Concatenated modular BK channel constructs reveal divergent stoichiometry in gating control by LRRC26 (γ1), pore, and selectivity filter
- Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, United States
Figures
Figure 1 with 1 supplement
Design of concatenated BKα subunit constructs that form functional channels.
(A) Schematic of the membrane topology and side view of the tetrameric 3D structure of the BKα subunit (PDB ID: 8GHF; cryo-EM structure in plasma membrane Tao et al., 2023) highlighting the three complementary separable regions in different colors. For clarity, the front and back subunits are shown in a partially transparent mode. (B) Schematic of the membrane topology for the BKαM module, concatenated dual- and quadruple-repeat constructs, and the complementary BKαΔM construct. (C) Immunoblot analysis of the V5-tagged αM(dual) (left, αM1M2; right, αM3M4) and BKαM(quad) constructs transiently expressed in HEK293 cells with an anti-V5 antibody. (D) Representative current traces from BK channels formed by intact, single repeat BKαM, dual-repeat BKαM(dual), and quadruple-repeat BKαM(quad) constructs in response to membrane depolarization from −80 mV in 20 mV steps at 0 and 10 µM intracellular free Ca2+. (E) Voltage dependence of BK channel activation for channels formed by the single (left), dual (middle), and quadruple (right) αM constructs in the absence and presence of 10 µM Ca2+. Electrophysiological recordings were repeated n=4–10, as indicated in Table 1. Error bars represent ± SEM.
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Figure 1—source data 1
Files containing original western blots for Figure 1C, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/107681/elife-107681-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for western blot analysis displayed in Figure 1C.
- https://cdn.elifesciences.org/articles/107681/elife-107681-fig1-data2-v1.zip
Figure 1—figure supplement 1
Complex formation between split BKα subunit constructs.
A FLAG-tagged αM construct was co-expressed with GFP-tagged αΔM or an empty vector in HEK293 cells. For comparison, equal amounts of total cellular protein from cell lysates were loaded onto SDS–PAGE. Notably, the presence of αM caused a reduction of the α△M expression, and the singular αM construct mostly oligomerized on SDS-PAGE.
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Figure 1—figure supplement 1—source data 1
Files containing original western blots for Figure 1—figure supplement 1 left panel, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/107681/elife-107681-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 1—figure supplement 1 left panel.
- https://cdn.elifesciences.org/articles/107681/elife-107681-fig1-figsupp1-data2-v1.zip
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Figure 1—figure supplement 1—source data 3
Files containing original western blots for Figure 1—figure supplement 1 right panel, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/107681/elife-107681-fig1-figsupp1-data3-v1.zip
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Figure 1—figure supplement 1—source data 4
Original files for western blot analysis displayed in Figure 1—figure supplement 1 right panel.
- https://cdn.elifesciences.org/articles/107681/elife-107681-fig1-figsupp1-data4-v1.zip
Figure 2
A single γ1 subunit per BK channel is sufficient for full modulation.
(A) Side view of the 3D structure of the BKα/γ1 channel complex (PDB ID: 7YO3 Yamanouchi et al., 2023) showing the γ1 subunit in purple and the three separable BKα regions in distinct colors. (B) Schematic of the membrane topology for the γ1 subunit and its fusion constructs, created by linking its C-terminus to the N-terminus of the αM dual- and quadruple-repeat constructs. (C) Immunoblot analysis of the V5-tagged γ1αM(dual) and γ1αM(quad) constructs expressed in HEK293 cells with an anti-V5 antibody. (D) Representative current traces from BK channels formed by co-expressing the γ1 subunit with the intact BKα or by γ1-fusion to the concatenated αM constructs (co-expressed with αΔM) in response to membrane depolarization from −80 mV in 20 mV steps in the virtual absence of Ca2+. (E) Voltage dependence of activation for channels formed by the γ1αM(dual) and γ1αM(quad) constructs co-expressed with αΔM. Electrophysiological recordings were repeated n=5–8, as indicated in Table 1. Error bars represent ± SEM.
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Figure 2—source data 1
Files containing original western blots for Figure 2C, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/107681/elife-107681-fig2-data1-v1.zip
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Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2C.
- https://cdn.elifesciences.org/articles/107681/elife-107681-fig2-data2-v1.zip
Figure 3
Stoichiometrically incremental effect of the L312A mutation on BK channel voltage gating and validation of functional integrity of the concatenated constructs.
(A) Side view of the BK channel pore structure (PDB ID: 8GHF Tao et al., 2023), highlighting the deep pore residue L312 and selectivity filter residues (stick and line modes). Only two diagonal pore domains are shown for clarity. (B) Representative current traces from BK channels formed by single αM and concatenated αM(dual) and αM(quad) constructs containing subunit-specific L312A mutations. Depolarizations from −80 mV were applied in 20 mV steps. Mutated subunits are indicated by filled circles and WT subunits by empty circles. (C) Voltage dependence of activation for channels formed by the indicated L312A mutant αM and αM(dual) constructs co-expressed with αΔM. Dashed lines show G-V curves of the corresponding non-mutated channels for comparison. (D) Voltage dependence of activation for channels formed by the αM(quad) constructs with different numbers of L312A mutations. A plot of the V1/2 vs. the number of mutated subunits is shown. (E) Plot of tail current decay rates (–120 mV) vs. number of L312A mutations, from αM(dual) and αM(quad) constructs. (F) Voltage dependence of activation for channels formed by co-expression of WT and L312A-mutant intact BKα subunits (n=6), or non-mutated and fully mutated αM(quad) constructs co-expressed with αΔM. (G) Representative current traces from channels formed by co-expressing the non-mutated and fully L312A mutated αM(quad) constructs (co-expressed with αΔM). Enlarged and fitted tail currents are shown below. Electrophysiological recordings were performed under Ca2+-free conditions and repeated n=4–10 as indicated in Table 1. Error bars represent ± SEM.
Figure 4
V288A-induced selectivity filter inactivation requires mutation of all subunits.
(A) Time-dependent inactivation of V288A-mutant BKα (intact) channels at –80 mV after a prolonged depolarization (160 mV for 200ms). Inactivation was assayed by monitoring the reduction in fast-activating currents (shown and compared in the middle) elicited by brief depolarization (160 mV for 10ms) after a prior –80 mV holding time of 10ms, 0.1 s, 0.2 s, 0.3 s, 0.4 s, 0.5 s, 0.6 s, and 0.7 s. The amplitudes of fast-activating currents are compared (middle) and plotted against time (right). (B) Representative current traces of V288A mutant BKα (intact) channels showing slowly developing depolarization-induced currents. (C) V288A mutant channel exhibited normal activation gating following recovery (160 mV for 100ms) from inactivation, as indicated by currents elicited by brief depolarization to different voltages after brief repolarization. (D) Representative current traces from channels formed by concatenated αM(dual) and αM(quad) constructs with subunit-specific V288A mutations (co-expressed with αΔM). Mutant and WT subunits are indicated as filled and empty circles, respectively. (E) Depolarization-induced current development rates for channels formed by non-mutated and V288A-mutant BKα (intact) and concatenated αM(dual) and αM(quad) constructs. Electrophysiological repeats: n=8 for α(intact)WT, 5 for α(intact)V288A, 4 for αMV288AMWT, 4 for αMV288AMV288A, 4 for αMV288AMWTMWTMWT, 4 for αMV288AMV288AMWTMWT, 3 for αMV288AMV288AMWTMV288A, and 4 for αMV288AMV288AMV288AMV288A. (F) Voltage dependence of depolarization-induced currents for BK channels formed by non-mutated and V288A-mutant BKα (intact) and concatenated αM(dual) and αM(quad) constructs. Electrophysiological repeats n=3–5 as indicated in Table 1. All recordings were performed using symmetric K+ (140 mM) solutions with 10 μM intracellular Ca2+. Error bars represent ± SEM.
Tables
Table 1
Boltzmann-fit parameters of the voltage-dependent concatenated tandem BK channel activation in the wildtype, mutants in the absence and presence of intracellular Ca2+.
| Expression* | Ca2+ (µM) | Boltzmann fit parameters | ||
|---|---|---|---|---|
| V1/2 (mV) | z | n† | ||
| αintact | 0 | 172±2 | 1.08±0.04 | 9 |
| αintact | 10 | 19±4 | 1.47±0.10 | 8 |
| αM | 0 | 191±1 | 1.11±0.13 | 4 |
| αM | 10 | 18±3 | 1.20±0.06 | 6 |
| αMM | 0 | 186±5 | 1.12±0.12 | 5 |
| αMM | 10 | 16±3 | 1.02±0.03 | 10 |
| αMMMM | 0 | 184±5 | 1.09±0.07 | 6 |
| αMMMM | 10 | 29±4 | 0.86±0.06 | 4 |
| αintact + γ1 | 0 | 35±2 | 1.56±0.11 | 8 |
| γ1αMM | 0 | 56±4 | 0.99±0.07 | 6 |
| γ1αMMMM | 0 | 42±3 | 1.13±0.13 | 5 |
| αML312A | 0 | 62±4 | 0.96±0.07 | 6 |
| αML312AMWT | 0 | 121±6 | 0.90±0.04 | 10 |
| αMWTML312A | 0 | 118±5 | 0.95±0.07 | 4 |
| αML312AML312A | 0 | 64±5 | 0.97±0.08 | 5 |
| αML312AM2WTM3WTM4WT | 0 | 153±4 | 0.96±0.10 | 5 |
| αML312AML312AMWTMWT | 0 | 133±2 | 0.77±0.05 | 4 |
| αML312AML312AML312AMWT | 0 | 98±5 | 0.90±0.11 | 5 |
| αML312AML312AML312AML312A | 0 | 61±6 | 1.05±0.09 | 5 |
| αML312AML312AML312AML312A + αMWTMWTMWTMWT | 10 | 180±13 (36%) | 0.90±0.52 | 5 |
| 65±8 (64%) | 1.01±0.13 | |||
| αintactV288A | 10 | 149±8 | 1.06±0.10 | 5 |
| αMV288AMWT | 10 | 31±2 | 1.14±0.09 | 5 |
| αMV288AMWTMWTMWT | 10 | 21±11 | 1.16±0.06 | 3 |
| αMV288AMV288AMWTMWT | 10 | 19±2 | 1.19±0.17 | 3 |
| αMV288AMV288AMWTMV288A | 10 | 38±3 | 1.05±0.06 | 3 |
| αMV288AMV288AMV288AMV288A | 10 | 127±10 | 0.75±0.21 | 3 |
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*
Except for the full-length BKαintact construct, all other listed (derivatives of αM) constructs were co-expressed with the complementary αΔM-GFP construct.
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†
The number of recorded excised inside-out patches from different HEK293 cells.
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Concatenated modular BK channel constructs reveal divergent stoichiometry in gating control by LRRC26 (γ1), pore, and selectivity filter
eLife 14:RP107681.
https://doi.org/10.7554/eLife.107681.3
