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. 2018 Dec 11;7:e40664. doi: 10.7554/eLife.40664

Voltage-dependent dynamics of the BK channel cytosolic gating ring are coupled to the membrane-embedded voltage sensor

Pablo Miranda 1,✉, Miguel Holmgren 1, Teresa Giraldez 2,3,✉
Editors: Richard Aldrich4, Richard Aldrich5
PMCID: PMC6301790  PMID: 30526860

Abstract

In humans, large conductance voltage- and calcium-dependent potassium (BK) channels are regulated allosterically by transmembrane voltage and intracellular Ca2+. Divalent cation binding sites reside within the gating ring formed by two Regulator of Conductance of Potassium (RCK) domains per subunit. Using patch-clamp fluorometry, we show that Ca2+ binding to the RCK1 domain triggers gating ring rearrangements that depend on transmembrane voltage. Because the gating ring is outside the electric field, this voltage sensitivity must originate from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of the voltage sensor, either by mutagenesis or regulation by auxiliary subunits, are paralleled by changes in the voltage dependence of the gating ring movements, whereas modifications of the relative open probability are not. These results strongly suggest that conformational changes of RCK1 domains are specifically coupled to the voltage sensor function during allosteric modulation of BK channels.

Research organism: Human, Xenopus

Introduction

The open probability of large conductance voltage-and Ca2+-activated K+ (BK or slo1) channels is regulated allosterically by voltage and intracellular concentration of divalent ions (Barrett et al., 1982; Moczydlowski and Latorre, 1983; Horrigan and Aldrich, 2002; Latorre et al., 2017). This feature makes BK channels important regulators of physiological processes such as neurotransmission and muscular function, where they couple membrane voltage and the intracellular concentration of Ca2+ (Robitaille and Charlton, 1992; Hu et al., 2001; Wang et al., 2001; Raffaelli et al., 2004). The BK channel is formed in the membrane as tetramers of α subunits, encoded by the KCNMA1 gene (Shen et al., 1994; Quirk and Reinhart, 2001). Each α subunit contains seven transmembrane domains (S0 to S6), a small extracellular N-terminal domain and a large intracellular C-terminal domain (Wallner et al., 1996; Meera et al., 1997; Tao et al., 2017) (Figure 2a). Similar to other voltage-gated channels, the voltage across the membrane is sensed by the voltage sensor domain (VSD), containing charged amino acids within transmembrane segments S2, S3 and S4 (Díaz et al., 1998; Ma et al., 2006; Pantazis and Olcese, 2012; Tao et al., 2017). The sensor for divalent cations is at the C-terminal region and is formed by two Regulator of Conductance for K+ domains (RCK1 and RCK2) per α subunit (Wei et al., 1994; Moss and Magleby, 2001; Xia et al., 2002; Zeng et al., 2005; Wu et al., 2010). In the tetramer, four RCK1-RCK2 tandems pack against each other in a large structure known as the gating ring (Wu et al., 2010; Yuan et al., 2011; Giraldez and Rothberg, 2017; Tao et al., 2017; Zhou et al., 2017). Two high-affinity Ca2+ binding sites are located in the RCK2 (also known as ‘Ca2+ bowl’) and RCK1 domains, respectively. Additionally, a site with low affinity for Mg2+ and Ca2+ is located at the interface between the VSD and the RCK1 domain (Shi and Cui, 2001; Zhang et al., 2001; Bao et al., 2002; Xia et al., 2002; Yang et al., 2007; Yang et al., 2008a; Tao et al., 2017) (Figure 2a). The high-affinity binding sites show structural dissimilarity (Zhang et al., 2010; Tao et al., 2017) and different affinity for divalent ions (Zeng et al., 2005). Apart from Ca2+, it has been described that Cd2+ selectively binds to the RCK1 site, whereas Ba2+ and Mg2+ show higher affinity for the RCK2 site (Xia et al., 2002; Zeng et al., 2005; Yang et al., 2008b; Zhou et al., 2012; Miranda et al., 2016). Thus, intracellular concentrations of Ca2+, Cd2+, Ba2+ or Mg2+ can shift the voltage dependence of BK activation towards more negative potentials. Using patch clamp fluorometry (PCF), we have shown that these cations trigger independent conformational changes of RCK1 and/or RCK2 within the gating ring, measured as large changes in the efficiency of Fluorescence Resonance Energy Transfer (FRET) between fluorophores introduced into specific sites in the BK tetramer. These rearrangements depend on the specific interaction of the divalent ions with their high-affinity binding sites, showing different dependences on cation concentration and membrane voltage (Miranda et al., 2013; Miranda et al., 2016). To date, the proposed transduction mechanism by which divalent ion binding increases channel open probability was a conformational change of the gating ring that leads to a physical pulling of the channel gate, where the linker between the S6 transmembrane domain and the RCK1 region acts like a passive spring (Niu et al., 2004). Such a mechanism would be analogous to channel activation by ligand binding in glutamate receptor or cyclic nucleotide-gated ion channels, also tetramers (Sobolevsky et al., 2009; James et al., 2017). Our previous results do not support this as the sole mechanism underlying coupling of divalent ion binding to channel opening, since the gating ring conformational changes that we have recorded: 1) are not strictly coupled to the opening of the channel’s gate, and 2) show different voltage dependence for each divalent ion. In addition, the recent cryo-EM structure of the full slo1 channel of Aplysia californica (Hite et al., 2017; Tao et al., 2017) shows that the RCK1 domain of the gating ring is in contact with the VSD, predicting that changes in the voltage sensor position could be reflected in the voltage dependent gating ring reorganizations.

Understanding the nature of the voltage dependence associated with individual rearrangements produced by binding of divalent ions to the gating ring is essential to untangle the mechanism underlying the role of such rearrangements in BK channel gating. To this end, we have now performed PCF measurements with human BK channels heterologously expressed in Xenopus oocytes, including a range of VSD mutations or co-expressed with different regulatory subunits. Here we provide evidence for a functional interaction between the gating ring and the voltage sensor in full-length, functional BK channels at the plasma membrane, in agreement with the structural data from Aplysia BK. Moreover, these data support a pathway that couples to divalent ion binding to channel opening through the voltage sensor.

Results

Voltage dependence of gating ring rearrangements is associated to activation of the RCK1 binding site

BK α subunits labeled with fluorescent proteins CFP and YFP in the linker between the RCK1 and RCK2 domains (position 667) retain the functional properties of wild-type BK channels (Miranda et al., 2013; Miranda et al., 2016). This allowed us to use PCF to detect conformational rearrangements of the gating ring measured as changes in FRET efficiency (E) between the fluorophores (Miranda et al., 2013; Miranda et al., 2016). Binding of Ca2+ ions to both high-affinity binding sites (RCK1 and Ca2+ bowl) produces an activation of BK channels, coincident with an increase in E from basal levels reaching saturating values at high Ca2+ concentrations (Miranda et al., 2013 and Figure 1a). In addition, we observed that the E signal has the remarkable property that in intermediate Ca2+ concentrations (from 4 µM to 55 µM) it shows voltage dependence besides its Ca2+ dependence (Miranda et al., 2013 and Figure 1a). As discussed previously (Miranda et al., 2013), these changes in E with voltage are not conformational dynamics of the gating ring that simply follow the voltage dependence of VSD. For instance, at 0 Ca2+ concentrations movements of the VSD occurs between 0 and +300 mV (Stefani et al., 1997; Horrigan et al., 1999; Horrigan and Aldrich, 2002; Zhang et al., 2014; Carrasquel-Ursulaez et al., 2015; Zhang et al., 2017). However, we do not observe changes in E between 0 and +240 mV (Figure 1a). Similarly, at 100 µM Ca2+, charge movement takes place between −100 and +150 mV (Carrasquel-Ursulaez et al., 2015), while our FRET signals at 95 µM Ca2+ do not vary within this voltage range (Figure 1a). Independent activation of high-affinity binding sites by other divalent ions (Ba2+, Cd2+, or Mg2+ (Miranda et al., 2016)) led us to postulate that Ca2+ activation has a site-dependent relation to voltage. To further evaluate the effect of individual high-affinity Ca2+ binding sites on the voltage-dependent component of the gating ring conformational changes we first selectively mutated the binding sites. Mutations D362A and D367A (Xia et al., 2002; Zeng et al., 2005) were introduced in the BK667CY construct (BK667CYD362A/D367A) to remove the high-affinity binding site located in the RCK1 domain. Figure 1b shows the relative conductance and E values for the BK667CYD362A/D367A construct at different membrane voltages for various Ca2+ concentrations. As described previously, the G-V curves show a significantly reduced shift to more negative potentials when Ca2+ is increased, as compared to the non-mutated BK667CY (Figure 1a–b, left panels). Specific activation of the Ca2+ bowl renders a smaller change in E values, which are not voltage-dependent within the voltage range tested (Figure 1b, right panel). To test the effect of eliminating the RCK2 Ca2+ binding site -the Ca2+ bowl- we mutated five aspartates to alanines (5D5A) (Bao et al., 2002). As expected, activation of only the RCK1 domain by Ca2+ reduced the Ca2+-dependent shift in the GV curves (Figure 1c, left panel). Even though the extent to which the E values changed with Ca2+ was reduced (Figure 1c), there was a persistent voltage dependence equivalent to that shown in Figure 1a corresponding to the non-mutated channel (most appreciable at 12 µM and 22 µM Ca2+ concentrations; Figure 1c, right panel) (Miranda et al., 2013). Further, at these two Ca2+ concentrations the changes in E occurred within the same voltage range (+60–120 mV) in channels with the Ca2+ bowl mutated (Figure 1c) or not (Figure 1a). This effect seems not to be attributable to Ca2+ binding to unknown binding sites in the channel, since the double mutation of the RCK1 and RCK2 sites abolishes the change in the FRET signal (Figure 1d). Altogether, these results indicate that the voltage-dependent component of the gating ring conformational changes triggered by Ca2+ in the BK667CY construct depends on activation of the RCK1 binding site. Because the gating ring is not within the transmembrane region, it is not expected to be directly influenced by the transmembrane voltage. Therefore, the voltage-dependent FRET signals must be coupled to the dynamics of the gate region associated with the opening and closing of the channel and/or those of the voltage sensor domain.

Figure 1. Voltage dependence of gating ring rearrangements is associated to activation of the RCK1 binding site.

Figure 1.

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G-V (left panels) and E-V curves (right panels) obtained simultaneously at several Ca2+concentrations from (a) the BK667CY construct, (b) mutation of the RCK1 high-affinity site (D362A/D367A), (c) mutation of the Ca2+ bowl (5D5A), or (d) both (D362A/D367A 5D5A). Note that the voltage dependence of the E signal is only abolished after mutating the RCK1 high-affinity binding site (b) or both (d). Data corresponding to each Ca2+ concentration are color-coded as indicated in the legend at the bottom. Solid curves in the G-V graphs represent Boltzmann fits. For reference, grey shadows in (a–d) left panels represent the full range of G-V curves corresponding to non-mutated BK667CY channels from 0 μM Ca2+ to 95 µM Ca2+ (indicated with colored dashed lines). Data points and error bars represent average ± SEM (n = 3–14, N = 2–8). Part of the data in (a, b and c) are taken from (Miranda et al., 2013) and (Miranda et al., 2016).

The voltage-dependent conformational changes of the gating ring are not related to the opening and closing of the pore domain

To test whether the voltage-dependent FRET signals relate to the opening and closing of the channel (intrinsic gating) we used two modifications of BK channel function in which the relative probability of opening is shifted in the voltage axis, yet the actual dynamics of voltage sensor are expected to be unaltered (Figure 2b). We reasoned that, if the voltage-dependent FRET signals of the gating ring are coupled to the opening and closing, they should follow a similar displacement with voltage. The first BK channel construct is the α subunit including the single point mutation F315A, which has been described to shift the voltage dependence of the relative conductance of the channel to more positive potentials, by uncoupling the voltage sensor activation from the gate opening (Figure 2c) (Carrasquel-Ursulaez et al., 2015). Figure 2d shows the relative conductance and E vs. voltage for the BK667CYF315A mutant at various Ca2+concentrations. Our results show that the shift of the relative probability of opening to more positive potentials (Figure 2d, left panel) does not lead to changes in the voltage dependence of the gating ring FRET signals (Figure 2d, right panel).

Figure 2. Modification of the voltage dependence of gate opening does not affect the gating ring voltage-dependent conformational changes.

Figure 2.

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(a) Topology of the BKα subunit where the voltage sensing domain (VSD), Ca2+ sensing domain (gating ring, GR) and pore domain (PD) are indicated by colored dashed lines boxes (see main text for a full description). (b) The three BK functional modules (VSD, PD, GR), schematically represented as colored boxes, interact allosterically. (c) Diagram representing the main effect of the F315A mutation, which is the uncoupling of the VSD to the PD. (d) G-V (left panel) and E-V curves (right panel) obtained simultaneously at several Ca2+concentrations after mutation of the F315 site to alanine (BK667CYF315A). It should be noted that the extent of the shifts induced by the mutation are smaller than previously reported (Carrasquel-Ursulaez et al., 2015), which could arise from the different experimental conditions and/or our fluorescent construct. (e) The interaction with the γ1 subunit favors the VSD-PD coupling mechanism (f) G-V (left) and E-V curves (right) of BK667CY α subunits co-expressed with γ1 subunits. In all panels, data corresponding to each Ca2+ concentration are color-coded as indicated in the bottom legend. Colored dashed lines represent the G-V and E-V curves corresponding to BK667CYα channels (Miranda et al., 2013; Miranda et al., 2016). The solid curves in the G-V graphs represent Boltzmann fits. The full range of G-V curves from 0 μM Ca2+ to 95 µM Ca2+ from BK667CY is represented as a grey shadow in left panels (d and f), for reference. Data points and error bars represent average ± SEM (n = 3–8; N = 2–3).

The second modification of BK function consisted in co-expressing the wild type α subunit with the auxiliary subunit γ1 (Yan and Aldrich, 2010; Yan and Aldrich, 2012; Gonzalez-Perez et al., 2014; Li and Yan, 2016). In this case, the relative probability of opening is shifted to more negative potentials by increasing the coupling between the voltage sensor and the gate of the channel (Figure 2e). This construct adds the advantage of representing a physiologically relevant modification of channel gating. Figure 2f shows the relative conductance and E vs. voltage in oocytes co-expressing the BK667CYα and γ1 at voltages ranging from −160 to +260 mV, with three [Ca2+] concentrations: nominal 0, 12 µM and 22 µM. As expected, the presence of the γ1 subunit drives the relative conductance curves to more negative potentials (Figure 2f, left panel) compared to the values obtained without γ1 (Figure 2f, dashed lines). Remarkably, the change in the voltage dependence of the relative conductance induced by γ1 does not alter the simultaneously recorded FRET signals (Figure 2f, right panel), which remains indistinguishable from that recorded with BK667CYα (Figure 2f, dashed lines).

The dynamics of the VSD are directly reflected in the gating ring conformation

Using the allosteric HA model of BK channel function, Horrigan and Aldrich (2002) proposed that Ca2+ binding to the Ca2+ bowl is coupled to the voltage sensor activation. Yet, the strength of that interaction (allosteric constant E) was smaller than those corresponding to Ca2+- or V-sensors with channel opening (Horrigan and Aldrich, 2002). Interestingly, when E was derived from gating currents data, a larger value was obtained (Carrasquel-Ursulaez et al., 2015). Further, Ca2+ binding to the RCK1 domain (but not to the Ca2+ bowl) is voltage-dependent (Sweet and Cox, 2008), which as the authors hypothesized might originate from physical interactions between the voltage sensors and the RCK1 domains. Additionally, using the cut-open oocyte voltage-clamp fluorometry approach, Savalli et al. (2012) showed that fluorescence emission from reporters within the VSD could change upon uncaged Ca2+ stimuli. This evidence indicates that the VSD is coupled to the gating ring, but none of these approaches directly monitored the conformational changes of the gating ring structure. Therefore, we decided to explore whether the voltage dependence of the gating ring movements is attributable to the voltage sensor activation. To this end we modified the voltage dependence of the VSD activation by co-expression with β auxiliary subunits or by introducing specific mutations in the VSD (Figure 3 and Figure 4). The effects of co-expressing BK α subunit with the four different types of auxiliary β subunits have been extensively studied (Tseng-Crank et al., 1996; Behrens et al., 2000; Brenner et al., 2000; Cox and Aldrich, 2000; Uebele et al., 2000; Lingle et al., 2001; Zeng et al., 2001; Bao and Cox, 2005; Orio and Latorre, 2005; Yang et al., 2008a; Sweet and Cox, 2009; Contreras et al., 2012; Li and Yan, 2016). β1 subunit has been previously proposed to alter the voltage sensor-related voltage dependence, as well as the intrinsic opening of the gate and Ca2+ sensitivity (Figure 3a) (Cox and Aldrich, 2000; Bao and Cox, 2005; Orio and Latorre, 2005; Sweet and Cox, 2009; Contreras et al., 2012; Castillo et al., 2015). Recordings from BK667CYα co-expressed with β1 subunits reveal the expected modifications in the voltage dependence of the relative conductance, that is an increase in the apparent Ca2+ sensitivity (Figure 3b, left panel) (Wallner et al., 1995; Cox and Aldrich, 2000; Bao and Cox, 2005; Orio and Latorre, 2005; Sweet and Cox, 2009; Contreras et al., 2012). In addition, it has been reported that β1 subunit alters the function of the VSD (Orio and Latorre, 2005; Castillo et al., 2015). Notably, the E-V curves are shifted to more negative potentials (Figure 3b, right panel), similarly to the described modification (Castillo et al., 2015). The structural determinants of the β1 subunit influence on the VSD reside within its N-terminus, which has been shown by engineering a chimera between the β3b subunit (which does not influence the VSD) and the N-terminus of the β1 (β3bNβ1) (Castillo et al., 2015). We recapitulated this strategy. First, we co-expressed BK667CY α subunits with β3b and observed the expected inactivation of the ionic currents at positive potentials, yet with different blockade kinetics (see Figure 3—figure supplement 1) (Uebele et al., 2000; Xia et al., 2000; Lingle et al., 2001). The relative open probability of this complex is like BK667CYα alone, except that at extreme positive potentials the values of relative conductance at the tails decrease due to inactivation (Figure 3—figure supplement 1b, left panel). The values of E vs V remained comparable to those observed for BK667CYα (Figure 3—figure supplement 1b, right panel). We then co-expressed the β3bNβ1 chimera (Castillo et al., 2015) with BK667CYα (Figure 3c). This complex did not modify the relative conductance vs. voltage relationship (Figure 3d, left panel) as compared with BK667CYα alone (Figure 3d, grey shadow). On the other hand, while the magnitude of the FRET change is the same as in BK667CYα, the voltage dependence of E values at [Ca2+] of 4 μM, 12 μM and 22 μM shifted to more negative potentials compared to the values of BK667CYα alone (Figure 3d, right panel, compare dashed to solid lines). Altogether, these results indicate that the alteration of the voltage dependence of the voltage sensor induced by the amino terminal of β1within the β3bNβ1 chimera underlies the modification of the voltage dependence of the gating ring conformational changes, reinforcing the hypothesis that this voltage dependence is directly related to VSD function.

Figure 3. Co-expression with β subunits.

(a) β1 subunits have been shown to directly regulate VSD function, shifting Vh(j) to more negative values (b) Left panel, G-V curves obtained at several Ca2+concentrations after co-expression of BK667CY with the β1 subunit, which induces a leftward shift in the E-V curves obtained simultaneously (right). (c) β3bNβ1 chimeras produce similar effects to β1 on VSD function, since they retain the N-terminal region of β1 (Castillo et al., 2015). (d) G-V (left) and E-V curves (right) of BK667CY α subunits co-expressed with the β3bNβ1 chimera. Data corresponding to each Ca2+ concentration are color-coded as indicated in the legend at the bottom. Colored dashed lines represent the G-V and E-V curves corresponding to BK667CYα channels (Miranda et al., 2013; Miranda et al., 2016). The solid curves in the G-V graphs represent Boltzmann fits. The full range of G-V curves from 0 μM Ca2+ to 95 µM Ca2+ from BK667CY is represented as a grey shadow in left panels (b and d), for reference. Data points and error bars represent average ± SEM (n = 3–10; N = 2–4).

Figure 3.

Figure 3—figure supplement 1. Co-expression with β3b subunits.

Figure 3—figure supplement 1.

(a) Representative currents obtained after applying depolarizing pulses to inside-out patches expressing BK667CYα (left) or BK667CYα + β3b channels, in the presence of 12 μM Ca2+. (b) Left panel, G-V curves obtained at several Ca2+concentrations after co-expression of BK667CY with β3b subunits, inducing no appreciable changes in the E-V curves obtained simultaneously (right). Note that when β3b is co-expressed with the BK667CY construct, the kinetics of inactivation are different than those observed with wild-type BK channels (Xia et al., 2000). At first glance, it appears as if the off rate of inactivation is largely increased. Note also that there is a substantial current reduction at the tails as blockade increased with very positive potentials, which is not observed with wild-type BK channels. The simplest interpretation is that the insertion of the fluorescent protein interferes with the kinetics of blockade mediated by the β3b NH2-terminal region (Lingle et al., 2001). Understanding this discrepancy will require further study.
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Figure 4. Mutation of charged residues of BK VSD.

Figure 4.

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VSD activation was altered by mutation of charged residues in the VSD that modify its voltage of half activation, Vh(j) (a) The R210E mutation induces a negative shift of Vh(j)(b) G-V (left panel) and E-V curves (right panel) obtained simultaneously from constructs BK667CY containing the R210E mutation at several Ca2+concentrations. (c) The E219R mutation produces a negative shift of Vh(j) (d) G-V (left panel) and E-V curves (right panel) obtained simultaneously from constructs BK667CY containing the E219R mutation at several Ca2+concentrations. (e) The R213E mutation induces a large positive shift of Vh(j) values. (f) G-V (left panel) and E-V curves (right panel) obtained simultaneously from constructs BK667CY containing the R213E mutation at several Ca2+concentrations. Data corresponding to each Ca2+ concentration are color-coded as indicated in the bottom legend. Colored dashed lines represent the G-V and E-V curves corresponding to non-mutated BK667CYα channels (Miranda et al., 2013; Miranda et al., 2016). The solid curves in the G-V graphs represent Boltzmann fits. The full range of G-V curves from 0 μM Ca2+ to 95 µM Ca2+ from BK667CY is represented as a grey shadow in left panels (b), (d and f), for reference. Data points and error bars represent average ± SEM (n = 4–10; N = 3–4).

VSD activation can also be altered by introducing single point mutations that modify the voltage of half activation of the voltage sensor, Vh(j). This parameter is determined by fitting data to the HA allosteric model (Ma et al., 2006) or directly from gating current measurements (Zhang et al., 2014). Mutations of charged amino acids on the VSD have been reported to produce different modifications in the Vh(j) values. In some cases, other parameters related to BK channel activation are additionally affected by the mutations. Mutation R210E shifts the Vh(j) value from +173 mV to +25 mV at 0 Ca2+ in BK channels (Figure 4a) (Ma et al., 2006). Consistent with this, introduction of this mutation in BK667CYα (BK667CYR210E) caused a shift of the relative conductance vs. voltage dependence towards more negative potentials (Figure 4b, left panel) as compared to BK667CY (Figure 4b, left panel, grey shadow). Simultaneously measured E values showed a negative shift in the voltage dependence of the FRET signal at intermediate Ca2+ concentrations (Figure 4b, right panel). Mutation E219R had been previously shown to produce a large negative shift in Vh(j) from +150 mV to +40 mV (ΔVh(j) = −110 mV; Figure 4c), additionally modifying the Ca2+ sensitivity and the coupling between the VSD and channel gate (Zhang et al., 2014). As previously reported, BK667CYE219R showed modified relative conductance vs. voltage relationships at different Ca2+ concentrations (Figure 4d, left panel) (Zhang et al., 2014). In addition, this construct revealed a shift to more negative potentials in the E vs. voltage dependence at intermediate Ca2+ concentrations (12 μM and 22 μM Ca2+; Figure 4d, right panel), paralleling the reported negative shift in Vh(j) (Ma et al., 2006; Zhang et al., 2014). Since mutations displacing the Vh(j) to more negative potentials induce equivalent shifts in the voltage dependence of the gating ring motion (measured as E), we tested if other mutations previously reported to induce positive shifts on Vh(j) (Ma et al., 2006) were also associated with changes of the E-V curves in the same direction. As shown by Ma et al., the largest effect on Vh(j) is induced by the R213E mutation, producing a shift of ΔVh(j)=+337 mV (Figure 4e) (Ma et al., 2006). The BK667CYR213E construct showed a significant shift in the voltage dependence of the relative conductance to more positive potentials (Figure 4f, left panel). Notably, this effect was paralleled by a large displacement in the E vs. voltage dependence towards more positive potentials (Figure 4f, right panel). Taken together, our data show that modifications of the Vh(j) values caused by mutating the VSD charged residues are reflected in equivalent changes in the voltage dependence of the gating ring conformational rearrangements, which occur in analogous directions and with proportional magnitudes at intermediate Ca2+ concentrations.

All these results on the VSD modifications and their corresponding changes in FRET signals support the existence of a direct coupling mechanism between the VSD function and the gating ring conformational changes.

Parallel alterations of the voltage dependence of VSD function and gating ring motions by selective activation of the RCK1 binding site

We have previously shown that specific interaction of Cd2+ with the RCK1 binding site leads to activation of the BK channel, which is accompanied by voltage-dependent changes in the E values at intermediate Cd2+ concentrations of 10 μM and 30 μM (Miranda et al., 2016). To further assess the role of the RCK1 binding site activation in the voltage dependence of the gating ring motions, we studied activation by Cd2+ of selected BK667CY VSD mutants (Figure 5). Addition of Cd2+ to the BK667CYE219R mutant (Figure 5a) shifted the voltage dependence of E towards more negative potentials at intermediate Cd2+ concentrations (10 μM and 30 μM; Figure 5b) when compared to non-mutated BK667CY (Figure 5b; dashed lines). This change in the E-V curves induced by selective activation of the RCK1 binding site with Cd2+ paralleled the large negative shift (ΔVh(j) = −110 mV) previously reported with the E219R mutant BK channels (Ma et al., 2006; Zhang et al., 2014). We also tested Cd2+ activation in the mutant BK667CYR201Q, which shifts the Vh(j) parameter by 47 mV towards positive potentials (Figure 5c) (Ma et al., 2006). Addition of Cd2+ rendered right-shifted E vs. voltage relationships (Figure 5d, right panel), following the direction of the predicted Vh(j) shift described for this mutant BK channel (Ma et al., 2006). Finally, addition of Cd2+ to the BK667CYF315A construct (Figure 5e) (Carrasquel-Ursulaez et al., 2015) did not have any effect on the E-V relationship (Figure 5f). These results are consistent with a mechanism in which specific binding of Cd2+ to the RCK1 binding site allows voltage-dependent conformational changes in the gating ring that are directly related to VSD activation.

Figure 5. Voltage dependence of gating ring rearrangements after specific activation of RCK1 high-affinity binding site by Cd2+.

Figure 5.

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(a) Effect of the VSD E219R mutation on the selective activation of RCK1 by Cd2+. (b) G-V (left panels) and E-V curves (right panels) obtained simultaneously at several Ca2+concentrations from constructs BK667CYE219R. (c) VSD R201Q mutation induces a positive shift of Vh(j) (d) G-V (left panels) and E-V curves (right panels) obtained simultaneously at several Cd2+concentrations from constructs BK667CYR201Q (e) Effect of the F315A mutation on the selective activation of RCK1 by Cd2+. (f) G-V (left panels) and E-V curves (right panels) obtained simultaneously at several Cd2+ concentrations from constructs BK667CYF315A. Data corresponding to each Cd2+ concentration are color-coded as indicated in the legend at the bottom. Colored dashed lines represent the G-V and E-V curves corresponding to BK667CYα channels (Miranda et al., 2013; Miranda et al., 2016). The solid curves in the G-V graphs represent Boltzmann fits. The full range of G-V curves from 0 μM Cd2+ to 100 µM Cd2+ corresponding to non-mutated BK667CY is represented as a grey shadow in left panels (b), (d), and (f), for reference. Data points and error bars represent average ± SEM (n = 3–4; N = 2).

Voltage dependence of Ba2+-induced gating ring movement is related to function of the channel gate

Ca2+, Mg2+ and Ba2+ bind to the Ca2+ bowl and trigger conformational changes of the gating ring region (Miranda et al., 2016). However, the effects of these ions on BK function and gating ring motions are fundamentally different. Notably, Ba2+ induces a rapid blockade of the BK current after a transient activation that is measurable at low Ba2+ concentrations (Zhou et al., 2012; Miranda et al., 2016) (Figure 6a). In addition, we previously showed that the gating ring conformational motions induced by Ba2+ show a voltage-dependent component, which is not observed when Ca2+ or Mg2+ bind to the Ca2+ bowl (Miranda et al., 2013; Miranda et al., 2016) (Figure 6b). We combined mutagenesis with the cation-specific activation strategy to identify the structural source of the voltage dependence in Ba2+-triggered gating ring motions. In this case, alteration of VSD function by mutating charged residues (Figure 6c and e) was not reflected in any change of the E vs. voltage relationships, as shown in Figure 6d and f for constructs BK667CYR210E and BK667CYR213E, respectively. These results indicate that the voltage dependence of Ba2+-induced gating ring conformational changes, unlike those induced by Ca2+ and Cd2+ through activation of the RCK1 binding site, may not be related to VSD activation. This conclusion is further supported by the lack of changes in Ba2+ responses when mutations in the VSD were made in a RCK1 Ca2+ binding site knockout (D362A D367A) background (Figure 6—figure supplement 1b & c). Next, we studied the effect of Ba2+ on BK667CY channels containing the F315A mutation (Figure 6g) (Carrasquel-Ursulaez et al., 2015). As shown in Figure 6h, the E values reached similar levels to those of non-mutated BK667CY channels at saturating Ba2+ concentrations. However, at intermediate concentrations of Ba2+ the E-V curves were shifted towards more positive potentials when compared with BK667CY channels (Figure 6h, dashed line). These results suggest that the voltage-dependent component of the conformational changes triggered by Ba2+ binding to the Ca2+ bowl are not directly related to VSD activation, but rather to the function of the channel gate.

Figure 6. Voltage dependence of gating ring movements triggered by Ba2+.

(a) The RCK2 site is selectively activated by Ba2+, which additionally induces pore block. (b) FRET efficiency (E) data obtained at several Ba2+ concentrations from BK667CY constructs (Miranda et al., 2016). (c) Effect of the VSD R210E mutation after selective activation of the RCK2 binding site by Ba2+. (d) E-V curves obtained at several Ba2+ concentrations from BK667CYR210E constructs. (e) Effect of the VSD R213E mutation after selective activation of the RCK2 binding site by Ba2+. (f) E-V curves obtained at several Ba2+ concentrations from BK667CYR213E constructs. (g) Effect of the F315A mutation after selective activation of the RCK2 binding site by Ba2+ (h) E-V curves obtained at several Ba2+ concentrations from BK667CYF315A constructs. Data corresponding to each Ba2+ concentration are color-coded according to the legend at the bottom. For reference, the curve corresponding to 100 μM Ba2+ from the BK667CY construct shown in (b) is also shown as a colored dashed line in panels (b, d, f and h). Data points and error bars represent average ± SEM (n = 4–6; N = 2–3).

Figure 6.

Figure 6—figure supplement 1. Additional experiments to characterize voltage dependence of gating ring movements triggered by Ba2+.

Figure 6—figure supplement 1.

FRET efficiency (E) data obtained at several Ba2+ concentrations from: (a) BK667CY constructs including D362A/D367A mutations to knockout the RCK1 binding site (BK667CYD362A D367A). (b) BK667CYD362A D367A with additional VSD R210E mutation (BK667CYD362A D367A R210E). (c) BK667CYD362A D367A with additional VSD E219R mutation (BK667CYD362A D367A E219R). For reference, the curve corresponding to 100 μM Ba2+ from the BK667CY construct shown in Figure 6a is also shown as a grey line in panels (a, b, and c). Ba2+ concentrations are indicated in legend of panel a. (d) E-V curves obtained simultaneously at several Ca2+concentrations (indicated in the legend) from constructs BK667CYD362A D367A after addition of 100 μM bbTBA. Data points and error bars in all panels represent average ± SEM (n = 3). Data in panel (a) were partly obtained from (Miranda et al., 2016).
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Discussion

Using fluorescently labeled BKα subunit constructs reporting protein dynamics between the RCK1 and RCK2 domains, we previously demonstrated that the channel high-affinity binding sites can be independently activated by different divalent ions, inducing energetically-additive rearrangements of the gating ring measured as changes in the FRET efficiency values, E (Miranda et al., 2013; Miranda et al., 2016). Further, the effects of Ca2+, Cd2+ and Ba2+ on the E values showed a voltage-dependent component, for which we could not provide an explanation. Voltage dependence of Ca2+-induced rearrangements seemed to be specifically related to RCK1 activation, since only the mutation of that binding site resulted in voltage-independent E signals (Miranda et al., 2016 and Figure 1). One possibility to explain this result is the existence of direct structural interactions of the RCK1 domain and the VSD. Interestingly, the recently obtained cryo-EM full BK structure from Aplysia californica revealed the existence of specific protein-protein interfaces formed by the amino terminal lobes of the RCK1 domains facing the transmembrane domain and the VSD/S4-S5 linkers (Hite et al., 2017). According to the structural data obtained in saturating Mg2+ and Ca2+ concentrations, gating of the channel by Ca2+ was proposed to be mediated, at least partly, by displacement of these interfaces causing the VSD/S4-S5 linkers to move, contributing to pore opening ((Hite et al., 2017; Tao et al., 2017); but see also (Zhou et al., 2017)). Our work provides functional data supporting this mechanism. Our data show that mutations altering the voltage dependence of BK VSD are reflected in the voltage dependence of the gating ring movements triggered by activation of the RCK1 binding site by Ca2+ or Cd2+. Mutations altering VSD function by inducing large leftward shifts in the Vh(j) values (Ma et al., 2006; Zhang et al., 2014) strongly correlate with negative shifts in the voltage dependence of the E signals. Likewise, mutations inducing positive shifts in the VSD voltage dependence of the voltage sensor function are reflected in E-V shifts towards more positive membrane voltages. Interestingly, we also observe a correlation between the changes in the slope of the G-V curves and that of the E-V curves (e.g. Figure 4f; see also Supplementary file 1), suggesting the existence of an interaction between the VSD and the gating ring. This idea is further supported by the effect of β1 which has been proposed to alter the voltage dependence of VSD function (Wallner et al., 1995; Cox and Aldrich, 2000; Nimigean and Magleby, 2000; Bao and Cox, 2005; Orio and Latorre, 2005; Contreras et al., 2012; Castillo et al., 2015). We observed that β1 and β3bNβ1 induce a leftward shift in the E-V curves. Conversely, two experimental strategies known to influence the G-V curves without direct interference with the VSD did not affect the voltage dependence of E. The lack of effect on the E-V curves of the mutation F315A can be explained because the shift in the G-V curves arises from the influence of this mutation in the C↔O transition with minor effects on the voltage dependence of the gating currents (Carrasquel-Ursulaez et al., 2015). Analogously, no change in the voltage dependence of E was observed after co-expression of BKα with the γ1 subunit, which shifts the voltage dependence of pore opening by enhancing its allosteric coupling with the voltage sensor activation (Yan and Aldrich, 2010). As with the mutation F315A, the presence of γ1 subunit produces a minor shift in the Q-V distributions, not paralleling the large shift in the G-V curves (Carrasquel-Ursulaez and Ramon Latorre, personal communication).

A puzzling result from our previous study was the observation that Ba2+ binding to the Ca2+ bowl triggers voltage-dependent conformational changes (Miranda et al., 2016). Even though we still do not know the mechanisms of this unique response to Ba2+, here we learned that it is not related to the dynamics of VSD, but rather influenced by perturbations affecting the opening and closing of the channel at the pore domain. Why Ba2+ but not Ca2+? A possible answer for this question is that Ba2+ has the additional property of blocking the permeation pathway (Miller, 1987; Neyton and Miller, 1988; Zhou et al., 2012), which could somehow be transmitted allosterically to the gating ring. If simply ion permeation blockade is what matters, then we might expect that blocking permeation with the high affinity quaternary ammonium derivative N-(4-[benzoyl]benzyl)-N,N,N-tributylammonium (bb-TBA) (Tang et al., 2009) should produce a voltage dependent FRET signal with Ca2+ activation. But, it does not (Figure 6—figure supplement 1d). Another possibility for the Ba2+ effect could be a direct allosteric interaction between the intrinsic gating in the pore and the divalent binding site in RCK2, which needs to be tested further.

Irrespectively of the fluorescent construct (Miranda et al., 2013) or the divalent ion used to activate the BK channel (Miranda et al., 2016), we have consistently observed that the conformational changes monitored as changes in the FRET efficiency are not strictly coupled to the intrinsic gating of the channel. In this study, we have found that the consequences of the voltage dependence of the intrinsic gating by manipulations of the VSD and the pore region are paralleled by the FRET efficiencies. These results rule out the possibilities that FRET signals derive from conformational changes in an unknown Ca2+ binding site or that they are completely uncoupled to the intrinsic gating.

In conclusion, our functional data show a strong correlation between the VSD function and the RCK1 conformational changes, suggesting a transduction mechanism from ion binding to change the channel activation. This transduction mechanism is in agreement with the existence of structural interactions between the RCK1 domain and the VSD. The correlation between VSD function and the RCK1 conformational changes is not observed between RCK2 and VSD, suggesting the existence of a different transduction mechanism that may include an indirect mechanism through the RCK1 or RCK1-S6 linker.

Materials and methods

Molecular biology and heterologous expression of tagged channels

Fluorescent BK α subunits were labelled with CFP or YFP using a transposon-based insertion method (Giraldez et al., 2005). Subunits labelled in the position 667 were subcloned into the pGEMHE oocyte expression vector (Liman et al., 1992). RNA was transcribed in vitro with T7 polymerase (Ambion, Thermo Fisher Scientific, Waltham, USA), and injected at a ratio 3:1 of CFP: YFP into Xenopus laevis oocytes, giving a population enriched in 3CFP:1YFP labelled tetramers (BK667CY) (Miranda et al., 2013; Miranda et al., 2016). Individualized Oocytes were obtained from Xenopus laevis extracted ovaries (Nasco, Fort Anderson, WI, USA). Neutralization of the Ca2+ bowl was achieved by mutating five consecutive aspartate residues to alanines (5D5A: 894–899) (Bao et al., 2002) on the BK667CY background. Elimination of RCK1 high-affinity Ca2+ sensitivity was achieved by double mutation D362A and D367A (Xia et al., 2002; Zeng et al., 2005; Zhang et al., 2010). Mutations were performed using standard procedures (Quickchange, Agilent Technologies, Santa Clara, USA). Auxiliary subunits (β3b, γ1 and chimera β3bNβ1) were co-injected with the BK667CFP/BK667YFP RNA mix at a 5:1 wt ratio, giving molar ratios above 20:1.

Patch-clamp fluorometry and FRET

Borosilicate pipettes with a large tip (0.7–1 MΩ in symmetrical K+) were used to obtain inside-out patches excised from Xenopus laevis oocytes expressing BK667CY. Currents were recorded with the Axopatch 200B amplifier and Clampex software (Axon Instruments, Molecular Devices, Sunnyvale, USA). Recording solutions contained (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 MgCl2 or BaCl2 to give the appropriate divalent concentration previously estimated using Maxchelator software (maxchelator.standford.edu) (Bers et al., 1994). Solutions containing Cd2+ were prepared with a bath solution containing KF instead of K-Mes to precipitate the contaminant Ca2+ previously to the administration of the proper concentration of CdCl2 estimated with Maxchelator. Solutions containing different ion concentrations were exchanged using a fast solution-exchange system (BioLogic, Claix, France). All experiments were performed in various batches of oocytes, using different Ca2+ solutions prepared over time.

Simultaneous fluorescent and electrophysiological recordings were obtained as previously described (Miranda et al., 2013; Miranda et al., 2016). Conductance-voltage (G-V) curves were obtained from tail currents using standard procedures. The G-V relations were fit with the Boltzmann function: G/Gmax = 1/(1 + exp (-zF(V-Vhalf)/RT), where Gmax is the maximum tail current, z is the voltage dependence of activation, Vhalf is the half-activation voltage of the ionic current. T is the absolute temperature (295K), F is the Faraday’s constant and R the universal gas constant. Fit parameters are provided in Supplementary file 1. Conformational changes of the gating ring were tracked as intersubunit changes of the FRET efficiency between CFP and YFP as previously reported (Miranda et al., 2013; Miranda et al., 2016). Analysis of the FRET signal was performed using emission spectra ratios. We calculated the FRET efficiency as E=(RatioA-RatioA0)/(RatioA1-RatioA0), where RatioA and RatioA0 are the emission spectra ratios for the FRET signal and the control only in the presence of acceptor respectively (Zheng and Zagotta, 2003); RatioA1 is the maximum emission ratio that we can measure in our system (Miranda et al., 2013; Miranda et al., 2016). This value of E is proportional to FRET efficiency (Zheng and Zagotta, 2003). The E value showed is an average of the E value corresponding to each tetramer present in the membrane patch and represent an estimation of the distance between the fluorophores located in the same position of the four subunits of the tetramer. Where possible, the E-V relations were fit with the Boltzmann function: E = 1/(1 + exp (-zF(V-Vhalf)/RT), where z is the voltage dependence of the gating ring movement (E) and Vhalf is the half-activation voltage of the fluorescent signal. Fit parameters are provided in Supplementary file 1.

Acknowledgments

MH and PM were supported by the intramural section of the National Institutes of Health (NINDS). TG was funded by the Spanish Ministry of Economy and Competitivity (grants SAF2013-50085-EXP and RyC-2012–11349) and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement 648936). We thank Deepa Srikumar for technical assistance and Andrew Plested for useful comments on the manuscript. The γ1 clone and the β3bNβ1 chimera were kind gifts from Chris Lingle and Ramon Latorre, respectively.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Pablo Miranda, Email: pablo.mirandafernandez2@nih.gov.

Teresa Giraldez, Email: giraldez@ull.edu.es.

Richard Aldrich, The University of Texas at Austin, United States.

Richard Aldrich, The University of Texas at Austin, United States.

Funding Information

This paper was supported by the following grants:

  • National Institute of Neurological Disorders and Stroke ZIA-NS002993 to Pablo Miranda, Miguel Holmgren.

  • H2020 European Research Council ERC-CoG-2014-648936 to Teresa Giraldez.

  • Ministerio de Economía y Competitividad SAF2013-50085-EXP to Teresa Giraldez.

  • Ministerio de Economía y Competitividad RyC-2012-11349 to Teresa Giraldez.

Additional information

Competing interests

No competing interests declared.

Author contributions

Conceptualization, Resources, Data curation, Formal analysis, Investigation, Visualization, Writing—original draft, Project administration, Writing—review and editing.

Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Visualization, Writing—original draft, Project administration, Writing—review and editing.

Conceptualization, Formal analysis, Supervision, Funding acquisition, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing.

Additional files

Supplementary file 1. Fit parameters of data shown in Figures 1–6.

The G-V and E-V relations were fit with Boltzmann functions G/Gmax = 1/(1 + exp (-zF(V-Vhalf)/RT), E = 1/(1 + exp (-zF(V-Vhalf)/RT), where Gmax is the maximum tail current, z is the voltage dependence of activation (G) or gating ring movement (E), Vhalf is the half-activation voltage of the ionic current or the fluorescent signal. T is the absolute temperature (295K), F is the Faraday’s constant and R the universal gas constant.

elife-40664-supp1.xlsx (23.2KB, xlsx)
DOI: 10.7554/eLife.40664.010
Transparent reporting form
elife-40664-transrepform.docx (246KB, docx)
DOI: 10.7554/eLife.40664.011

Data availability

All data generated and analysed during this study are included in the manuscript.

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Decision letter

Editor: Richard Aldrich1

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "The BK channel gating ring is strongly coupled to the voltage sensor" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Richard Aldrich as the Senior and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal his identity: Christopher J Lingle (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Essential revisions:

1) Modify the Title

Reviewers thought the title was vague and could be modified to better convey the message of the manuscript. The term "strongly coupled" also needs to be modified or justified since it implies a quantitative assessment of coupling that was not addressed.

2) Address previous work and novelty

The authors should provide a more complete comparison with previous work, especially with regards to VSD/gating-ring coupling or interaction, to help clarify "what is new" and whether the results are consistent or inconsistent with previous results.

3) Provide more guidance for interpreting the results

Indicating the expected status of voltage sensors under different conditions and manipulations would help readers to interpret the corresponding FRET data. Reviewers also desired more guidance on how to interpret the quantitative relationship between VSD activation and the voltage-dependence of FRET as well as several interesting or puzzling features of the data including the effect of Ba2+, the remarkably steep Ca2+ dependence of FRET, and the lack of voltage-dependence of FRET in 0 Ca2+. Mechanistic or quantitative interpretation is lacking, and ideally should be improved, if only to provide some framework for future evaluation and tests. Proposing a model (i.e. a kinetic scheme) to account for the major features of the relation between Ca2+, voltage, and FRET efficiency (i.e. lack of FRET in 0 Ca2+ or high Ca2+, and correlations observed in the different mutants) would greatly strengthen the conclusions and enhance the significance of the results. While developing a model to account for all features of the data may be beyond the scope of this study, the authors should at minimum indicate which features of the data can or cannot be accounted for by previously proposed models (i.e. Miranda 2013) and whether their data provide any new insight into mechanism that must be included in gating models. This should be possible to do without incorporating additional experiments.

4) Reconcile discrepancies and apparent inconsistencies in the data

The authors should address the apparent inconsistency between the effect of the γ1 subunit on FRET and its predicted effect on VSD activation, as well as discrepancies between some of the results and data previously published.

Results which seem to contradict earlier results include: a) GV shift magnitude of F315A in 100 Ca2+; b) Lack of a GV shift for the β3Nβ1 construct; c) Whether they have adequate expression of β3b.

Because the individual reviews include several important points they are included here for you reference.

Reviewer #1:

The manuscript by Miranda et al. examines energetic coupling in BK channels between its modular domains that underlie voltage sensing and calcium sensing, using patch-clamp fluorometry. The authors show here that the FRET efficiency (E) depends strongly on the integrity of the Ca2+ binding site in RCK1 (determined by D362/D367), and relatively much less on integrity of the Ca2+ bowl site. It is further shown that movements in the gating ring that are tracked by the authors' FRET measurements are not directly linked to channel opening, but instead to voltage-sensor dynamics. The greater relative contribution of the RCK1 site to VSD coupling is corroborated by selective activation of the site by Cd2+. However (interestingly), selective activation of the Ca2+ bowl site by Ba2+ also exhibits voltage-dependence in the FRET response, which is attributed energetic coupling with the movement of the gate. These results thus substantially extend previous work on preferential coupling of Ca2+ binding at the RCK1 site with voltage-sensor activation.

Overall, the manuscript is written in a logical format and the data, as presented, are of high quality. This is an outstanding contribution to our understanding of BK channels, forming a bridge between structural and functional studies. I have only a few comments aimed at improving the presentation.

1) The title seems a little vague; maybe the authors could think of one that more strongly conveys the message of this paper, which appears to be that the coupling is preferential, or selective, among the Ca2+-activation sites.

2) Unfortunately the authors measurement of FRET efficiency is represented by "E", and the allosteric coupling between the gating ring and voltage sensor from the Horrigan model is also "E". This makes things a bit confusing, but I'm not sure what can be done about it.

3) In Discussion (paragraph 1):

• "inducing additive rearrangements" should probably read "inducing energetically-additive rearrangements".

• " Further, the effects of Ca2+, Cd2+ and Ba2+ on the E values showed a voltage-dependent component, for which we could not provide a structural basis." This statement seems a little misleading; there seems to be some structural basis for voltage-dependence of Ca2+ and Cd2+ effects, because the RCK1 sight is physically somewhat close to the VSD (as the authors reason later on), and it is the Ba2+ effect that is more puzzling.

4) It seems like the interpretation of the Ba2+ effect may require more study, or more comment; if Ba2+ really is acting solely through the Ca2+ bowl, then the E-V relation observed with Ba and the S4 mutants (Figure 6D and F) should resemble the E-V relations with an RCK1 site knockout (D362A/D367A) combined with S4 mutation. Is this the case? If not, then this might argue against Ba2+ acting solely through the Ca2+ bowl, and suggest the Ba2+ is having other effects.

Reviewer #2:

This team has for several years been probing conformational changes in the cytosolic gating ring of the BK channel using sets of genetic encoded fluorescent tags to generate FRET signals that change with conformational status. Previous results had revealed an unexplained result that the FRET-reported signals exhibited voltage-dependence, despite the fact that the gating ring is not within the electric field across the membrane. This tended to contradict a standard model of BK activation in which coupling between voltage-sensors (VSDs) and the cytosolic domain (CTD) was thought to be rather weak. Furthermore, previous results from this team indicated that the two high affinity Ca2+ binding sites in the gating ring may behave differently in regards to voltage-sensitivity. The present paper nicely extends these earlier results with a number of functional tests that lead to the conclusion that, in the presence of Ca2+, the status of the voltage-sensors (VSDs) can influence the conformational status of the gating ring. Furthermore, this largely involves Ca2+ ligation at the RCK1 domain, but not the Ca2+ bowl. In sum, the authors find that the previously reported voltage-dependent conformational change of the BK gating ring induced by Ca2+-binding to the RCK1 high-affinity Ca2+ binding site is strongly correlated with the activation of the BK VSD. Manipulations that shift the activation-voltage relationship of the BK VSD, including mutations of key residues in the BK VSD or co-expression with β-1 accessory subunits, also shift the fluorescence-voltage relationship of the BK gating ring toward same direction. Manipulations that change other aspect of BK channel gating, such as F315A mutation that reduces the intrinsic BK gating equilibrium constant, the coupling between the BK VSD and BK gate, and the coupling the BK VSD and BK gating ring, or co-expression with the γ-1 subunit that enhances the coupling between the BK VSD and BK gate, do not significantly change the fluorescence-voltage relationship of the BK gating ring.

Overall, the paper is interesting, addresses an important, unresolved question, and is clearly presented (albeit with some suggestions below). There are some details on particular experiments which may be somewhat inconsistent with some earlier results and may require clarification. This particularly applies to some of the β subunit tests.

Guidance regarding status of voltage sensors under different conditions. Some readers might also wonder about what the voltage-sensor status is under various manipulations for which GVs and EVs are generated. Although explicit measurements of gating currents would be too much to ask, there are situations where it might be useful for the readers to be reminded whether VSDs are likely to be entirely resting or mostly activated, for example. The authors are essentially assuming that the E/V measurements are a surrogate for VSD activation, but unfortunately this is largely an untested assumption, although not unreasonable. For the F315A construct, the earlier Carrasquel, 2015 paper does provide comparable Q/V curves for human F380A, which support the view that the VSD equilibrium is not altered by this mutation. However, the Vh for activation of the human F380A construct in that paper at 100 μM Ca2+ is about 232 mV, much more positive than what is reported (less than +100 mV) in the present manuscript for mouse F315A. Reasons for this rather major discrepancy are not clear and not mentioned.

Are CTD and VSD totally uncoupled in absence of ligand? There is one aspect of the data that seems to pose an interesting limiting condition regarding the overall mechanism that the authors might consider discussing. Specifically, in the absence of Ca2+, any conformational change involving VSDs and pore-gate-domain (PGD) activation seems entirely uncoupled from the gating ring. It seems impressive that for every construct that is examined, at 0 Ca2+, even when full activation is shifted to potentials negative to +100 mV (e.g., with γ1), there is never any detectable FRET signal. This seems to say that Ca2+ is required for conformational coupling between VSD and CTD, or, to say it another way, that VSD movements do not couple to the CTD in the absence of Ca2+, or Ca2+ is obligatory for VSD-CTD coupling.

Is there a way to compare Ca2+-dependence of EV measurements to other approaches? Another interesting aspect of the data about which little is said is the rather astonishingly steep Ca2+-dependence of FRET signal generation at any single voltage. This seems consistent across constructs, and is reduced in the single site constructs with Cd2+ and Ba2+. Maybe this fits with what we know about Ca2+-dependence of activation, but it seems unusually steep. Although it may be difficult to extract from the E./V data, it seems like there is some information regarding apparent voltage-dependence of ligand affinity embedded in the data, that might be interesting to contrast to earlier results, e.g., from Sweet and Cox.

Various aspects of the β subunit experiments are confusing. First, in Figure 3D, the β3bNβ1 construct is producing no effect on GVs at all. Yet, in the Supplementary Information of Castillo et al., 2015, Vh is reported to shift negatively about 50 mV for presumably the same construct. Part of their argument was that the positive residues in the β1 N-terminus are responsible not only for the effect on voltage-sensors, but also the gating shift. Mutation of the K3K4 charged residues in the N-terminus abolishes the β1 gating shift, while mutation of K10R11 retained the gating shift.

Figure 3—figure supplement 1. Presumably the β3b construct is the human form, but this should be clarified, since the mouse form does not cause rapid inactivation. The results in Figure 3—figure supplement 1 are only partially supportive of the idea that adequate expression of β3b has been achieved. The main argument the authors make is apparently the roll-over in the GV relationship seen at 12 and 22 microM Ca2+. But it is very odd that this roll-over is not seen at 95 and 4 microM. The Materials and methods state that all GVs were generated from tail currents. Because the β3b inactivation is very low affinity, it has been shown that recovery from block is essentially complete at the peak of the usual tail currents, so tail current GVs are not particularly affected by this block (Xia et al., 2000. Figure 6C vs. 6D.). Yet steady-state conductance with adequate β3b expression shows marked block, which is really not so readily apparent in the example traces in panel a. The β3 subunit, of all β subunits, is known to be a bit challenging to express and the results here are not convincing in that regards. This is critical, when the observed results are really not any different from BK667CY α alone. The fact that no gating shift was observed with the chimeric β3bNβ1 construct also raises some concern in that regard. Positive controls that these β subunit constructs are being adequately expressed are lacking.

The scale bars in panel A of Figure 3—figure supplement 1 are also likely to be in error. And the time base of these recordings really doesn't provide any chance of seeing the distinctive features of patches with β3b containing channels.

Resolution of the issues raised above would impact on the discussion about these experiments presented in the subsection “The dynamics of the VSD are directly reflected in the gating ring conformation”.

Reviewer #3:

This study examines the interaction between transmembrane and cytoplasmic domains of BK channels by determining the effects of mutations in each domain, or co-assembly with regulatory subunits, on conformational changes in the cytoplasmic gating ring. The authors have previously demonstrated that gating ring conformation can be monitored by measuring FRET efficiency between CFP- and YFP-tagged Slo1 subunits. FRET efficiency is increased by the binding of Ca2+ and other divalent cations to two previously identified high-affinity binding sites, and at intermediate Ca2+ concentrations by membrane depolarization. The effect of voltage on FRET is consistent with the suggestion, most recently based on BK channel structure, that functional interactions occur between the RCK1 domain of the gating ring and VSD. Here, this hypothesis is supported by showing that (a) voltage-dependent FRET is abolished by mutating the RCK1 Ca2+ binding site and (b) changes in voltage-dependent FRET induced by mutations or regulatory subunits are correlated with VSD activation rather than channel opening.

The manuscript is well written and the data clearly described. However, the conclusions are not particularly novel and I have concerns about the lack of comparison to previous work, and the extent to which the results provide new insight.

The idea that functional interactions occur between the VSD and gating ring in BK channels has already been addressed by several previous studies such as Horrigan and Aldrich, 2002, Sweet and Cox, 2008, and Savalli et al., 2012. Only the first of these is referenced in the manuscript, and that one is mischaracterized. The authors seem to suggest that an interaction between VSD and Ca2+-binding sites was included in the Horrigan Aldrich model simply for completeness (i.e. because it couldn't be ruled out). In fact, that study observed clear evidence for an interaction based on the effects of Ca2+ on VSD activation (gating current). These results led to an estimate of the interaction energy between Ca2+- and V-sensors and the conclusion that the interaction was not altered by channel opening. In addition, Horrigan and Aldrich concluded that the interaction was weak, not because they couldn't measure it, but because the interaction energy was small compared to that between Ca2+- or V-sensors and channel opening. Sweet and Cox 2008 then tested a prediction that the interaction is bi-directional, by confirming that the apparent Kd for Ca2+-binding is reduced by altered by membrane depolarization. Furthermore, they concluded that the interaction occurred with RCK1 rather than RCK2, based on the effect of mutating the different Ca2+-binding sites. Finally, Savalli et al., 2012 used patch clamp fluorometry (on the VSD) to confirm that Ca2+ binding to the gating ring alters VSD activation. Thus, while the detailed approach used in the manuscript is new, neither the conclusions or even the use of patch clamp fluorometry appear to be novel. That said, the results could be valuable if they provide new insight into the nature of the interaction or its properties. But here too I have doubts. As the authors point out, the results show a strong correlation between effects on voltage-sensor activation and changes in the voltage-dependence of FRET. However, the results are described in very qualitative terms that don't seem to provide much insight beyond the basic conclusion. At minimum, I think the authors need to discuss whether the results are consistent or inconsistent with previous results, and make clear what is new.

One specific concern is whether the lack of effect of the γ1 subunit on FRET efficiency in Figure 2F is indeed consistent with the hypothesis that voltage-dependent FRET reflects VSD activation. The authors typically reference the effect of various mutations and regulatory subunits on the half-activation voltage of the VSD, Vh(j). Strictly speaking, Vh(j) is the half-activation voltage when channels are closed, and for most of the experiments may be an appropriate parameter to look at since voltage-dependent FRET is being measured under condition where a large fraction of channels are closed. However, in the case of γ1, voltage-dependent FRET is occurring over a range where channels are open (i.e. rel. conduct. In Figure 2F is saturated). While the authors are correct that Yan and Aldrich, 2010 proposed that Vh(j) is unaltered by γ1, their model predicts a large shift in VSD half activation voltage when channels are open (because when channels are open, VSD activation depends on VSD-gate coupling, which is enhance by γ1). Therefore, is seems like one should expect γ1 to shift v-dependent FRET to more negative voltages if it was simply detecting VSD activation.

eLife. 2018 Dec 11;7:e40664. doi: 10.7554/eLife.40664.014

Author response

Essential revisions:

1) Modify the Title

Reviewers thought the title was vague and could be modified to better convey the message of the manuscript. The term "strongly coupled" also needs to be modified or justified since it implies a quantitative assessment of coupling that was not addressed.

We thank the reviewers for this suggestion. We have now changed the title to ‘Voltage-dependent dynamics of the BK channel cytosolic gating ring are coupled to the membrane-embedded voltage sensor’.

2) Address previous work and novelty

The authors should provide a more complete comparison with previous work, especially with regards to VSD/gating-ring coupling or interaction, to help clarify "what is new" and whether the results are consistent or inconsistent with previous results.

We agree with the reviewers and we have added a paragraph to describe previous work and better clarify the novelty in our study:

‘Using the allosteric HA model of BK channel function, Horrigan and Aldrich (2002) proposed that Ca2+ binding to the Ca2+ bowl is coupled to the voltage sensor activation. […] This evidence indicates that the VSD is coupled to the gating ring, but none of these approaches directly monitored the conformational changes of the gating ring structure.’

3) Provide more guidance for interpreting the results:

Indicating the expected status of voltage sensors under different conditions and manipulations would help readers to interpret the corresponding FRET data.

We are grateful to the reviewers for this recommendation. A new paragraph in has been included in the manuscript:

‘As discussed previously (Miranda et al., 2013), these changes in E with voltage are not conformational dynamics of the gating ring that simply follow the voltage dependence of VSD. […] However, we do not observe changes in E between 0 and +240 mV (Figure 1A). Similarly, at 100 µM Ca2+, charge movement takes place between -100 and +150 mV (Carrasquel-Ursulaez et al., 2015), while our FRET signals at 95 µM Ca2+ do not vary within this voltage range (Figure 1A).’

Provide more guidance for interpreting the results: Reviewers also desired more guidance on how to interpret the quantitative relationship between VSD activation and the voltage-dependence of FRET.

We agree with the reviewers in that a description of the quantitative relationship between VSD activation and voltage-dependence of FRET is lacking. Unfortunately, we have not found a satisfactory simultaneous fit of VSD activation (reflected by the voltage dependence of the macroscopic conductance) and the E-V data (see Miranda et al., 2013). One main limitation of our approach is the dynamic range of the E-[Ca2+] data. As in any FRET approach, information is restricted to a narrow window around the R0 value for the pair, which in our case is 50Å (see Miranda et al., 2013). In other words, any distance between fluorophores of >100 Å would result in a value of E close to 0, and similarly, any distance between the fluorophores shorter than 50 Å will give a value of E around 0.5 (this lower end distance is limited by the location of the fluorophore within the barrel of the protein). To provide data access to the readers, we have now generated a table with all values corresponding to the Boltzmann fits of G-V and E-V data, which has been added as Supplementary file 1. In addition, we are now indicating within the main text that there is a consistent correlation between the changes in the slope of the G-Vs data from S4 mutants with the changes in the steepness of the E-V relationship: ‘Interestingly, we also observe a correlation between the changes in the slope of the G-V curves and that of the E-V curves (see for instance, Figure 4F; see also Supplementary file 1)’.

Provide more guidance for interpreting the results: As well as several interesting or puzzling features of the data including the effect of Ba2+.

We agree with the observations of the reviewers and appreciate their suggestions regarding the effect of Ba2+. We have performed the experiments proposed by the reviewers, which are included in a new figure supplement (Figure 6—figure supplement 1). Our new results show that: 1) the E-V relations observed with Ba2+ and the S4 mutants (Figure 6) resemble the E-V relations with the RCK1 Ca2+ binding site knockout (D362A D367A) containing S4 mutations (BK667CYD362A D367A R210E and BK667CYD362A D367A E219R). These observations confirm that the observed voltage-dependence of the Ba2+ is not related to the RCK1 site. These results are shown in Figure 6—figure supplement 1 and described in the revised manuscript version:

‘This conclusion is further supported by the lack of changes in Ba2+ responses when mutations in the VSD were made in a RCK1 Ca2+ binding site knockout (D362A D367A) background (Figure 6—figure supplement 1B and C.’

2) no shift in the voltage dependence of the FRET signal is observed after addition of the high affinity blocker bbTBA. Results are shown in Figure 6—figure supplement 1 and a new paragraph has been added to Discussion:

‘Why Ba2+ but not Ca2+? A possible answer for this question is that Ba2+ has the additional property of blocking the permeation pathway (Miller, 1987; Neyton and Miller, 1988; Zhou et al., 2012), which could somehow be transmitted allosterically to the gating ring. If simply ion permeation blockade is what matters, then we might expect that blocking permeation with the high affinity quaternary ammonium derivative N-(4-[benzoyl]benzyl)-N,N,N-tributylammonium (bb-TBA) (Tang et al., 2009) should produce a voltage dependent FRET signal with Ca2+ activation. But, it does not (Figure 6—figure supplement 1D). Another possibility for the Ba2+ effect could be a direct allosteric interaction between the intrinsic gating in the pore and the divalent binding site in RCK2, which needs to be tested further.’

Provide more guidance for interpreting the results: The remarkably steep Ca2+ dependence of FRET.

We thank the reviewers for this observation. To better appreciate the Ca2+-dependence of the FRET signal, we have looked at the E and G/Gmax data as a function of [Ca2+]. In Author response image 1, we have converted the data from Figure 1A-D to [Ca2+] dose-response curves. The E-[Ca2+] curves corresponding to the D362A/D367A and D362A/D367A 5D5A mutants are shown as average of all curves obtained at the various voltages. This representation evidences that the voltage effect on the E signal is observed within a specific range of Ca2+ concentrations (from around 4 to 50 μM). Within this range, the steepness of the E-[Ca2+] curves is similar to that of the G/Gmax-[Ca2+] data. We believe, as explained above, that the dynamic range of our E-[Ca2+] data is limited to a narrow window by technical constrains of the FRET technique.

Author response image 1.

Author response image 1.

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Provide more guidance for interpreting the results: And the lack of voltage-dependence of FRET in 0 Ca2+.

As we have discussed above, we believe that the dynamic range of our E-[Ca2+] data is limited to a narrow window by technical constrains of the FRET technique. We have included the following in the manuscript: ‘As discussed previously (Miranda et al., 2013), these changes in E with voltage are not conformational dynamics of the gating ring that simply follow the voltage dependence of VSD. […] Similarly, at 100 µM Ca2+, charge movement takes place between -100 and +150 mV (Carrasquel-Ursulaez et al., 2015), while our FRET signals at 95 µM Ca2+ do not vary within this voltage range (Figure 1A).’

Provide more guidance for interpreting the results: Mechanistic or quantitative interpretation is lacking, and ideally should be improved, if only to provide some framework for future evaluation and tests. Proposing a model (i.e. a kinetic scheme) to account for the major features of the relation between Ca2+, voltage, and FRET efficiency (i.e. lack of FRET in 0 Ca2+ or high Ca2+, and correlations observed in the different mutants) would greatly strengthen the conclusions and enhance the significance of the results. While developing a model to account for all features of the data may be beyond the scope of this study, the authors should at minimum indicate which features of the data can or cannot be accounted for by previously proposed models (i.e. Miranda 2013) and whether their data provide any new insight into mechanism that must be included in gating models. This should be possible to do without incorporating additional experiments.

We agree with the reviewers that a model would provide some framework for future evaluation and tests. Unfortunately, we have been unsuccessful to adapt the HA model to describe the voltage dependencies of the E and G signals simultaneously. As described in Miranda et al., 2013, we explored a large number of modifications but all failed to describe the data. If we were to adopt a model, it should be reminiscent of, but simpler than, the one proposed by Savalli et al., 2012). In this model two different allosteric constants would account for the coupling of the gating ring to the PD, the VSD would only be allosterically coupled to the RCK1 (E’) and the RCK domains would be related by a new constant F. Notably, our data support E’ and C1’, but we have no information on C2’ or F. Given the premature nature of our understanding, we agree with the reviewers in that developing a model is beyond the scope of this study.

Author response image 2.

Author response image 2.

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4) Reconcile discrepancies and apparent inconsistencies in the data

The authors should address the apparent inconsistency between the effect of the γ1 subunit on FRET and its predicted effect on VSD activation.

We thank the reviewers for bringing up this apparent inconsistency. We agree that it was not adequately discussed in the manuscript. It is correct that, in the simplest scenario of the HA allosteric model, “a large shift in VSD half activation voltage when channels are open” (reviewer #3) would be expected. However, we would argue that a precise understanding of the allosteric coupling between the VSD and the pore, defined by D, should arise from the gating currents and their charge distributions and how they relate to the G-Vs and the presence or absence of γ1. We have had access to unpublished data, kindly provided by Carrasquel-Ursulaez and Latorre, showing gating currents, Q-V and G-V distributions in the presence and absence of gamma 1 subunit. Their findings support our statement “Analogously, no change in the voltage dependence of E was observed after co-expression of BKα with the γ1 subunit, which shifts the voltage dependence of pore opening by enhancing its allosteric coupling with the voltage sensor activation (Yan and Aldrich, 2010). As with the mutation F315A, the presence of γ1 subunit produces a minor shift in the Q-V distributions, not paralleling the large shift in the G-V curves (Carrasquel-Ursulaez and Ramon Latorre, personal communication).”, which has been included in the revised manuscript version.

As well as discrepancies between some of the results and data previously published.

Results which seem to contradict earlier results include: a) GV shift magnitude of F315A in 100 Ca2+;

We agree with the reviewers that our results show smaller G-V shifts than those previously reported. The mutation F315A used in our study was introduced in the background of a human BK channel (GenBank accession no. U11058) construct, and is equivalent to the F380A mutant described in the Carrasquel-Ursulaez article, 2015. The differences in the observed G-V curves may be due, at least in part, to the use of different electrophysiological protocols and conditions than those described in Carrasquel-Ursulaez et al., specifically: 1) we use 300 ms long pulses instead of the 2 ms pulses used by Carrasquel-Ursulaez et al. 2) We use 40 mM symmetrical K+ rather than 110 mM K+ used by Carrasquel-Ursulaez et al. In addition, our construct has a fluorescent protein inserted. To what extent any of these differences contribute to a smaller shift in the GV, we do not know. We do not think this is a major issue, because we do observe large shifts (between ∆Vhalf of +100 to +200 mV, depending on Ca2+ concentrations) in the mutant, which are not accompanied by a shift in the E-V, and that is what it is important for this work. We have added a note in the legend of Figure 2 to make the reader aware of this apparent discrepancy: ‘It should be noted that the extent of the shifts induced by the mutation are smaller than previously reported (Carrasquel-Ursulaez et al., 2015), which could arise from the different experimental conditions and/or our fluorescent construct.’

b) Lack of a GV shift for the β3Nβ1 construct;

We are slightly puzzled by this comment. Data shown in Figure 3D indicate that β3Nβ1 induces a negative shift of the E-V curve. This is consistent with the Q-V data by Castillo et al., 2015; Figure 3 and Table S2, showing Q-V data for the different chimeras). To our knowledge, no G-V curves analysis data is shown in that paper. Why should we expect a shift in the G-Vs? Most amino acids of this chimera are from β3b, with just a few from the intracellular N-terminus of β1.

c) Whether they have adequate expression of β3b.

We thank the reviewers for this observation. First, we would like to state that we do have adequate expression of β3b. β3b RNA was always co-injected at molar ratios that were 25X larger that the BKa RNAs. This information is now provided in the Materials and methods section: ‘Auxiliary subunits (β3b, γ1 and chimera β3bNβ1) were co-injected with the BK667CFP/BK667YFP RNA mix at a 5:1 weight ratio, giving molar ratios above 20:1.’. To convince Dr, Lingle, we provide in Author response image 3 comparing the ionic currents obtained after co-expressing β3b plus non-fluorescently labelled BKα subunits (WT BKα) and β3b plus the BK667CY construct. Each panel showing ionic currents corresponds to a different construct indicated at the top. In each panel, current trace in black represents the ionic current in response to the most positive voltage step (+240 mV). These data show that when β3b is co-expressed with WT BKα we observe no difference with the effect described in the literature (e.g., Xia et al., 2000). Note that there is not an effect of blockade on the tail currents (see also G-Vs on the right, in yellow). Once β3b is co-expressed with the BK667CY construct, the kinetics of inactivation changed. It appears as if the off rate of inactivation is largely increased at positive potentials. Note also that there is a substantial current reduction at the tails as blockade increased with very positive potentials (see also G-V in blue). We use this behavior as an indication of β3b expression. The simplest interpretation is that the insertion of the fluorescent protein interferes with the kinetics of blockade mediated by the β3b NH2-terminal region (Lingle et al., 2001). Understanding this discrepancy will require further study. In any event, we consider this issue not affecting our conclusions. In this context, we agree that the main text needed to be modified to: ‘First, we co-expressed BK667CY α subunits with β3b and observed the expected inactivation of the ionic currents at positive potentials, yet with different blockade kinetics (see Figure 3—figure supplement 1) (Uebele et al., 2000; Xia et al., 2000; Lingle et al., 2001). […] The simplest interpretation is that the insertion of the fluorescent protein interferes with the kinetics of blockade mediated by the β3b NH2-terminal region (Lingle et al., 2001). Understanding this discrepancy will require further study.’

Author response image 3.

Author response image 3.

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Supplementary file 1. Fit parameters of data shown in Figures 1–6.

    The G-V and E-V relations were fit with Boltzmann functions G/Gmax = 1/(1 + exp (-zF(V-Vhalf)/RT), E = 1/(1 + exp (-zF(V-Vhalf)/RT), where Gmax is the maximum tail current, z is the voltage dependence of activation (G) or gating ring movement (E), Vhalf is the half-activation voltage of the ionic current or the fluorescent signal. T is the absolute temperature (295K), F is the Faraday’s constant and R the universal gas constant.

    elife-40664-supp1.xlsx (23.2KB, xlsx)
    DOI: 10.7554/eLife.40664.010
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    elife-40664-transrepform.docx (246KB, docx)
    DOI: 10.7554/eLife.40664.011

    Data Availability Statement

    All data generated and analysed during this study are included in the manuscript.

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