Molecular mechanism of pharmacological activation of BK channels

Abstract

Large-conductance voltage- and Ca(2+)-activated K(+) (Slo1 BK) channels serve numerous cellular functions, and their dysregulation is implicated in various diseases. Drugs activating BK channels therefore bear substantial therapeutic potential, but their deployment has been hindered in part because the mode of action remains obscure. Here we provide mechanistic insight into how the dehydroabietic acid derivative Cym04 activates BK channels. As a representative of NS1619-like BK openers, Cym04 reversibly left-shifts the half-activation voltage of Slo1 BK channels. Using an established allosteric BK gating model, the Cym04 effect can be simulated by a shift of the voltage sensor and the ion conduction gate equilibria toward the activated and open state, respectively. BK activation by Cym04 occurs in a splice variant-specific manner; it does not occur in such Slo1 BK channels using an alternative neuronal exon 9, which codes for the linker connecting the transmembrane segment S6 and the cytosolic RCK1 domain--the S6/RCK linker. In addition, Cym04 does not affect Slo1 BK channels with a two-residue deletion within this linker. Mutagenesis and model-based gating analysis revealed that BK openers, such as Cym04 and NS1619 but not mallotoxin, activate BK channels by functionally interacting with the S6/RCK linker, mimicking site-specific shortening of this purported passive spring, which transmits force from the cytosolic gating ring structure to open the channel's gate.

Fig. 1.

Discrimination of BK channel openers based on the influence of alternative exon 9. (A–C) Comparison of the BK openers Cym04 (A), NS1619 (B), and MTx (C). Upper: Representative current traces upon depolarization to 110 mV (Slo1) or 50 mV (Slo1_9a) before (black) and after (gray) compound application to the cytosolic side. Lower: Tail currents before (open symbols) and after application of the indicated BK opener (gray filled symbols) are shown as a function of test potential. Data were normalized to the maximal control values. Incubation times and concentrations used were: 2 min 10 μM Cym04 (A), 2 min 30 μM NS1619 (B), and 10 min 2.5 μM MTx (C). Data points are mean ± SEM (n = 4–7). Continuous curves are data fits according to Eq. 1. The blue dashed curve in B denotes data for 10 μM NS1619 illustrating that, at equivalent concentration, NS1619 elicits a clearly smaller left-shift of Slo1 channels compared with Cym04 (A). (D) Mean shift in half-maximal activation voltage (ΔV_0.5) as well as changes in the slope factors (ΔV_e) induced by the indicated openers for Slo1 and Slo1_9a channels. Data points are mean ± SEM, n indicated in parentheses. ***P < 0.001, Slo1 vs. Slo_9a.

Fig. 2.

Characterization of BK channel activation by Cym04. (A) Slo1 current traces showing channel activation (Upper) and deactivation (Lower) at the indicated voltages before (Left) and after application of 10 μM Cym04 (Right). The superimposed colored curves are results of single-exponential fits. (B) Current traces at 50 and −50 mV before (black) and after (red) application of 10 μM Cym04. Bars left of the current traces indicate the current levels corresponding to closed channels (“c”) or increasing numbers of open channels (o₁, o₂). (C) Whole-cell gating currents (Left) upon depolarization to the indicated voltages before (black) and after (red) application of 10 μM Cym04. Corresponding gating charge (Q) was normalized to the maximal value (Q_max), averaged (n = 9) and plotted against voltage (Right). Continuous curves are Boltzmann fits. All data in A–C were recorded in the absence of intracellular Ca²⁺. (D) Normalized conductance-voltage plots for Slo1 currents from inside-out patches measured in the presence of 100 μM Ca²⁺ (squares) or 10 mM Mg²⁺ (circles) before (open symbols) and after (red filled symbols) application of 10 μM Cym04. Data points are mean ± SEM (n = 6), and continuous curves are data fits according to Eq. 1. (E) Cym04-induced shifts in half-maximal activation voltage (ΔV_0.5) and slope factor (ΔV_e) for experiments as shown in D at different [Ca²⁺]_i and [Mg²⁺]_i, as well as in the absence of divalent cations for Slo1 coexpressed with Slo β1 subunit and for BK channels in LNCaP cells. Vertical red lines indicate control values for Slo1 channels in the absence of divalent cations. Data points are mean ± SEM, n indicated in parentheses. *P < 0.05; **P < 0.01, comparing with Slo1 in zero [Ca²⁺]_i.

Fig. 3.

The S6/RCK linker is a molecular determinant of BK channel activation by Cym04. (A) A plausible 3D structure of a tetrameric Slo1 BK channel. The transmembrane segment (green) is based on the homology model based on the Kv channel structure according to Yuan et al. (20) (Protein Data Bank #2R9R), and the cytosolic domain (blue) is according to Wu et al. (21) (Protein Data Bank #3NAF). The image was created using MacPyMOL v0.99. The arrow points to the probable location of the S6/RCK linker segment, which is not resolved in the available crystallographic structures. For size comparison one Cym04 molecule is also shown. (B) Comparison of amino acid sequences encoded by Slo1 exon 9 (Slo1) and the alternative exon 9 (Slo1_9a). Grouping of residues (“End S6,” “Proximal linker,” “Distal linker”) indicates the three areas for chimera construction. Dots represent conserved identical residues. S6 is assumed to end at I326; +++ denotes a cluster of positive charges and ΔΔ a site of deletion. (C) Mean half-maximal activation voltage (V_0.5, Left) and slope factors (V_e, Center Left) of Slo1 BK channel variants under control conditions obtained from Boltzmann fits to normalized tail current recordings. Corresponding shifts in half-maximal activation voltage (ΔV_0.5, Center Right) and slope factors (ΔV_e, Right) induced by 10 μM Cym04. Data are mean ± SEM (n in parentheses). *P < 0.05; **P < 0.01; ***P < 0.001, comparing with Slo1 (999) channels according to two-sided unpaired Student t tests and Holm-Bonferroni correction.

Fig. 4.

The S6/RCK linker specifically determines whether Cym04 activates BK channels. (A) Current recordings of the indicated Slo1 mutants with altered linker length before (black) and after application of 10 μM Cym04 (gray). (B) Half-maximal activation voltages (V_0.5, Left) and slope factors (V_e, Center Left) for the indicated constructs obtained from Boltzmann fits to normalized tail current recordings. Corresponding shifts in V_0.5 (Center Right) and V_e (Right) upon application of 10 μM Cym04. Data are mean ± SEM (n in parentheses). *P < 0.05; ***P < 0.001, comparing with Slo1 channels according to two-sided unpaired Student t tests and Holm-Bonferroni correction.