The BK channel gating ring is strongly coupled to the voltage sensor

The open probability of large conductance voltage- and calcium-dependent potassium (BK) channels is regulated allosterically by changes in the transmembrane voltage and intracellular concentration of divalent ions (Ca2+ and Mg2+). The divalent cation sensors reside within the gating ring formed by eight Regulator of Conductance of Potassium (RCK) domains, two from each of the four channel subunits. Overall, the gating ring contains 12 sites that can bind Ca2+ with different affinities. Using patch-clamp fluorometry, we have shown robust changes in FRET signals within the gating ring in response to divalent ions and voltage, which do not directly track open probability. Only the conformational changes triggered through the RCK1 binding site are voltage-dependent in presence of Ca2+. Because the gating ring is outside the electric field, it must gain voltage sensitivity from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of voltage sensor dynamics known to shift gating currents produce a cognate shift in the gating ring voltage dependence, whereas changing BK channels’ relative probability of opening had little effect. These results strongly suggest that the conformational changes of the RCK1 domain of the gating ring are tightly coupled to the voltage sensor function, and this interaction is central to the allosteric modulation of BK channels.

Apart from Ca 2+ , it has been described that Cd 2+ selectively binds to the RCK1 site, whereas Ba 2+ and Mg 2+ show higher affinity for the RCK2 site (2,4,25,26,(28)(29)(30). Thus, intracellular concentrations of Ca 2+ , Cd 2+ , Ba 2+ or Mg 2+ 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 (30,31). 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 (32). Such a mechanism would be analogous to channel activation by ligand binding in glutamate receptor or cyclic nucleotide-gated ion channels, also tetramers (33, 34). 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 (15,22) 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 to 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 now performed PCF measurements with BK channels including a range of VSD mutations or coexpressed 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 of 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 (30,31). This allowed us to use PCF to detect conformational rearrangements of the gating ring measured as changes in FRET efficiency (E) between the fluorophores (30,31). Binding of Ca 2+ ions to both high-affinity binding sites (RCK1 and Ca 2+ bowl) produces an activation of BK channels, coincident with an increase in E from basal levels reaching saturating values at high Ca 2+ concentrations ((31) and Fig. 1a). In addition, we observed that the E signal has the remarkable property that in intermediate Ca 2+ concentrations it shows voltage dependence besides its Ca 2+ dependence ((31) and Fig. 1a). Independent activation of high-affinity binding sites by other divalent ions (Ba 2+ , Cd 2+ , or Mg 2+ (30)) led us to postulate that Ca 2+ activation has a site-dependent relation to voltage. To further evaluate the effect of individual high-affinity Ca 2+ binding sites on the voltage-dependent component of the gating ring conformational changes we first selectively mutated the binding sites. Mutations D362A and D367A (2,4) were introduced in the BK667CY construct (BK667CY D362A/D367A ) to remove the high-affinity binding site located in the RCK1 domain. Fig. 1b shows the relative conductance and E values for the BK667CY D362A/D367A construct at different membrane voltages for various Ca 2+ concentrations.
As described previously, the G-V curves show a significantly reduced shift to more negative potentials when Ca 2+ is increased, as compared to the non-mutated BK667CY ( Fig. 1a-b, left panels). Specific activation of the Ca 2+ bowl renders a smaller change in E values, which are not voltage-dependent within the voltage range tested (Fig. 1b, right panel). To test the effect of eliminating the RCK2 Ca 2+ binding site -the Ca 2+ bowl-we mutated five aspartates to alanines (5D5A) (28). As expected, activation of only the RCK1 domain by Ca 2+ reduced the Ca 2+dependent shift in the GV curves (Fig. 1c, left panel). Even though the extent to which the E values changed with Ca 2+ was reduced (Fig. 1c), there was a persistent voltage dependence equivalent to that detected in the non-mutated channel (Fig. 1c, right panel) (31). This effect seems not to be attributable to Ca 2+ 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 (Fig. 1d).
Altogether, these results indicate that the voltage-dependent component of the gating ring conformational changes triggered by Ca 2+ 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 voltagedependent 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. 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 the intrinsic gating are expected to be unaltered (Fig. 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 ( Fig. 2c) (35). Fig. 2d shows the relative conductance and E vs. The second modification of BK function consisted in co-expressing the wild type α subunit with the auxiliary subunit γ1 (36-39). 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 (Fig. 2e). This construct adds the advantage of representing a physiologically relevant modification of channel gating. Fig. 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 [Ca 2+ ] 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 (Fig. 2f, left panel) compared to the values obtained without γ1 (Fig. 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 (Fig 2f, right panel), which remains indistinguishable from that recorded with BK667CYα (Fig. 2f, dashed lines).

The dynamics of the VSD are directly reflected in the gating ring conformation
In the allosteric model of BK channel function, Ca 2+ binding to the Ca 2+ bowl is weakly coupled to the voltage sensor activation (reflected in the low value of the allosteric constant E) (3). Nevertheless, the model does not discount some level of interaction between the VSD and the gating ring, so we decided to explore if the voltage dependence of the gating ring conformational change is attributable to the voltage sensor activation. To this end, we modified the voltage dependence of the voltage sensor activation by co-expression with β auxiliary subunits or by introducing specific mutations in the VSD ( Fig. 3 and Fig. 4). The effects of co-expressing BK α subunit with the four different types of auxiliary β subunits have been extensively studied (37, 40-49). β1 subunit has been previously proposed to alter the voltage sensor-related voltage dependence, as well as the intrinsic opening of the gate and Ca 2+ sensitivity ( Fig. 3a) (42-44, 46, 47, 50). Recordings from BK667CYα co-expressed with β1 subunits reveal the expected modifications in the voltage dependence of the relative conductance, i.e. an increase in the apparent Ca 2+ sensitivity (Fig. 3b, left panel) (42-44, 46, 47, 51). In addition it has been reported that β1 subunit alters the function of the VSD (42, 50). Notably, the E-V curves are shifted to more negative potentials (Fig. 3b, right panel), similarly to the described modification (50). 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) (50). We recapitulated this strategy.  Figure 1, right panel). We then co-expressed the β3bNβ1 chimera (50) with BK667CYα (Fig. 3c). This complex did not modify the relative conductance vs. voltage relationship (Fig. 3d, left panel) as compared with BK667CYα alone (Fig. 3d,  VSD activation can also be altered by introducing single point mutations that modify the voltage of half activation of the voltage sensor, V h (j). This parameter is determined by fitting data to the HA allosteric model (19) or directly from gating current measurements (55). Mutations of charged amino acids on the VSD have been reported to produce different modifications in the V h (j) values. In some cases, other parameters related to BK channel activation are additionally affected by the mutations. Mutation R210E shifts the V h (j) value from +173 mV to +25 mV at 0 Ca 2+ in BK channels ( Fig. 4a) (19). Consistently, introduction of this mutation in BK667CYα (BK667CY R210E ) caused a shift of the relative conductance vs. voltage dependence towards more negative potentials (Fig. 4b, left panel) as compared to BK667CY (Fig. 4b, (Fig. 4b, right panel). Mutation E219R had been previously shown to produce a large negative shift in V h (j) from +150 mV to +40 mV (ΔV h (j) = -110 mV; Fig. 4c), additionally modifying the Ca 2+ sensitivity and the coupling between the VSD and channel gate (55). As previously reported, BK667CY E219R showed modified relative conductance vs. voltage relationships at different Ca 2+ concentrations (Fig. 4d, left panel) (55). In addition, this construct revealed shift to more negative potentials in the E vs.  (19,55). Since mutations displacing the V h (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 V h (j) (19) were also associated to changes of the E-V curves in the same direction. As shown by Ma et al., the largest effect on V h (j) is induced by the R213E mutation, producing a shift of ΔV h (j)=+337mV (Fig. 4e) (19). The BK667CY R213E construct showed a significant shift in the voltage dependence of the relative conductance to more positive potentials (Fig. 4f = -110 mV) previously reported with the E219R mutant BK channels (19,55). We also tested Cd 2+ activation in the mutant BK667CY R201Q , which shifts the V h (j) parameter by 47 mV towards positive potentials (Fig. 5c) (19). Addition of Cd 2+ rendered right-shifted E vs. voltage relationships (Fig.5d, right panel), following the direction of the predicted V h (j) shift described for this mutant BK channel (19). Finally, addition of Cd 2+ to the BK667CY F315A construct (Fig.   5e) (35) did not have any effect on the E-V relationship (Fig. 5f). These results are consistent with a mechanism in which specific binding of Cd 2+ to the RCK1 binding site allows voltagedependent conformational changes in the gating ring that are directly related to VSD activation.
Voltage dependence of Ba 2+ -induced gating ring movement is related to function of the channel gate Ca 2+ , Mg 2+ and Ba 2+ bind to the Ca 2+ bowl and trigger conformational changes of the gating ring region (30). However, the effects of these ions on BK function and gating ring motions are fundamentally different. Notably, Ba 2+ induces a rapid blockade of the BK current after a transient activation that is measurable at low Ba 2+ concentrations (29,30) (Fig. 6a). In addition, we previously showed that the gating ring conformational motions induced by Ba 2+ show a voltage-dependent component, which is not observed when Ca 2+ or Mg 2+ bind to the Ca 2+ bowl (30, 31) (Fig. 6b). We combined mutagenesis with the cation-specific activation strategy to identify the structural source of the voltage dependence in Ba 2+ -triggered gating ring motions. In this case, alteration of VSD function by mutating charged residues ( Fig. 6c and 6e) was not reflected in any change of the E vs. voltage relationships, as shown in Fig. 6d and 6f for constructs BK667CY R210E and BK667CY R213E , respectively. These results indicate that the voltage dependence of Ba 2+ -induced gating ring conformational changes, unlike those induced by Ca 2+ and Cd 2+ through activation of the RCK1 binding site, may not be related to VSD activation. To further test this hypothesis, we studied the effect of Ba 2+ on BK667CY channels containing the F315A mutation (Fig. 6g) (35). As shown in Fig. 6h, the E values reached similar levels to those of non-mutated BK667CY channels at saturating Ba 2+ concentrations. However, at intermediate concentrations of Ba 2+ the E-V curves were shifted towards more positive potentials when compared with BK667CY channels (Fig. 6h, dashed line). These results suggest that the voltage-dependent component of the conformational changes triggered by Ba 2+ binding to the Ca 2+ bowl are not directly related to VSD activation, but rather to the function of the channel gate. According to the structural data, gating of the channel by Ca 2+ 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 (15,22). Our work now provides functional data supporting this mechanism. Our data show that mutations altering the voltage dependence of BK VSD are when BK667CY channels are coexpressed with the β3bNβ1 chimera, which affects VSD function without altering other parameters related to gating (50). Therefore, the voltagedependent component of the E signal seems to be related strictly to VSD function, as further demonstrated by the lack of effect of the γ1 subunit, which has been shown to shift the voltage dependence of gate opening by enhancing the allosteric coupling of voltage sensor activation without affecting VSD operation (36). Similar lack of effect on the voltage dependence of E is observed after introducing into BK667CY channels the mutation F315A, which has been also associated to uncoupling VSD function from pore opening (35).

DISCUSSION
A puzzling result from our previous study was the observation that Ba 2+ binding to the Ca 2+ bowl triggers voltage-dependent conformational changes (30). Even though we still do not know the mechanisms of this unique response to Ba 2+ , 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. A possible explanation for this result is that Ba 2+ block of the permeation pathway (16,29,56) is somehow transmitted allosterically to the gating ring. Alternatively, there could be a direct allosteric interaction between the intrinsic gating region and the divalent binding site in RCK2.
Irrespectively of the fluorescent construct (31) or the divalent ion used to activate the BK channel (30), 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 Ca 2+ 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 strong 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.

Molecular biology and heterologous expression of tagged channels.
Fluorescent BK α subunits were labelled with CFP or YFP using a transposon based insertion method (57). Subunits labelled in the position 667 were subcloned into the pGEMHE oocyte expression vector (58). 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) (30,31).

Patch-clamp fluorometry and FRET.
Borosilicate pipettes with a large tip (0.7-1 MΩ in symmetrical K + ) were used to obtain inside- Simultaneous fluorescent and electrophysiological recordings were obtained as previously described (30,31). Conductance-voltage (G-V) curves were obtained from tail currents using standard procedures. Conformational changes of the gating ring were tracked as intersubunit changes of the FRET efficiency between CFP and YFP as previously reported (30,31). Analysis of the FRET signal was performed using emission spectra ratios. We calculated the FRET efficiency as E=(RatioA-RatioA 0 )/(RatioA 1 -RatioA 0 ), where RatioA and RatioA 0 are the emission spectra ratios for the FRET signal and the control only in the presence of acceptor respectively (60); RatioA 1 is the maximum emission ratio that we can measure in our system (30,31). This value of E is proportional to FRET efficiency (60). 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.     to BK667CYα channels (30,31). The solid curves in the G-V graphs represent Boltzmann fits.
The full range of G-V curves from 0 µM Cd 2+ to 100 µM Cd 2+ 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=3-10).   c d e f