Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K+ channels for atrial fibrillation treatment

Abstract

The Ca²⁺-activated SK4 K⁺ channel is gated by Ca²⁺-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K⁺ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca²⁺-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K⁺ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.

Keywords: KCa3.1; PIP2; atrial fibrillation; calmodulin; potassium channel.

Fig. 1.

Inside-out macropatch recordings reveal the importance of calcium and PIP2 for SK4 K⁺ channel gating. (A) Representative traces of WT SK4 currents recorded from a transfected CHO cell exposed to different intracellular free Ca²⁺ concentrations under inside-out patch-clamp configuration. Currents are recorded by 10 repetitive 1-s-duration voltage ramps from −100 mV to +100 mV from a holding potential of 0 mV. (B) Dose-dependent activation of WT SK4 channels by intracellular free Ca²⁺ in the presence (n = 5) and absence (n = 6) of BA69b 10 µM, yielding EC₅₀s of 435 nM and 65 nM, respectively. (C) Representative traces of WT SK4 currents before (black) and after PLL (50 µg/mL) internal application (red). (D) Internal application of PLL (50 µg/mL) decreases the SK4 current by 68 ∓ 4% (n = 22; two-tailed paired t test, t = 15.71, df = 21, ****P < 0.0001). (E) WT SK4 current is enhanced in response to increasing diC8–PIP2 concentrations after prior depletion of endogenous PIP2 by PLL. The experiment is performed with internal 1 µM free Ca²⁺ concentration. (F) Dose-dependent activation of WT SK4 channels coexpressed WT CaM (n = 6) or CaM T79D (n = 7) by internal application of diC8-PIP2, yielding EC₅₀s of 154 nM and 871 nM, respectively.

Fig. 2.

PIP2–calmodulin interface and SK4 channel activation. (A) Effects of increased PIP2 levels by cotransfection with PIP4,5 kinase on WT and mutant SK4 channels. Whole-cell SK4 K⁺ currents are activated using a voltage ramp protocol from −100 mV to +60 mV for 150 ms. PIP4,5 kinase significantly increases WT SK4 and mutant Q353A currents by 3.7- and 3.5-fold, respectively (n = 41 and n = 29, respectively; one-way ANOVA, F (15, 224) = 17.74, Sidak's multiple comparisons test P < 0.0001); all other mutants are not activated by PIP4,5 kinase (n = 5–36). (B) Internal purified CaM T79D (3 µM) produces significantly lower SK4 currents than with WT CaM (n = 10–11, two-tailed unpaired t test, t = 2.099, df = 19, P < 0.0454). (C) PIP4,5 kinase significantly increases SK4 currents cotransfected with WT CaM, while no effects are observed with CaM mutants K75A and T79D (n = 8–19). (D) Cotransfection of WT SK4 channels with CK2α subunit enzyme significantly decreases current density by 47 ∓ 6% (n = 9–12, two-tailed unpaired t test, t = 2.433, df = 19, *P = 0.0250). (E) Docking of PIP2 (cyan/orange stick) to the Ca²⁺-bound state I (6CNN) of the SK4 channel cryo-EM structure; the S1–S4 helices, the CaM, and the SK4 proximal carboxyl terminus (helices A and B) are shown in deep purple, gray, and light cyan, respectively. (F) Zoom-in of the PIP2-binding pocket showing the interacting residues.

Fig. 3.

Effects of small allosteric modulators on SK4 channel activation. (A) Chemical structure of 1-EBIO. (B) Representative trace of WT SK4 currents in the absence and presence of 10 µM BA-40, showing activation of ∼1.4-fold. Whole-cell SK4 K⁺ currents are activated using a voltage ramp protocol from −100 mV to +60 mV for 150 ms. (C) Representative trace of WT SK4 currents in the absence and presence of 20 µM BA6b, showing an inhibition of ∼25%. (D) Statistical summary of the pharmacological effects of 10 µM BA-40, 10 µM BA-100, and 20 µM BA6b on WT SK4 currents with 42 ∓ 8% activation (n = 14), 28 ∓ 6% activation (n = 9), and 25 ∓ 2% inhibition (n = 6), respectively. (E) Representative trace of WT SK4 currents in the absence and presence of 20 µM BA6b9, displaying an inhibition of ∼56%. (F) Dose-dependent inhibition of WT SK4 channels by BA69b yielding an apparent IC₅₀ of 8.6 ∓ 0.8 µM (n = 6). (G) Representative traces of an inside-out macropatch from a CHO cell expressing WT SK4 channels in the absence and presence of 10 µM BA6b9 under internal 1 µM free Ca²⁺ concentration. Currents are recorded by 10 repetitive 1-s-duration voltage ramps from −100 mV to +100 mV from a holding potential of 0 mV. (H) Representative trace of WT SK4 currents in the absence and presence of 0.5 µM Tram-34, demonstrating an inhibition of ∼86%. (I) Statistical summary of the pharmacological effects of 0.5 µM Tram-34 and 20 µM BA6b9 on WT SK4 currents with 86 ∓ 4% (n = 5) and 56 ∓ 1% (n = 41) inhibition, respectively.

Fig. 4.

Molecular docking of BA6b9 and functional validation in transfected CHO cells. (A) Representative trace of WT SK4 currents in the absence and presence of 20 µM BA6b9. (B) Representative trace of the SK4 mutant H192A current in the absence and presence of 20 µM BA6b9, showing the significant lower inhibition (16%) compared with that obtained for WT SK4 (56%). (C) Representative trace of WT SK4 channel cotransfected with CaM mutant M72A in the absence and presence of 20 µM BA6b9, showing complete insensitivity to inhibition by BA6b9. (D) SK4 channel mutants R352Q, R191A, and H192A are significantly less sensitive to the inhibitory effect of 20 µM BA6b9 compared with WT SK4 (one-way ANOVA, F (10, 165) = 10.27; Dunnett's multiple comparisons test; n = 14, P < 0.0003, n = 16, P < 0.0001 and n = 11, P < 0.0001, and n = 62, respectively). (E) Statistical summary of the pharmacological effects of 20 µM BA6b9 on WT SK4 channel cotransfected with CaM mutants M72A and M76A, displaying their significant lower sensitivity to BA6b9 inhibition (one-way ANOVA, F(2, 35) = 25.59, Dunnett's multiple comparisons test; respectively, 0 ∓ 3% inhibition n = 7, P < 0.0001 and 19 ∓ 9% inhibition n = 7, P = 0.0008) as compared with WT CaM (47% inhibition, n = 24). (F) BA6b9-binding pocket showing the interacting residues. BA6b9 is docked to the Ca²⁺-bound state I (6CNN) of the SK4 channel cryo-EM structure; BA6b9, PIP2, S1 helix and S4–S5 linker, CaM, and the SK4 proximal carboxyl terminus (helix B) are represented by green stick, cyan/orange stick, deep purple, gray, and cyan, respectively.

Fig. 5.

Immunohistochemistry of SK4 channel and α-actinin expression in healthy rat cardiac tissue. Representative left atrial (LA) and left ventricular (LV) paraffin embedded sections of a healthy rat heart. (A and B) Sections are processed and stained with secondary antibodies as a negative control. (C and D) Sections are labeled exclusively with SK4 monoclonal antibody and stained with Congo Red as described in “Materials and Methods”. Note the enhanced intensity and scatter of the red staining in the LA cardiomyocytes in (C) compared with the LV in (D). Note also, in (D), the positive staining of SK4 channel of a blood vessel. (E and F) Sections are exclusively labeled with α-actinin polyclonal antibody and stained with 3,3'-diaminobenzidine as described in “Materials and Methods”. Notice the sarcomeric striated Z-band pattern of α-actinin.

Fig. 6.

Double staining immunohistochemistry of SK4 channel and α-actinin expression in healthy rat cardiac tissue. (A and B) Double-stained LA with SK4 channel and α-actinin antibodies, showing SK4 channel localization at the sarcolemma as well as in the intracellular membrane network with a sarcomeric striated Z-band pattern in (B). (C and D) Double-stained LV with SK4 channel and α-actinin antibodies showing localization not only in the sarcolemma and striations across the cells but also at the intercalated discs between ventricular myocytes in (D) (black arrows).

Fig. 7.

Tram-34 and BA6b9 effects on the electrophysiological properties of the isolated rat hearts. Data are analyzed by two-tailed paired t test, except in (D) and (J), by two-tailed Mann Whitney test. (A and G) AERP and AVERP are significantly prolonged by 10 µM Tram-34 (n = 12, t = 3.293, df = 11, P = 0.0072 and t = 4.197, df = 11, P = 0.0015, respectively) and 10 µM BA6b9 (n = 16, t = 2.822, df = 15, P = 0.0129 and n = 15, t = 4.706, df = 14, P = 0.0003, respectively). (B and H) Heart rate is significantly decreased by 10 µM Tram-34 (n = 21, t = 5.816, df = 20, P < 0.0001) and 10 µM BA6b9 (n = 19, t = 6.209, df = 18, P < 0.0001). (C and I) PR interval is significantly increased by 10 µM Tram-34 (n = 11, t = 5.997, df = 10, P = 0.0001) and 10 µM BA6b9 (n = 17, t = 5.592, df = 16, P < 0.0001). (D and J) Ten micromolar Tram-34 and 10 µM BA6b9 significantly lower AFIS (see Materials and Methods) by 54% and 48%, respectively, compared with carbachol alone (n = 7, P = 0.0023 and n = 10, P = 0.0218, respectively). (E and K) Ten micromolar Tram-34 and 10 µM BA6b9 prevent sustainability (see Materials and Methods) in 28% (2/7) and 30% (3/10) of heart preparations, respectively. (F1) Representative recording showing AF induced after 10-min incubation with 0.3 µM carbachol by burst pacing through a quadripolar electrode attached to the high right atrium. (F2) Higher time resolution recording of the area marked by horizontal bar in (F1).