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The tremorgenic fungal alkaloid paxilline (PAX) is a commonly used specific inhibitor of the large-conductance, voltage- and Ca2+-dependent BK-type K⁺ channel. PAX inhibits BK channels by selective interaction with closed states. BK inhibition by PAX is best characterized by the idea that PAX gains access to the channel through the central cavity of the BK channel, and that only a single PAX molecule can interact with the BK channel at a time. The notion that PAX reaches its binding site via the central cavity and involves only a single PAX molecule would be consistent with binding on the axis of the permeation pathway, similar to classical open channel block and inconsistent with the observation that PAX selectively inhibits closed channels. To explore the potential sites of interaction of PAX with the BK channel, we undertook a computational analysis of the interaction of PAX with the BK channel pore gate domain guided by recently available liganded (open) and metal-free (closed) Aplysia BK channel structures. The analysis unambiguously identified a preferred position of PAX occupancy that accounts for all previously described features of PAX inhibition, including state dependence, G311 sensitivity, stoichiometry, and central cavity accessibility. This PAX-binding pose in closed BK channels is supported by additional functional results.

Context SLO1and SLO3 are similar voltage-gated K + channels. However, SLO3 expression is sperm specific and plays an important role in the hyperpolarization of the sperm membrane potential that is crucial for sperm fertilization. This makes SLO3 an excellent molecular target for the development of male contraceptives, and computational methods can facilitate structural insights for this drug development. Here, we evaluated the differential molecular interactions between the human SLO3 (hSLO3) and SLO1 (hSLO1) potassium channels and iberiotoxin (IbTX), a toxin that selectively blocks SLO channels. To do this, molecular docking and dynamics were implemented on the channel-toxin complexes to help elucidate atomistic details of their interaction and binding energy. Our analysis found that IbTX has a similar binding energy to both channels but interacts in a distinct manner with them. Particularly, Trp14 and Arg25 residues of IbTX diverges in their interaction with the residues Val283 and Asn260 residues of hSLO3 and the corresponding residues Tyr359 and Ala336 of hSLO1. Knowledge of key residues in the molecular interface of IbTX blockage can help guide and hasten non-hormonal contraceptive development. Our results encourage the use of toxins as scaffolds for specific SLO3 blockers. Methods Atomistic molecular dynamics were implemented on the channel-toxin complexes. To generate the complexes, IbTX was docked to the channels using HADDOCK. CHARMM-GUI was used to generate simulation systems. GROMACS v2023.1 was used to run the simulations for 500 ns in an NPT ensemble at 297.26 K employing the CHARMM36 force field. Binding energy was evaluated by molecular mechanics generalized born surface area (MM/GBSA) with gmxMMPBGBSA.py. Graphical Abstract

BK-type K⁺ channels are activated by voltage and intracellular Ca²⁺, which is important in modulating muscle contraction, neural transmission, and circadian pacemaker output. Previous studies suggest that the cytosolic domain of BK channels contains two different Ca²⁺ binding sites, but the molecular composition of one of the sites is not completely known. Here we report, by systematic mutagenesis studies, the identification of E535 as part of this Ca²⁺ binding site. This site is specific for binding to Ca²⁺ but not Cd²⁺. Experimental results and molecular modeling based on the X-ray crystallographic structures of the BK channel cytosolic domain suggest that the binding of Ca²⁺ by the side chains of E535 and the previously identified D367 changes the conformation around the binding site and turns the side chain of M513 into a hydrophobic core, providing a basis to understand how Ca²⁺ binding at this site opens the activation gate of the channel that is remotely located in the membrane.

Large-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca 2+ . The only covalent connection between the cytosolic Ca 2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue ‘C-linker’. To determine the linker’s role in human BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combining atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.

Patients suffering from a variety of neurological diseases such as spinal cord injury, Parkinson's disease, and multiple sclerosis often develop neurogenic detrusor overactivity (NDO), which currently lacks a universally effective therapy. Here, we tested the hypothesis that NDO is associated with changes in detrusor smooth muscle (DSM) large conductance Ca(2+)-activated K(+) (BK) channel expression and function. DSM tissue samples from 33 patients were obtained during open bladder surgeries. NDO patients were clinically characterized preoperatively with pressure-flow urodynamics demonstrating detrusor overactivity, in the setting of a clinically relevant neurological condition. Control patients did not have overactive bladder and did not have a clinically relevant neurological disease. We conducted quantitative polymerase chain reactions (qPCR), perforated patch-clamp electrophysiology on freshly-isolated DSM cells, and functional studies on DSM contractility. qPCR experiments revealed that DSM samples from NDO patients showed decreased BK channel mRNA expression in comparison to controls. Patch-clamp experiments demonstrated reduced whole cell and transient BK currents (TBKCs) in freshly-isolated DSM cells from NDO patients. Functional studies on DSM contractility showed that spontaneous phasic contractions had a decreased sensitivity to iberiotoxin, a selective BK channel inhibitor, in DSM strips isolated from NDO patients. These results reveal the novel finding that NDO is associated with decreased DSM BK channel expression and function leading to increased DSM excitability and contractility. BK channel openers or BK channel gene transfer could be an alternative strategy to control NDO. Future clinical trials are needed to evaluate the value of BK channel opening drugs or gene therapies for NDO treatment and to identify any possible adverse effects.

The large-conductance calcium-activated potassium (BK Ca ) channel, which is crucial for urinary bladder smooth muscle relaxation, is a potential target for overactive bladder treatment. Our prior work unveiled CTIBD as a promising BK Ca channel activator, altering V 1/2 and G max . This study investigates CTIBD’s activation mechanism, revealing its independence from the Ca 2+ and membrane voltage sensing of the BK Ca channel. Cryo-electron microscopy disclosed that two CTIBD molecules bind to hydrophobic regions on the extracellular side of the lipid bilayer. Key residues (W22, W203, and F266) are important for CTIBD binding, and their replacement with alanine reduces CTIBD-mediated channel activation. The triple-mutant (W22A/W203A/F266A) channel showed the smallest V 1/2 shift with a minimal impact on activation and deactivation kinetics by CTIBD. At the single-channel level, CTIBD treatment was much less effective at increasing P o in the triple mutant, mainly because of a drastically increased dissociation rate compared with the WT. These findings highlight CTIBD’s mechanism, offering crucial insights for developing small-molecule treatments for BK Ca -related pathophysiological conditions.

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.

Large-conductance voltage- and Ca2+-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.

Inactivation is an intrinsic property of several voltage-dependent ion channels, closing the conduction pathway during membrane depolarization and dynamically regulating neuronal activity. BK K + channels undergo N-type inactivation via their β2 subunit, but the physiological significance is not clear. Here, we report that inactivating BK currents predominate during the day in the suprachiasmatic nucleus, the brain’s intrinsic clock circuit, reducing steady-state current levels. At night inactivation is diminished, resulting in larger BK currents. Loss of β2 eliminates inactivation, abolishing the diurnal variation in both BK current magnitude and SCN firing, and disrupting behavioural rhythmicity. Selective restoration of inactivation via the β2 N-terminal ‘ball-and-chain’ domain rescues BK current levels and firing rate, unexpectedly contributing to the subthreshold membrane properties that shift SCN neurons into the daytime ‘upstate’. Our study reveals the clock employs inactivation gating as a biophysical switch to set the diurnal variation in suprachiasmatic nucleus excitability that underlies circadian rhythm. BK potassium channels have been previously shown to mediate SCN circadian firing, although the precise mechanisms are unclear. Here, using knockout and rescue approaches, the authors find that the ß2 ‘ball-and-chain’ confers BK channel inactivation during the day, promoting SCN electrical upstate.

The allosteric communication between the pore domain, voltage sensors, and Ca 2+ binding sites in the calcium- and voltage-activated K + channel (BK) underlies its physiological role as the preeminent signal integrator in excitable systems. BK displays shallow voltage sensitivity with very fast gating charge kinetics, yet little is known about the molecular underpinnings of this distinctive behavior. Here, we explore the mechanistic basis of coupling between voltage-sensing domains (VSDs) and calcium sensors in Aplysia BK by locking the VSDs in their activated (R196Q and R199Q) and resting (R202Q) states, with or without calcium. Cryo-EM structures of these mutants reveal unique tilts at the S4 C-terminal end, together with large side-chain rotameric excursions of the gating charges. Notably, the VSD resting structure (R202Q) also revealed BK in its elusive, fully closed state, highlighting the reciprocal relation between calcium and voltage sensors. These structures provide a plausible path where voltage and Ca 2+ binding couple energetically and define the conformation of the pore domain and, thus, BK’s full functional range. The allosteric communication between the pore domain, voltage sensors, and Ca2+ binding sites in BK channels underlies its physiological role. Here, cryo-EM structures provide a plausible path where voltage and Ca2+ binding define the conformation of the pore domain.