XB-ART-53038
Proc Natl Acad Sci U S A
2016 Jun 07;11323:E3231-9. doi: 10.1073/pnas.1606381113.
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β1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel.
Castillo JP
,
Sánchez-Rodríguez JE
,
Hyde HC
,
Zaelzer CA
,
Aguayo D
,
Sepúlveda RV
,
Luk LY
,
Kent SB
,
Gonzalez-Nilo FD
,
Bezanilla F
,
Latorre R
.
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Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.
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Species referenced: Xenopus laevis
Genes referenced: vsig1
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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). |
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