-dimensional electron density profile along the central axis of the filter reflects the number of electrons in the filter, we compared the one-dimensional electron density profiles and estimated an increase of 46 e− compared with the Na+ complex. This is roughly equivalent to the replacement of a Na+ ion with a Ba2+ ion, suggesting that a single Ba2+ ion bound in the filter of the Ba2+–Na+ complex. Piasta et al. (2011) also determined that a single Ba2+ ion binds in and blocks the channel based on their blockade experiments, consistent with our estimation from the crystal structure. At the wavelength used for data collection (0.9786 Å), anomalous scattering of Ba2+ dominated in the protein (Table 2). We then calculated an anomalous difference Fourier map of the Ba2+–Na+ complex and observed nonequivalent Ba2+ occupancy at the four sites of the MthK filter (Fig. 4 A). The anomalous signal at site 2 is much stronger than that at sites 3 and 4, with no substantial signal observed at site 1. This suggests that the Ba2+ ion predominately occupies site 2 at the Ba2+–Na+ environment and has no binding at site 1.
The same analysis of the Ba2+–K+ complex revealed that the electron density in the filter of the Ba2+–K+ complex is substantially stronger than that of the K+ complex (in 100 mM K+; Fig. 3, D and E) but weaker than that of the Ba2+–Na+ complex (Fig. 3, C–E), indicating a partial Ba2+ ion bound in the filter of the Ba2+–K+ complex. The anomalous difference Fourier map of the Ba2+–K+ complex further revealed nonequivalent Ba2+ occupancy at the four sites of the MthK filter. However, different to that in the Ba2+–Na+ environment, Ba2+ ions predominately occupy sites 3 and 4 in the Ba2+–K+ environment and have no binding at sites 1 and 2 (Fig. 4, B and C).
In this study, we characterized the Ba2+ blockade property of the MthK K+ channel at the single-channel level and further determined the positions of Ba2+ ions in the pore of the MthK K+ channel at two different ionic environments (Ba2+–Na+ and Ba2+–K+). We concluded that the MthK K+ channel shares a similar Ba2+ blockade property with other K+ channels. Our main experimental observation is that, in the Ba2+–Na+ environment, a single Ba2+ ion shares occupancy among sites 2–4, predominately at site 2, and has no binding at site 1, whereas in the Ba2+–K+ environment, partial Ba2+ ion occupies sites 3 and 4 and has no binding at sites 1 and 2. This allows us to analyze the interactions of Ba2+ ions and the permeation ions and to provide structural interpretations of Ba2+ blockade of K+ channels relating to the physical origins of K+ channel selectivity.
Site 1 as a high energy barrier for Ba2+ ions
Our structural data are highly consistent with functional data of Ba2+ blockade on K+ channels. The electrophysiological recordings showed that externally added Ba2+ also blocks the MthK K+ channel, but at a concentration on the order of 10 mM, ∼50-fold higher than that required for internal block. Moreover, the external K+ lock-in properties of external Ba2+ blocked states are identical to those induced by internal Ba2+, indicating that Ba2+ enters the K+ channel selectivity filter to induce block, but the energy barrier for entering the site is higher from the external solution than from the internal solution. This observation agrees well with the functional data on Ca2+-activated K+ channel showing that a concentration of 10 mM is required for external Ba2+ block, whereas a concentration of only 100 nM, ∼10,000-fold lower than that required for external block, is required for internal Ba2+ block (Vergara and Latorre, 1983). Similar asymmetric Ba2+ block was also observed in the delayed rectifier K+ channel of squid (Armstrong and Taylor, 1980; Armstrong et al., 1982), the inward rectifier of muscle (Standen and Stanfield, 1978a,b), and KcsA (Piasta et al., 2011). Consistently, the ionic configurations deduced by x-ray crystallographic analysis on two ionic environments (Ba2+–K+ and Ba2+–Na+) revealed that Ba2+ rarely binds at site 1 of the MthK K+ channel selectivity filter, indicative of a very low affinity of Ba2+ binding at site 1 close to the channel’s outer mouth, and revealed a high energy barrier that limits Ba2+ to enter from, or exit to, the external side. Such a barrier does not seem to exist in the inner mouth of the channel, as the ionic configurations revealed Ba2+ bindings at sites 2–4. Interestingly, Ba2+ binding at site 1 was observed in NaK2K (see Lam et al. in this issue); correspondingly, external Ba2+ block in NaK2K is stronger than that in MthK (unpublished data).
A single Ba2+ ion binds in the K+ channel selectivity filter
The estimation of a single Ba2+ ion bound in the filter of the Ba2+–Na+ complex raised an interesting question related to the Ba2+-blocking mechanism of the K+ channel. Why does only a single Ba2+ ion bind in the filter, instead of two, like that of alkali metal ions K+, Rb+, or Cs+? Charge balance provides an explanation. The selectivity filter contains 20 oxygen atoms surrounding a 12-Å-long narrow pore. Each oxygen atom is associated with a partially negative charge. A charge balance exists between 20 partially negative oxygen atoms and two monovalent K+ ions or one divalent Ba2+ ion. In addition, K+ ions have been shown to simultaneously occupy 1,3 or 2,4 positions in the filter, but this binding configuration would seem to be unstable, as the repulsive force between two Ba2+ ions is fourfold that of K+ ions. Therefore, Ba2+ ions cannot simultaneously occupy 1,3 or 2,4 positions in the filter, with 1,4 positions becoming possible. The very low affinity of Ba2+ binding at site 1 would then result in such a single Ba2+ ion binding in the filter.
When a single divalent Ba2+ ion binds in the MthK filter, can a monovalent Na+ reside in the filter? It is difficult to distinguish Na+ ions from water molecules based on the electron density map, as both have an equal number of electrons. However, the Na+ binding at site 1 of the MthK filter adopts a pyramidal Na+ coordination (Ye et al., 2010). This unique coordination has not been observed in the Ba2+–Na+ complex structure, indicating that Ba2+ binding predominately at site 2 drives most of the Na+ ions out of site 1. Because the repulsive force between a Ba2+ ion and a Na+ ion is twofold that of K+ ions, Ba2+ and Na+ ions simultaneously occupying 1,3 or 2,4 positions in the filter would seem to be an unstable binding configuration for electrostatic reasons. Therefore, with a single divalent Ba2+ ion bound in the MthK filter, only a partial Na+ ion binds in the filter.
Interaction of Ba2+ and K+ ions in the K+ channel selectivity filter
A central feature of all K+ channels is that their selectivity filters have a preference for K+ ions over other cations. Among all four sites of the K+ channel selectivity filter, site 1 is more highly selective for K+ ions than other cations, especially Na+ ions, as at a low K+/high Na+ condition, K+ prefers sites 1 and 3 in MthK (Ye et al., 2010) and sites 1 and 2 in NaK2K (Sauer et al., 2013) K+ channels. In the Ba2+–K+ environment, K+ ions dominate the MthK filter, leaving a partial Ba2+ ion bound mainly at sites 3 and 4. This ionic configuration allows us to deduce a molecular basis for external lock-in and enhancement effects. At low K+ concentration, a K+ ion enters the MthK filter from external solution and binds preferably at site 1. The repulsive force between a K+ ion and a Ba2+ ion is twice that of two K+ ions, which drives the Ba2+ ion to move internally and mainly binds at site 4. However, at high K+ concentration, K+ ion further enters site 2, or even site 3. K+ and Ba2+ ions simultaneously occupying 2,4 positions in the filter is a relatively unstable binding configuration, which explains why the effective Kd for achieving the external enhancement effect is nearly 500 mM (Neyton and Miller, 1988a). Such an unstable binding configuration forces Ba2+ dissociation to the internal side by the electrostatic repulsion. The ionic configuration in the Ba2+–Na+ environment also suggests a molecular basis for the internal enhancement effect. Monovalent cation, either Na+ or K+, resides at the central cavity, effectively driving the Ba2+ ion to predominately bind at site 2 through the electrostatic repulsion.
A few recent studies proposed different interpretations of the external K+ lock-in effect. Piasta et al. (2011) suggested that the K+ ion binds at site 1 and locks the Ba2+ ion preferably at site 2. However, K+ and Ba2+ ions simultaneously binding to adjacent sites 1 and 2 will have strong electrostatic repulsion in between and are highly unstable. Computational studies suggested that the K+ ion binds at site 0 and locks Ba2+ ion preferably at site 2 (Kim and Allen, 2011; Rowley and Roux, 2013). We think site 0 is unlikely to be the lock-in site based on three reasons. First, site 0 does not have the square antiprism configuration and is unlikely to be selective for K+. Second, site 0 is a very weak ion-binding site. The electron density at site 0 is much weaker than that of sites 1–4 in KcsA filter (Zhou et al., 2001), whereas there is no density at site 0 in MthK (Ye et al., 2010), indicating weak K+ binding at site 0. Third, the voltage dependence suggests that the external lock-in site resides ∼15% inside (Neyton and Miller, 1988a).
Our high resolution structural data allow us to clearly define the internal and external lock-in and enhancement effects (Neyton and Miller, 1988a,b). As shown in Fig. 5, without Ba2+, two K+ ions bind equivalently in the 1,3 and 2,4 configurations. The electrostatic repulsion between them destabilizes the ions and lowers the K+ ion–binding affinity. This allows an additional K+ ion to enter the filter and to expel a bound K+ ion from the opposite end, warranting the efficient conduction of K+. With the addition of Ba2+ from the internal side, Ba2+ enters the filter and initiates block by occupying the S4 site. The electrostatic repulsion between Ba2+ and K+ allows the Ba2+ ion to effectively drive out K+ ions from the filter, leaving a single Ba2+ ion alternating between S2, S3, and S4 sites and blocking the passage of K+ ions. Ba2+ ions may leave the filter either by dissociation back to the internal side or proceeding to the external side by overcoming the high energy barrier at site 1. The addition of external K+ in low concentration causes K+ ion binding at its high affinity site 1, locking Ba2+ at site 4 and preventing Ba2+ from exiting to the external side. Although external K+ in high concentration allows the K+ ion to enter site 2, it forces Ba2+ ion dissociation to the internal side by the electrostatic repulsion. The internal K+, or other permeant ions, can occupy the central cavity, locking the Ba2+ ion mainly at site 2 and preventing the Ba2+ ion from exiting to the internal side.
We thank Youxing Jiang for discussions and critical reviews of the manuscript and S. Huang and J. He at the Shanghai Synchrotron Radiation Facility for on-site assistance.
This work was supported in part by funds from the National Natural Science Foundation of China (grant 31370721), the Ministry of Science and Technology (grants 2014CB910300 and 2011CB910500), the Natural Science Foundation of Zhejiang Province (grant R2100439), the Specialized Research Fund for the Doctoral Program of Higher Education (grant 20110101110122), and the Fundamental Research Funds for the Central Universities (grant to S. Ye).
The authors declare no competing financial interests.
Kenton J. Swartz served as editor.