Castillo JP , Sánchez-Rodríguez JE , Hyde HC , Zaelzer CA , Aguayo D , Sepúlveda RV , Luk LY , Kent SB , Gonzalez-Nilo FD , Bezanilla F , Latorre R .

R01 GM030376 NIGMS NIH HHS , R37 GM030376 NIGMS NIH HHS , U54 GM087519 NIGMS NIH HHS

	Fig. 1. LRET strategy in the BK channel. (A) LBT binds a luminescent Tb3+ ion with high affinity, acting as a LRET donor. Highlighted in blue is the tryptophan, which serves as an antenna that sensitizes Tb3+ to become excited by a 266-nm laser pulse. (B) Fluorescent probe bodipy interacts via LRET with the LBT-chelated Tb3+ with an R0 value of 39.7 å. The bodipy molecule is covalently conjugated to IbTX at position D19C. (C) Due to the homotetrameric symmetry of the BK channel, each of the four donors (D) can transfer energy via LRET to the single acceptor (A) that is offset from the pore axis (dotted black arrows). The four different distances result in multiexponential decay of SE. (D) LBT constructs were engineered (one LBT at a time) within the BK α-subunit: NT; on the extracellular side of transmembrane segments S0, S1, and S2; and within the BK β1-subunit on the extracellular side of transmembrane segments TM1 and TM2. RCK, Regulator of Conductance of K+.
	Fig. S1. α-LBT constructs expressed in the membrane of X. laevis oocytes. (A) Ionic current of inside-out membrane patches containing the four different α-LBT constructs in response to the indicated voltage pulse protocol. (B) Normalized tail current vs. voltage plots. Small changes in the slope and half-activation voltage suggest slight disruption of the voltage sensor structure in these four constructs.
	Fig. S2. α-LBT constructs (NT, S0, S1, S2) coexpressed with the β1-subunit and α-BK coexpressed with β1-LBT constructs (TM1, TM2). Ionic currents in response to the indicated voltage pulse protocol show the characteristic slowed activation and deactivation kinetics produced by the presence of the β1-subunit (compare with Fig. S1).
	Fig. 2. LRET measurements of LBT-tagged BK constructs. (A) Representative DO emission recording from an oocyte expressing the S0 construct. The red solid line represents a three-exponential fit. The slowest component with a time constant of ≈2.5 ms (red dotted line) corresponds to the decay from Tb3+ bound to LBT (Inset). (B) SE of the acceptor recorded after addition of IbTX-bodipy from the same oocyte of A. (Inset) Attached fluorophore acts as an LRET acceptor. Gray traces in A and B correspond to DO and SE control experiments, respectively, using WT BK α-subunit (α-BK). (C) Representative SE recorded from NT, S0, S1, and S2 constructs exhibited characteristic decay kinetics. Because there are four donors and one acceptor per channel, SE decays are composed of four energy transfer-based exponential components. Faster SE kinetics indicate that the four donors are positioned in closer proximity to the acceptor. a.u., arbitrary units.
	Fig. 3. LRET-based measurements reveal a rearrangement of the BK α-subunit when coexpressed with the β1-subunit. Representative SE decays recorded from oocytes independently expressing the α-LBT constructs coexpressed with the β1-subunit: NT (A), S0 (B), S1 (C), and S2 (D). For comparison, each panel shows in gray a representative SE trace of the corresponding construct expressed without the β1-subunit. Visual inspection shows that the most dramatic changes of LRET measurements occur in the NT construct. (E) SE from oocytes independently coexpressing the WT BK α-subunit with TM1 and TM2. Different kinetics indicate that the donors of these constructs are located in different parts of the channel structure.
	Fig. 4. Geometric model used to fit experimental SE traces. (A) Three cylindrical coordinates, r (red arrow), θ (black arc), and z (blue arrow), determine the position of the four donors (red circles) with respect to the acceptor position (black circle). The SNPS fitting program calculates four effective distances (d1 through d4, orange lines) that determine the shape of the theoretical SE decay according to Eqs. S1–S3 (SI Text, S1. LRET Calculations). The black cross is the projection of the acceptor cloud center of mass onto the x–y plane containing the four donors. (B) Geometric fit to a single S0 construct SE decay (Top) and weighted (wt.) residuals (Bottom). Goodness of fit is evaluated with the reduced chi square (χ2RG) value. a.u., arbitrary units. (C) Two equivalent geometric solutions are possible from a single fit, because the rotation angle may be ±θ with respect to the acceptor position.
	Fig. 5. Spatial map of the extracellular face of the BK VSD obtained from the geometric fit to SE traces. The position of each donor is represented by colored filled circles. The 95% confidence isosurfaces are shown as transparent volumes. For the sake of clarity, the donors from only one subunit are shown. The reference point is the acceptor (black circle) near the fourfold symmetry axis. The crystal structure of the pore section from the Kv1.2/2.1 paddle chimera (transparent gray) and the modeled IbTX-bodipy (purple) are presented in the background as references. The tentative area where the bulk of the BK VSD may be located is encircled by a black line. The space-filled boundary of the Kv1.2/2.1 paddle chimera is shown as a fainter gray line. (A and B) Top and side views, respectively, of the spatial map for BK constructs in the absence of the β1-subunit. (C and D) Top and side views, respectively, of the spatial map for BK constructs in the presence of the β1-subunit. Donors from the β1-LBT constructs TM1 and TM2 are shown as cyan and orange circles, respectively. For completeness, both solution types are shown for S0 + β1, TM1, and TM2 constructs. However, the less likely solutions are rendered transparent to reinforce the fact that they appear nonparsimonious.
	Fig. S3. Alternative solutions for the geometric model obtained by fitting the SE recordings. Donor positions are represented as colored spheres with their corresponding 95% confidence isosurfaces. Solution type 1 is within the transparent blue area, whereas solution type 2 is within the transparent red area. The central black segment from the origin is the symmetry line separating solution types 1 and 2. The other two black segments delimit the spatial region where solutions are allowed for Donor1, given the tetrameric fourfold symmetry of the geometric model. (A) Geometric solutions for NT, S0, S1, and S2 constructs in the absence of the β1-subunit. (B) Geometric solutions for NT, S0, S1, and S2 constructs coexpressed with the β1-subunit, and also for the β1-TM1 and β1-TM2 constructs. The location of the acceptor (black circle) is referenced to the IbTX-bodipy model (shown in purple). The reference structure of the Kv1.2/2.1 paddle chimera is shown in transparent gray for comparison purposes. Transmembrane segments S1 and S2 of the paddle chimera are colored in red and green, respectively.
	Fig. S4. Effect of the acceptor position in the geometric model for BK-LBT constructs in the presence of the β1-subunit. (A) The z-coordinate of all donors except S1 clash with the upper boundary equal to the height of the acceptor (37.4 å) when the acceptor radial shift is equal to 0, which corresponds to the best acceptor position for the data in absence of the β1-subunit (4.75 å radial from the pore axis). We thus explored the possibility of a slight β1-induced acceptor perturbation by varying the acceptor radial distance to achieve a more structurally meaningful spread of z-coordinates among LBT constructs. A positive shift means further away from the z axis. When acceptor radial shift is >1 å, the S1 donor drops below the extracellular face of the membrane (15 å, dashed line). (B) Fit error (χ2RG) vs. acceptor radial shift. χ2RG decreases for all constructs as the acceptor is displaced away from the z axis. The average fit error of the entire dataset is shown as black diamonds.
	Fig. 6. Change in the donor position due to the presence of the β1-subunit. (A and B) Top and side views, respectively, of the changes in the donor positions as a consequence of coexpression with the β1-subunit. The 95% confidence isosurfaces are not displayed for clarity. Colored arrows indicate the direction of the change in position.
	Fig. 7.  LRET-restrained molecular model of the BK channel. LBT insertions were sequentially included (one at a time) in LRET-restrained MD simulations used to construct the BK model. (A) Top view of the BK model in the absence of the β1-subunit. (B) Top view of the BK model in the presence of the β1-subunit. (C and D) Top and side views, respectively, of the BK model in the presence of the β1-subunit (solid white) compared with the model in its absence (transparent pink). The side view (D) includes only one subunit for clarity. The TM1 and TM2 segments were docked to match best the donor position obtained from β1-LBT constructs. LBT-labeled helices are colored consistent with LBT construct colors used throughout the paper.
	Fig. S5. Synthesis and characterization of synthetic bodipy-labeled IbTX. (A) Native chemical ligation-based synthesis scheme for [D19C]IbTX labeled with bodipy. (Inset) Structural model of toxin-dye conjugate, displaying all cysteine residues and its three disulfide bonds. (B) Analytical liquid chromatography (LC)-MS data for the purified and folded molecule [D19C]IbTX with N-terminal conversion to WT pyroglutamate, designated as product 1. (Inset) Electrospray mass spectrometry (ESMS) data: observed, 4,218.9 ± 0.3 Da; calculated, 4,219.0 Da [average (avg.) isotopes] with three disulfide bonds. (C) Analytical LC-MS data for the purified molecule [D19C]IbTX-bodipy, designated as product 2. (Inset) ESMS data: observed, 4,479.0 ± 0.3 Da (avg. isotopes); calculated, 4,479.1 Da (avg. isotopes). The MS data shown were collected across the main UV-absorbing peak in the chromatogram. We observed that 1 F− was lost from the IbTX-bodipy molecule during the ionization process, which was also observed in MS measurement of bodipy alone.
	Fig. S6. Toxin-fluorophore conjugate and diffusive volume of the acceptor fluorophore. (A) Schematic of bodipy conjugated to IbTX at residue D19C. ψ1–ψ4 are the four dihedral angles that separate the chromophore from the toxin backbone. (B) Homology-based model of IbTX-bodipy bound to the BK pore. The bodipy full structure is shown as a ball and stick representation. The model was made using the CTX-bound Kv1.2/2.1 crystal structure (PDB ID code 4JTA) from Banerjee et al. (22). (C) Conformational space of bodipy attached to IbTX was modeled by rotating each of the four dihedral angles ψ1–ψ4 by 30° for a total of 20,376 conformations. All conformations that gave atomic distances less than 1.4 å or with vdW potential energy >1,000 kcal/mol were discarded, resulting in a final set of 3,290 valid conformations. The final acceptor cloud is composed of discrete points, each of which represents the position of bodipy’s transition dipole moment per valid conformation (estimated as the geometric center of the chromophore’s central ring).

Banerjee, Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K(+) channel. 2013, Pubmed

Banerjee, Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K(+) channel. 2013, Pubmed
Bao, Gating and ionic currents reveal how the BKCa channel's Ca2+ sensitivity is enhanced by its beta1 subunit. 2005, Pubmed , Xenbase
Brenner, Cloning and functional characterization of novel large conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4. 2000, Pubmed
Candia, Mode of action of iberiotoxin, a potent blocker of the large conductance Ca(2+)-activated K+ channel. 1992, Pubmed
Carvacho, Intrinsic electrostatic potential in the BK channel pore: role in determining single channel conductance and block. 2008, Pubmed , Xenbase
Castillo, Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels. 2015, Pubmed , Xenbase
Cha, Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy. 1999, Pubmed
Contreras, A BK (Slo1) channel journey from molecule to physiology. 2013, Pubmed
Contreras, Modulation of BK channel voltage gating by different auxiliary β subunits. 2012, Pubmed , Xenbase
Cox, Role of the beta1 subunit in large-conductance Ca(2+)-activated K(+) channel gating energetics. Mechanisms of enhanced Ca(2+) sensitivity. 2000, Pubmed , Xenbase
Cui, Allosteric linkage between voltage and Ca(2+)-dependent activation of BK-type mslo1 K(+) channels. 2000, Pubmed
Cui, Molecular mechanisms of BK channel activation. 2009, Pubmed
Dworetzky, Phenotypic alteration of a human BK (hSlo) channel by hSlobeta subunit coexpression: changes in blocker sensitivity, activation/relaxation and inactivation kinetics, and protein kinase A modulation. 1996, Pubmed
Díaz, Role of the S4 segment in a voltage-dependent calcium-sensitive potassium (hSlo) channel. 1998, Pubmed , Xenbase
Galvez, Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. 1990, Pubmed
Giangiacomo, Novel alpha-KTx sites in the BK channel and comparative sequence analysis reveal distinguishing features of the BK and KV channel outer pore. 2008, Pubmed
Hackeng, Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology. 1999, Pubmed
Heyduk, Luminescence energy transfer with lanthanide chelates: interpretation of sensitized acceptor decay amplitudes. 2001, Pubmed
Hyde, Nano-positioning system for structural analysis of functional homomeric proteins in multiple conformations. 2012, Pubmed , Xenbase
Johnson, Determination of the three-dimensional structure of iberiotoxin in solution by 1H nuclear magnetic resonance spectroscopy. 1992, Pubmed
Jones, Improving the accuracy of transmembrane protein topology prediction using evolutionary information. 2007, Pubmed
Klauda, Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. 2010, Pubmed
Knaus, Primary sequence and immunological characterization of beta-subunit of high conductance Ca(2+)-activated K+ channel from smooth muscle. 1994, Pubmed
Koval, A role for the S0 transmembrane segment in voltage-dependent gating of BK channels. 2007, Pubmed
Krogh, Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. 2001, Pubmed
Latorre, Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage. 2006, Pubmed
Liu, Location of modulatory beta subunits in BK potassium channels. 2010, Pubmed
Liu, Position and role of the BK channel alpha subunit S0 helix inferred from disulfide crosslinking. 2008, Pubmed
Liu, Locations of the beta1 transmembrane helices in the BK potassium channel. 2008, Pubmed
Long, Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. 2007, Pubmed
Lu, MaxiK channel partners: physiological impact. 2006, Pubmed
Ma, Role of charged residues in the S1-S4 voltage sensor of BK channels. 2006, Pubmed , Xenbase
McGuffin, The PSIPRED protein structure prediction server. 2000, Pubmed
McManus, Functional role of the beta subunit of high conductance calcium-activated potassium channels. 1995, Pubmed , Xenbase
Meera, Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus. 1997, Pubmed
Meera, A calcium switch for the functional coupling between alpha (hslo) and beta subunits (Kv,cabeta) of maxi K channels. 1996, Pubmed
Morrow, Defining the BK channel domains required for beta1-subunit modulation. 2006, Pubmed
Nimigean, Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism. 2000, Pubmed
Nitz, Structural origin of the high affinity of a chemically evolved lanthanide-binding peptide. 2004, Pubmed
Nugent, Transmembrane protein topology prediction using support vector machines. 2009, Pubmed
Orio, New disguises for an old channel: MaxiK channel beta-subunits. 2002, Pubmed
Orio, Structural determinants for functional coupling between the beta and alpha subunits in the Ca2+-activated K+ (BK) channel. 2006, Pubmed , Xenbase
Orio, Differential effects of beta 1 and beta 2 subunits on BK channel activity. 2005, Pubmed , Xenbase
Pantazis, Relative transmembrane segment rearrangements during BK channel activation resolved by structurally assigned fluorophore-quencher pairing. 2012, Pubmed , Xenbase
Pantazis, Relative motion of transmembrane segments S0 and S4 during voltage sensor activation in the human BK(Ca) channel. 2010, Pubmed , Xenbase
Pantazis, Operation of the voltage sensor of a human voltage- and Ca2+-activated K+ channel. 2010, Pubmed
Phillips, Scalable molecular dynamics with NAMD. 2005, Pubmed
Posson, Extent of voltage sensor movement during gating of shaker K+ channels. 2008, Pubmed , Xenbase
Posson, Small vertical movement of a K+ channel voltage sensor measured with luminescence energy transfer. 2005, Pubmed , Xenbase
Posson, The voltage-dependent gate in MthK potassium channels is located at the selectivity filter. 2013, Pubmed
Purwaha, An artifact in LC-MS/MS measurement of glutamine and glutamic acid: in-source cyclization to pyroglutamic acid. 2014, Pubmed
Richardson, Distance measurements reveal a common topology of prokaryotic voltage-gated ion channels in the lipid bilayer. 2006, Pubmed
Rosenthal, Direct detection of nitroxyl in aqueous solution using a tripodal copper(II) BODIPY complex. 2010, Pubmed
Sali, Comparative protein modelling by satisfaction of spatial restraints. 1993, Pubmed
Sandtner, In vivo measurement of intramolecular distances using genetically encoded reporters. 2007, Pubmed , Xenbase
Schnölzer, In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. 1992, Pubmed
Selvin, Principles and biophysical applications of lanthanide-based probes. 2002, Pubmed
Semenova, Bimane fluorescence scanning suggests secondary structure near the S3-S4 linker of BK channels. 2009, Pubmed
Song, Orientation of fluorescent lipid analogue BODIPY-PC to probe lipid membrane properties: insights from molecular dynamics simulations. 2011, Pubmed
Sun, Regulation of Voltage-Activated K(+) Channel Gating by Transmembrane β Subunits. 2012, Pubmed
Toro, Maxi-K(Ca), a Unique Member of the Voltage-Gated K Channel Superfamily. 1998, Pubmed
Torres, A marriage of convenience: beta-subunits and voltage-dependent K+ channels. 2007, Pubmed
Wallner, Characterization of and modulation by a beta-subunit of a human maxi KCa channel cloned from myometrium. 1995, Pubmed , Xenbase
Wallner, Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca(2+)-sensitive K+ channels: an additional transmembrane region at the N terminus. 1996, Pubmed
Wang, Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy. 2009, Pubmed
Wells, Optimization of the molecular dynamics method for simulations of DNA and ion transport through biological nanopores. 2012, Pubmed
Yan, BK potassium channel modulation by leucine-rich repeat-containing proteins. 2012, Pubmed
Yan, LRRC26 auxiliary protein allows BK channel activation at resting voltage without calcium. 2010, Pubmed