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 (3)) 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 BKαM(dual) and BKαM(quad) constructs transiently expressed in HEK293 cells. (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) BKαM 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.

Boltzmann-fit parameters of the voltage-dependent concatenated tandem BK channel activation in the wildtype, mutants in the absence and presence of intracellular Ca2+.

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 (23)) showing the γ1 subunit in purple and the three separable BKα regions in distinct colors. (B) Schematic of the membrane topology for the BKγ1 subunit and its fusion constructs, created by linking its C-terminus to the N-terminus of the BKαM dual- and quadruple-repeat constructs. (C) Immunoblot analysis of the BKγ1αM(dual) and BKγ1αM(quad) constructs expressed in HEK293 cells. (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 BKαM constructs (co-expressed with BKαΔM) 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 BKγ1αM(dual) and BKγ1αM(quad) constructs co-expressed with BKαΔM. Electrophysiological recordings were repeated n = 5 – 8, as indicated in Table 1. Error bars represent ± SEM.

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 (3)), 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 BKαM and concatenated BKαM(dual) and BKαM(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 BKαM and BKαM(dual) constructs co-expressed with BKαΔM. Dashed lines show G-V curves of the corresponding non-mutated channels for comparison. (D) Voltage dependence of activation for channels formed by the BKαM(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 BKαM(dual) and BKαM(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 BKαM(quad) constructs co-expressed with BKαΔM. (G) Representative current traces from channels formed by co-expressing the non-mutated and fully L312A mutated BKαM(quad) constructs (co-expressed with BKαΔM). 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.

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. Inactivation was assayed monitoring the reduction in fast-activating currents (shown and compared in the middle) elicited by brief depolarization at different intervals. 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 100 ms) 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 BKαM(dual) and BKαM(quad) constructs with subunit-specific V288A mutations (co-expressed with BKαΔM). 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 BKαM(dual) and BKαM(quad) constructs. Electrophysiological repeats: n = 8 for BKα(intact)WT, 5 for BKα(intact)V288A, 4 for BKαMV288AMWT, 4 for BKαMV288AMV288A, 4 for BKαMV288AMWTMWTMWT, 4 for BKαMV288AMV288AMWTMWT, 3 for BKαMV288AMV288AMWTMV288A, and 4 for BKαMV288AMV288AMV288AMV288A. (F) Voltage dependence of depolarization-induced currents for BK channels formed by non-mutated and V288A-mutant BKα (intact) and concatenated BKαM(dual) and BKαM(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.