Structural bases for blockade and activation of BK channels by Ba2+ ions

We studied the impact of Ba²⁺ ions on the function and structure of large conductance potassium (BK) channels. Ion composition has played a crucial role in the physiological studies of BK channels due to their ability to couple ion composition and membrane voltage signaling. Unlike Ca²⁺, which activates BK channels through all Regulator of K⁺ Conductance (RCK) domains, Ba²⁺ has been described as specifically interacting with the RCK2 domain. It has been shown that Ba²⁺ also blocks potassium permeation by binding to the channel’s selectivity filter. The Cryo-EM structure of the Aplysia BK channel in the presence of high concentration Ba²⁺ here presented (PDBID: 7RJT) revealed that Ba²⁺ occupies the K⁺ S3 site in the selectivity filter. Densities attributed to K⁺ ions were observed at sites S2 and S4. Ba²⁺ ions were also found bound to the high-affinity Ca²⁺ binding sites RCK1 and RCK2, which agrees with functional work suggesting that the Ba²⁺ increases open probability through the Ca²⁺ bowl site (RCK2). A comparative analysis with a second structure here presented (PDBID: 7RK6), obtained without additional Ba²⁺, shows localized changes between the RCK1 and RCK2 domains, suggestive of coordinated dynamics between the RCK ion binding sites with possible relevance for the activation/blockade of the channel. The observed densities attributed to Ba²⁺ at RCK1 and RCK2 sites and the selectivity filter contribute to a deeper understanding of the structural basis for Ba²⁺'s dual role in BK channel modulation, adding to the existing knowledge in this field.

Introduction

The voltage-dependent and Ca²⁺-activated potassium (BK) channels are unique, given their large single-channel conductance, allowing a detailed characterization of their properties. BK channels couple Ca²⁺ with membrane voltage signaling, playing essential roles in various physiological functions (Latorre et al., 2017). BK channels are tetramers forming a large intracellular structure with eight high-affinity Ca²⁺ sites (Hite et al., 2017; Tao et al., 2017) called the gating ring (Jiang et al., 2002). Each Ca²⁺ site is embedded in a regulator of potassium conductance (RCK) domain (Jiang et al., 2001). Ca²⁺ ions interact with BK channels’ RCK1 and RCK2 domains. Aplysia BK channels Ca²⁺ bowl site uses two side-chain carboxylates (Asp905 and Asp907) (Hite et al., 2017; Tao et al., 2017) and two main-chain carboxyl oxygens (Gln899 and Asp902) of the RCK2 domain, and the side-chain of Asn438 from the RCK1 domain of the neighboring subunit to coordinate a Ca²⁺ ion. Ca²⁺ at the high-affinity site of the RCK1 domain is interacting with two side-chain carboxylates (Asp356 and Glu525) and three main-chain carboxylates (Glu591, Arg503, Gly523). Many divalent ion species (Mn²⁺, Ni²⁺, Mg²⁺, and Co²⁺) do not activate BK channels through the high-affinity binding sites (Zeng et al., 2005), while Sr²⁺, as Ca²⁺, can activate BK channels through the RCK1 and the Ca²⁺ bowl sites (Zeng et al., 2005). Cd²⁺ activates through the RCK1 domain (Zeng et al., 2005; Zhang et al., 2010). Ba²⁺ properties are more complex. Ba²⁺ has been described as a potent blocker of BK channels (Vergara and Latorre, 1983), and detailed kinetic studies at the single-channel level provided consistent evidence that blockade likely occurred within the narrowest region of the permeation pathway. Access of Ba²⁺ from the inside and the outside to the blocking site is voltage-dependent (Vergara and Latorre, 1983). Increasing K⁺ concentration diminishes Ba²⁺ on-rate to its site (Vergara and Latorre, 1983). Access of Ba²⁺ to its location is through an open channel (Miller et al., 1987), and closing the channel with a Ba²⁺ bound can trap the blocker for minutes (Miller, 1987). By a thorough study of the influences of intracellular and extracellular K⁺ on the kinetics of Ba²⁺ blockade (Neyton and Miller, 1988a; b), it was proposed that the Ba²⁺ blocking site is wrapped between two K⁺ ions lock-in sites in the narrow region of the permeation pathway. More recently, Ba²⁺ was also found to activate BK channels through the Ca²⁺ bowl site (Zhou et al., 2012; Miranda et al., 2016), yet a unified model integrating the functional properties of Ba²⁺ has yet to be obtained. Here, we present two cryoEM structures of Aplysia Slo1 BK channels with Ba²⁺ incorporated into nanodiscs. These structures reveal that Ba²⁺ can occupy nine sites within the BK channel tetrameric complex, eight in the gating ring, and one at site S3 of the selectivity filter. Because these structures were obtained with BK channels exposed to different concentrations of Ba²⁺, comparative analysis revealed differences at the gating ring and the selectivity filter, providing details of the mechanisms involved in blockade and activation.

Methods

Expression, purification, and reconstitution of Aplysia Slo1

The full-length Slo1 gene from Aplysia californica was expressed in Sf9 cells with a C-terminal GFP/rho-1D4 tag cleavable by precision protease. Baculovirus was generated in Sf9 cells using the Bac-to-Bac system. Cells were harvested and lysed, and membranes were collected by centrifugation. The membrane pellet was homogenized and treated with DDM and CHS for protein extraction. After ultracentrifugation, the supernatant was affinity purified using a GFP nanobody resin. The protein was cleaved and further purified by size-exclusion chromatography. Slo1 tetramers were incorporated into lipid nanodiscs with MSP1E3D1 (see Supplementary Material and method section).

CryoEM

Samples were prepared following the standard workflow for cryoEM SPA. Grids were vitrified using a Vitrobot MK IV (Thermo Fisher Scientific). CryoEM data were collected on a Titan Krios electron microscope (Thermo Fisher Scientific) with a K2 detector (Gatan) and processed using cryoSPARC (Punjani et al., 2020) and RELION (Scheres, 2012). Aplysia Slo1 structure (PDBID:5tj6) was docked into maps using ChimeraX (Pettersen et al., 2021). Models were built in COOT (Emsley and Cowtan, 2004) and refined in PHENIX.real_space_refine (Adams et al., 2010). See Supplementary Material for a more details.

Electrophysiological characterization of Ba²⁺ dual-functional effects on BK channels

Full-length Slo1 from Aplysia was subcloned into the pGemHE vector (Liman et al., 1992). Next, the plasmid was linearized with NheI before performing RNA in vitro T7 polymerase transcription with Ambion, Thermo Fisher Scientific kit. Finally, 50 ng of synthesized RNA was injected into each Xenopus laevis oocyte. Oocytes were purchased from Ecocyte Bioscience. Excised inside-out patches were obtained using borosilicate pipettes (VWR 53432–921) with tips ranging between 0.7–1 MΩ, using recording solutions containing (in mM): pipette, 40 KMeSO₃, 100 N-methylglucamine–MeSO₃, 20 HEPES, 2 KCl, 2 MgCl₂, 100 μM CaCl₂ (pH = 7.4); bath solution, 40 KMeSO₃, 100 N-methylglucamine–MeSO₃, 20 HEPES, 2 KCl, 1 EGTA, and free BaCl₂ concentrations varying between 1–100 µM, previously estimated using Maxchelator (Bers et al., 1994). Currents were recorded with an Axopatch 200B amplifier using Clampex software (Axon Instruments, Molecular Devices). Solutions containing different ion concentrations were exchanged using a fast solution exchange (BioLogic RSC-200).

Results

Activation and blockade of Aplysia Slo1 by Ba²⁺

Ba²⁺ was initially reported to be a potent BK channel blocker (Vergara and Latorre, 1983). However, within the last decade, Ba²⁺ has also been shown to activate BK channels (Zhou et al., 2012; Miranda et al., 2016). To monitor these two functional properties of Ba²⁺ in Aplysia Slo1 channels, we recorded macroscopic currents using excised inside-out patches and repeatedly applied 20-ms voltage steps to +120 mV from a holding potential of −70 mV at a duty cycle of 1 pulse per 300 ms. Figure 1A shows the steady-state normalized current before and after (arrow) four different Ba²⁺ concentration exposures to the same patch. In the presence of 100 µM Ba²⁺, a fast blockade of the permeation pathway is observed (open circles). However, exposing the channels to lower Ba²⁺ concentrations, the blockade is slower, unveiling activation by Ba²⁺ that preceded the block (black, cyan, and red circles for 10, 5, and 1 µM Ba²⁺, respectively). Figure 1B shows representative current traces before Ba²⁺ activation (thick black traces), during activation (thin black traces), and after blockade (thick gray traces) for exposures to 10, 5, and 1 µM Ba²⁺. These results confirm that Ba²⁺ can block and activate Aplysia Slo1 with similar features as the human Slo1 (Zhou et al., 2012; Miranda et al., 2016).

Figure 1

Figure 1. Ba²⁺ activation and blockade of BK current. (A) Time course of normalized K⁺ currents in response to 1 μM (red), 5 μM (cyan), 10 μM (full black circle) or 100 μM (open circle) Ba²⁺ applied to an inside-out patch containing Aplysia BK channels. Each dot corresponds to the average current of the last 10 ms of a 20 ms pulse to 120 mV from a holding potential of −70 mV in 300 ms intervals. The arrow represents the moment of the Ba²⁺ addition. (B) Representative current traces in 10 μM Ba²⁺ (top panel), 5 μM Ba²⁺ (middle panel) and 1 μM Ba²⁺ (bottom panel) from Figure 1A. The current in the absence of Ba²⁺ (bold black trace) increases during Ba²⁺ activation (thin black trace) and reduces after Ba²⁺ blockade (gray trace).

Structures of Aplysia Slo1 in the presence of Ba²⁺

Our first structure (PDBID: 7RK6) was obtained from Aplysia Slo1 channel protein purified in the presence of 40 mM Ba²⁺ to ensure the replacement of any remaining Ca²⁺ ions from the expression system, but after nanodisc incorporation, it was run through the size exclusion chromatography column preequilibrated with a buffer lacking any divalent ions. We will refer to this structure as the Low-Barium structure (∼1.4 mM Ba²⁺, see Supplementary Method Section). The Low-Barium structure helps us identify the initial Ba²⁺ binding geometry at low ion concentration, i.e., the number and locations of densities attributed to Ba²⁺ ions. A second structure was obtained by adding 10 mM Ba²⁺ throughout the purification process. We will refer to this structure as the High-Barium structure (PDBID: 7RJT). The overall arrangement of these new structures is similar to those previously published Aplysia BK channel structures (Hite et al., 2017; Tao et al., 2017). Eight sites with strong map densities within the gating rings, attributed to Ba²⁺ ions, can be seen in both structures. The High-Barium structure has an additional density attributed to Ba²⁺ within the selectivity filter at the traditional K⁺ site S3 (Figure 2A). In this structure, three densities can be attributed to K⁺ ions at sites S2 and S4 and, interestingly, a third one at the entrance of the selectivity filter from the intracellular side (Figure 2A). The selectivity filter of the Low-Barium structure only has densities attributed to K⁺ ions, which are observed at sites S2, S3, and S4 (Figure 2B).

Figure 2

Figure 2. Overview of the High-Barium (light brown) and Low-Barium (light blue) structures. (A) The High-Barium structure has a density attributed to Ba²⁺ at site S3. K⁺ ions, in purple, at sites S2 and S4 and, a third one at the entrance of the selectivity filter from the intracellular side. (B) The selectivity filter of the Low-Barium structure shows densities attributed to K⁺ ions at sites S2, S3, and S4. (C) The distance between Lys320 Ca atoms across the pore axis (40.7 Å) is shown as a red circle for the Low-Barium structure and in blue (44.7 Å) for High-Barium structure. (D) Helix aB shifts towards its C-terminal end in the High-Barium structure (arrows). (E) Shift of the Glu388 within the BC strand (arrows). (F) General view of the BK channel structures, indicating regions of interest. Green box corresponding to Figure 2D. Red box area is presented in Figures 2G, H. (G) The methionine cluster is within the blue box. (H) RCK1 - RCK2 region in two views rotated 30 Deg to facilitate the visualization of contacts. See supplementary video for details.

Influence of Ba²⁺ on Aplysia Slo1

In the Ca²⁺/Mg²⁺ structure, divalent ions introduce a new bend of the S6 helices at Gly302 (Hite et al., 2017; Tao et al., 2017). Since this residue is near the inner end of the selectivity filter, this conformational change is likely a consequential movement linked to the opening of the permeation gate in the Aplysia Slo1 channel. This bend at Gly302 induces an expansion of the RCK1 N-lobes of ∼14 Å (monitored at the Cα atom of Lys320) relative to the structural model in the presence of 1 mM EDTA (Hite et al., 2017; Tao et al., 2017). In the presence of Ba²⁺, our structural models do not show the bend at Gly302. Nonetheless, we do observe that the distance between Cα atoms of the Lys320 across the pore axis was 40.7 Å for the Low-Barium and 44.7 Å for High-Barium structures, respectively (Figure 2C). As previously (Hite et al., 2017; Tao et al., 2017), the RCK1 N-lobe showed much larger motions in our two structures than the RCK1 C-lobe. The helix αB appears to shift towards its C-terminal end in the High-Barium structure (Figure 2D). This movement is driven by the displacement of the intracellular domain relative to the trans-membrane domain with the helix αB in close proximity to the interphase between the two domains. As a result of this movement, one of the Mg²⁺ coordinating residues, Glu388 within the βC strand, moves towards the position found in the Ca²⁺/Mg²⁺ structure (Hite et al., 2017; Tao et al., 2017) (Figure 2E). These observations help explain previous functional data showing that the Mg²⁺ affinity is higher in open Slo1 channels than in closed ones (Shi and Cui, 2001) and that the ability of Mg²⁺ to enhance channel activation is by affecting the close→open transition (Zhang et al., 2001).

Contrary to the RCK1, comparative analysis of the RCK2 domain displayed little differences between our structural models.

Ba²⁺ ions at the high-affinity divalent binding sites of the gating ring

Ba²⁺ activates BK channels by interacting with the Ca²⁺ bowl (Zhou et al., 2012; Miranda et al., 2016). Yet, the Low-Barium and High-Barium structures have eight densities within the gating ring. Most residues interacting with Ca²⁺ at the RCK1 and RCK2 sites in the Ca²⁺/Mg²⁺ structure (Tao et al., 2017) interact with Ba²⁺ (Figures 2F–H). In the Ca²⁺ bowl, the small linker between helices 3₁₀ and αR, containing Asp907, is quite flexible. Consequently, the side chain of Asp907 does not face the Ca²⁺ bowl binding pocket in the Low-Barium structure while in the High-Barium structure, it does (Figures 2G, H). In the RCK1 binding site, all side chains coordinating Ba²⁺ remain in a similar orientation in both the Low-Barium and High-Barium structures. The movement of Asp907 in response to saturating Ba²⁺, triggers an intricate series of concerted changes suggesting coordinated dynamics of the two binding sites within the same subunit (Supplementary Video S1), as previously suggested (Hite et al., 2017; Tao et al., 2017). Key residues involved in this flexible corridor are Pro909, Tyr914, Arg503, the Methionine cluster involving Met511, Met 519, and Met915, and an Asn438 from a neighboring subunit (Figures 2G, H). Some of these residues have been previously identified in functional studies (Shi et al., 2002; Zhang et al., 2010; Bukiya et al., 2014; Li et al., 2018). This rearrangement is facilitated by the rotation of the methionine cluster between the two binding sites (Supplementary Video). Beneath the flexible corridor is a rigid beta-strand structure impeding changes in the direction perpendicular to the membrane (Figure 2G). The general architecture of this flexible corridor is maintained in the hBK channels (Tao and MacKinnon, 2019).

The two structures here presented suggest the selective nature of Ba²⁺ activation is not the result of the absence of Ba²⁺ at the RCK1 site, yet we may speculate that the contribution of Asp356 to the coordination of a divalent ion at the RCK1 site (Hite et al., 2017; Tao et al., 2017; Tao and MacKinnon, 2019) is crucial to trigger the conformational changes responsible for the activation of the gating ring through the RCK1 domain. This residue shows a rearrangement between our two structures, possibly compensatory of the Arg503 displacement and partial disengagement from the Ba²⁺ coordination sphere at low ion concentration, suggesting a complex molecular choreography connecting the two domains.

Discussion

The interactions of Ba²⁺ ions with BK channels are unique among divalent ions because they activate the channel by increasing the open channel probability and block K⁺ permeation. Here we presented two cryoEM structures of Aplysia Slo1 BK channels with Ba²⁺ incorporated into nanodiscs, that integrate the dual functional properties of Ba²⁺ in BK channels. On the one hand, in the High-Barium structure, a density attributed to Ba²⁺ was found in the selectivity filter at the K⁺ site S3, and two additional densities attributable to K⁺ ions at S2 and S4. This Ba²⁺ explains blockade, which was functionally observed more than four decades ago (Vergara and Latorre, 1983). Further, through a series of insightful experiments assessing the influence of intracellular and extracellular K⁺ on the kinetics of Ba²⁺ blockade, it was proposed the presence of K⁺ lock-in sites (Neyton and Miller, 1988a; b). In the High-Barium structure, K⁺ at S2 would be consistent with the external lock-in site while K⁺ ions at S4 and the intracellular entrance of the selectivity filter would be consistent with the internal lock-in site. On the other hand, eight Ba²⁺ ions were found within the gating ring, occupying the four high-affinity binding sites of the RCK1 domains and the four sites of the Ca²⁺ bowls (RCK2). These Ba²⁺ ions explain activation of BK channels. Similarly, in the Low-Barium structure, there were eight densities in the gating ring that are attributed to Ba²⁺ ions, but none at the selectivity filter. These results are consistent with experimental observations where Ba²⁺-activation at low Ba²⁺ concentrations precede Ba²⁺ block (Zhou et al., 2012; Miranda et al., 2016), presumably because the apparent affinity of Ba²⁺ at the selectivity filter is lower than at the high affinity binding sites of the gating ring. Why is it that we observed eight densities within the gating ring while functional work has shown that Ba²⁺ activates BK channels selectively through the Ca²⁺ bowl (Zhou et al., 2012; Miranda et al., 2016)? Even though a definitive answer is beyond the information provided by our structures, they both represent different stages of channel activation. Yet, the differences in the Ba²⁺ bound high affinity divalent sites between our structures were exclusively observed in the Ca²⁺ bowl site, suggesting that this site is readily sensitive to the changes in Ba²⁺ concentrations exposed by our experimental conditions.

Even though the selectivity filters from homologous K⁺ channels are very similar, divalent ions can occupy different sites (Jiang and MacKinnon, 2000; Guo et al., 2014; Lam et al., 2014; Rohaim et al., 2020). Although poorly understood, these differences can sometimes be associated with experimental conditions like the presence or absence of K⁺ combined with co-crystallization or soaking with Ba²⁺. For example, in crystal structures of the membrane-spanning domain of MthK K⁺ channels, a bacterial relative of BK channels, Ba²⁺ is found predominantly in the S3 and S4 in the presence of K⁺ ions, while in the presence of Na⁺, Ba²⁺ is found at S2 (Guo et al., 2014). Recently, it was demonstrated that Ba²⁺ is an open channel blocker of KcsA channels, and a divalent ion at the selectivity filter occupying the K⁺ site S4 has been reported (Rohaim et al., 2020). In Aplysia Slo1 channels, however, Ba²⁺ was found at site S3 in the High-Barium structure and K⁺ ions at sites S2 and S4, making High-Barium the first structure with this ion distribution.

There are three conformational changes between our structures that merit some discussion in the context of previous work. First, the expansion at the N-terminus of the gating ring between Cα atoms of Lys320 from opposite subunits is smaller than those observed previously (Hite et al., 2017; Tao et al., 2017). Nonetheless, in both cases the direction of the change is the same from a less to a more activated state of the channel. In addition, the absolute value of this distance in the 10 mM Ca²⁺/10 mM Mg²⁺ structure is 52.2 Å, larger than in the High-Ba²⁺ structure (44.7 Å), even though both models presumably represent their own maximal channel activation. This could be explained by the bend at Gly302 in the Ca²⁺ and Mg²⁺structural model, which is not present in ours. It is tempting to speculate that this bend at Gly302 is also responsible for the functional differences in the activation strength between Ca²⁺ and Ba²⁺ observed in human Slo1 (Zhou et al., 2012). Second, in our High-Ba²⁺ structure we observed a displacement of the βC strand containing Glu388, towards the position found in the Ca²⁺/Mg²⁺ structure (Hite et al., 2017; Tao et al., 2017). These results indicate that the final position of Glu388 when coordinating Mg²⁺ is not entirely induced by the presence of Mg²⁺. In addition, they are consistent with previous functional work showing that open mouse BK channels have higher apparent Mg²⁺ affinity than those closed (Shi and Cui, 2001). Third, the Asp907 side chain faces into the binding pocket in the High-Ba²⁺ structure while facing outward in the Low-Ba²⁺ structure. This same trend was also observed between the 10 mM Ca²⁺/10 mM Mg²⁺ and the 1 mM EDTA structures (Hite et al., 2017; Tao et al., 2017), indicating that the “face-in ↔ face-out” dynamics of Asp907 are not strictly dependent on the presence or complete absence of Ba²⁺. These dynamics do reveal a flexible corridor connecting the two high affinity binding sites as described here (Supplementary Video S1). These rearrangements have also been observed previously with Aplysia Slo1 (Hite et al., 2017; Tao et al., 2017) and in human Slo1 (Tao and MacKinnon, 2019), even though the latter has important differences in the amino acid sequences of the RCK1/RCK2 corridor, suggesting common dynamics among BK channels and different divalent ion species involved.

Data availability statement

The datasets presented in this study can be found in online repositories. The repositories and accession numbers can be found below: https://www.wwpdb.org/, 7RJT and 7RK6; https://www.ebi.ac.uk/emdb/, EMD-24490 and EMD-24493.

Author contributions

SS: Data curation, Formal Analysis, Visualization, Writing–review and editing. PM: Data curation, Formal Analysis, Visualization, Writing–review and editing. TG: Data curation, Formal Analysis, Visualization, Writing–review and editing. JZ: Data curation, Formal Analysis, Software, Visualization, Writing–review and editing. RC: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing–original draft, Writing–review and editing. MH: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing–original draft, Writing–review and editing.

Funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. MH, PM and SS were supported by the intramural section of the National Institutes of Health (NINDS, 1ZIANS002993). REC and JZ were supported by the intramural section of the National Institutes of Health (NIAID, 75N95023A000001).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Publisher’s note

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmolb.2024.1454273/full#supplementary-material

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