BK Channel Pore Research Articles

On the localization and composition of BK channel pore gate: Selectivity filter vs. lipid occlusion of deep pore

On the localization and composition of BK channel pore gate: Selectivity filter vs. lipid occlusion of deep pore

Sweetening K-channels: what sugar taught us about permeation and gating.

Because they enable for the modification of both viscosity and osmolarity, sugars have been used as a biophysical probe of voltage-gated K-channels for a while. Viscosity variations made it possible to measure the pore sizes in large and small conductance K-channels using techniques similar to those used in the 1980s to study the gramicidin A channel. These analyses led to the finding that the size of the internal mouth appears to be the primary cause of the conductance differences between Shaker-like channels and large conductance BK-channels. As an osmotic agent, adding sugar unilaterally causes streaming potentials that indicate H2O/K+ cotransport across the BK-channel pore. Osmotic experiments on Shaker K-channels suggest that the pore gate operation and the slow inactivation displace comparable amounts of water. Functionally isolated voltage sensors allow estimation of individual osmotic work for each voltage sensing charge during voltage-activation, reporting dramatic internal and external remodeling of the Voltage Sensing Domain´s solvent exposed surfaces. Remarkably, each charge of the VSD appears to take a unique trajectory. Thus, manipulation of viscosity and osmolarity, together with 3D structures, brings in solid grounds to harmonize function and structure in membrane proteins such as K-channels and, in a wider scope, other structurally dynamic proteins.

Homology modeling and molecular docking simulation of martentoxin as a specific inhibitor of the BK channel.

BackgroundLarge conductance calcium-activated potassium channel (BK channel) is gated by both voltage and calcium ions and is widely distributed in excitable and nonexcitable cells. BK channel plays an important role in epilepsy and other diseases, but BK channel subtype-specific drugs are still extremely rare. Martentoxin was previously isolated from the venom of members of Scorpionidae and shown to be composed of 37 amino acids. Research has shown that the pharmacological selectivity of martentoxin to the BK channel is higher than that to other potassium channels. Therefore, it is of great significance to study the mechanism of interaction between martentoxin and BK channels.MethodsThe three-dimensional structure of BK channel pore region was constructed by homologous modeling method, and the key amino acid sites of BK channel interaction with martentoxin were analyzed by protein-protein docking, molecular dynamic simulation and virtual alanine mutation.ResultsBased on homologous modeling of BK channel pore structure and protein-protein docking analysis, Phe1, Lys28 and Arg35 of martentoxin were found to be key amino acids in toxin BK channel interaction.ConclusionsThis study reveals the structural basis of martentoxin interaction with BK channel. These results will contribute to the design of BK channel specific blockers based on the structure of martentoxin.

The functionally relevant site for paxilline inhibition of BK channels

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.

Traumatic Brain Injury Alters BK Channel Subunit Expression, Localization, and Function in Cerebral Arterial Myocytes

Traumatic brain injury (TBI) induces acute changes in cerebrovascular function, which include alterations to cerebral artery myogenic tone and autoregulation. In arterial smooth muscle cells (myocytes), large‐conductance calcium‐activated potassium (BK) channels play a vital role in modulating plasma membrane potential and myogenic tone. Here, we examined BK channel expression and function in cerebral resistance size arteries one week following moderately‐severe TBI induced by controlled cortical impact with craniotomy. Total BKα (pore‐forming) and β1 (regulatory) subunit expression were reduced one week post‐TBI. Biotinylation of plasma membrane‐bound proteins revealed a compensatory increase of β1 subunits to the plasma membrane following TBI, which normalized single BK channel activation as assessed using patch‐clamp electrophysiology. However, the loss of total BKα subunits resulted in diminished whole cell, transient BK currents. Pressurized cerebral arteries from TBI rats exhibited greater myogenic tone at 60 mmHg and a diminished response to iberiotoxin, a BK channel pore blocker. In conclusion, moderately‐severe TBI enhances cerebral artery contractility, in part, through reductions in arterial myocyte BK channel subunit expression and function. Moreover, these TBI‐induced alterations in cerebral artery function can persist well beyond the initial 24–48 hour period post‐injury.Support or Funding InformationFlorida State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Solution structure of extracellular loop of human \u03b24 subunit of BK channel and its biological implication on ChTX sensitivity

Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming α subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BKα coexpression with auxiliary β (β1-β4) subunits, as well as newly defined γ subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched β4 subunit (hβ4) alters the conductance-voltage curve, slows activation and deactivation time courses of BK channels. Its extracellular loop (hβ4-loop) specifically impedes ChTX to bind BK channel pore. However, the structure of β4 subunit’s extracellular loop and the molecular mechanism for gating kinetics, toxin sensitivity of BK channels regulated by β4 are still unclear. To address them, here, we first identified four disulfide bonds in hβ4-loop by mass spectroscopy and NMR techniques. Then we determined its three-dimensional solution structure, performed NMR titration and electrophysiological analysis, and found that residue Asn123 of β4 subunit regulated the gating and pharmacological characteristics of BK channel. Finally, by constructing structure models of BKα/β4 and thermodynamic double-mutant cycle analysis, we proposed that BKα subunit might interact with β4 subunit through the conserved residue Glu264(BKα) coupling with residue Asn123(β4).

Gating ring strengthens marriage of BK channel voltage sensing to pore opening.

A new JGP study shows that the gating ring helps the voltage-sensing domain open the BK channel's pore.

An old probe sheds new light on BK channel pore structure.

In many scientific areas, including drug discovery, the greatest challenge is picking the best targets and questions to pursue. To develop improved treatments using target-based therapeutics, a better understanding of the roles of specific channel types in normal and pathophysiological function is

A charged residue in S4 regulates coupling among the activation gate, voltage, and Ca2+ sensors in BK channels.

Coupling between the activation gate and sensors of physiological stimuli during ion channel activation is an important, but not well-understood, molecular process. One difficulty in studying sensor-gate coupling is to distinguish whether a structural perturbation alters the function of the sensor, the gate, or their coupling. BK channels are activated by membrane voltage and intracellular Ca(2+) via allosteric mechanisms with coupling among the activation gate and sensors quantitatively defined, providing an excellent model system for studying sensor-gate coupling. By studying BK channels expressed in Xenopus oocytes, here we show that mutation E219R in S4 alters channel function by two independent mechanisms: one is to change voltage sensor activation, shifting voltage dependence, and increase valence of gating charge movements; the other is to regulate coupling among the activation gate, voltage sensor, and Ca(2+) binding via electrostatic interactions with E321/E324 located in the cytosolic side of S6 in a neighboring subunit, resulting in a shift of the voltage dependence of channel opening and increased Ca(2+) sensitivity. These results suggest a structural arrangement of the inner pore of BK channels differing from that in other voltage gated channels.

Single Aspartate Substitutions at Multiple Deep-Pore Positions Lead to Constitutively Open BK Channels

The pore-lining amino acids of ion channel proteins reside on the interface between a polar (the pore) and a non-polar environment (the rest of the protein). Manipulating the charge states of their side-chains, by tilting the energy balance between a buried and a pore-exposed conformation, can reveal important details about gating-related structural dynamics. For example, charged side-chains at the M314 position in the BK channel stabilize an open conformation. The M314D mutant, in particular, led to constitutive channel activities. We have now scanned the S6 region (I308 to N328) with aspartate replacement. We found 3 other positions where aspartate substitutions led to constitutively open BK channels in nominal zero calcium and neutral pH. These mutants are L312D, A313D and A316D. Together with M314, these positions are all located within the deep-pore region of the channel, which is immediately intracellular to the selectivity filter but more extracellular to the lower-pore region that aligns with the Kv channel “bundling crossing”. These findings provide further evidence that the BK channel pore is different from that of Kv channels, where aspartate substitution at a single position (as opposed to multiple positions in BK) in the lower-pore region (as opposed to the deep-pore region in BK) led to constitutive channel activities. The aspartate mediated constitutive activities in Kv channels is believed to result from the disruption of specific hydrophobic interactions in the “bundle-crossing” gate of a closed channel. Our observations with BK channels are unlikely to result from disruption of specific interactions, as multiple positions in the BK deep-pore, some of them adjacent along the S6 helix, showed aspartate mediated constitutive activities.

Probing the Allosteric Interactions that Mediate Voltage-Dependent BK (Slo1) Channel Activation with a Pore Blocker

Like many transmembrane voltage-activated proteins, BK channels possess conserved voltage-sensing domains (VSD) which activate upon membrane depolarization. VSDs are structurally and functionally associated with the channel pore domain, such that VSD activation facilitates pore opening. BK channels are selectively blocked by Iberiotoxin (IbTX), a peptide component of scorpion venom which binds to the BK pore domain, preventing K+ flux. To elucidate the allosteric consequences of toxin binding to the BK channel pore, we probed the operation of the BK VSD using cut-open oocyte voltage clamp fluorometry, whereby a fluorophore labeling the BK VSD helix S0 reported the conformational rearrangements, and therefore the state of activation, of the VSD. This was performed in conducting channels, as well as channels blocked by external IbTX (10 μM) perfusion. The blockade regime caused a >95% decrease of macroscopic K+ conductance and an apparent functional impairment of the VSD, as suggested by the half-activation shift to depolarized potentials by ∼35 mV and effective charge reduction by ∼20%. Since IbTX is a BK channel pore blocker, we propose that the observed impairment of the VSD must be due to the allosteric coupling between the pore and the voltage sensing domains. To test and quantify this interpretation, we used a statistical-mechanical model of BK channel activation, which accounted for the blockade-induced impairment of the BK VSD by a large reduction (2.1x10−7 to 1.4x10−9) of the intrinsic equilibrium constant for channel opening at 0 mV (L0), suggesting that IbTX blockade caused the pore to “lock” into a shut state, which resulted in the observed perturbation of the voltage-sensing apparatus, mediated by the strong (−75 meV) allosteric linkage between the two domains.

Cysteine scanning and modification reveal major differences between BK channels and Kv channels in the inner pore region

BK channels are regulated by two distinct physiological signals, transmembrane potential and intracellular Ca(2+), each acting through independent modular sensor domains. However, despite a presumably central role in the coupling of sensor activation to channel gating, the pore-lining S6 transmembrane segment has not been systematically studied. Here, cysteine substitution and modification studies of the BK S6 point to substantial differences between BK and Kv channels in the structure and function of the S6-lined inner pore. Gating shifts caused by introduction of cysteines define a pattern and direction of free energy changes in BK S6 distinct from Shaker. Modification of BK S6 residues identifies pore-facing residues that occur at different linear positions along aligned BK and Kv S6 segments. Periodicity analysis suggests that one factor contributing to these differences may be a disruption of the BK S6 α-helix from the unique diglycine motif at the position of the Kv hinge glycine. State-dependent MTS accessibility reveals that, even in closed states, modification can occur. Furthermore, the inner pore of BK channels is much larger than that of K(+) channels with solved crystal structures. The results suggest caution in the use of Kv channel structures as templates for BK homology models, at least in the pore-gate domain.

Intersubunit Coupling in the Pore of BK Channels

The structural basis underlying the gating of large conductance Ca(2+)-activated K(+) (BK) channels remains elusive. We found that substitution of Leu-312 in the S6 transmembrane segment of mSlo1 BK channels with hydrophilic amino acids of smaller side-chain volume favored the open state. The sensitivities of channels to calcium and voltage were modified by some mutations and completely abolished by others. Interpretation of the results in terms of an allosteric model suggests that the calcium-insensitive mutants greatly destabilize the closed relative to the open conformation and may also disrupt the allosteric coupling between Ca(2+) or voltage sensors and the gate. Some Phe-315 mutations also favor the open state, suggesting that Leu-312 and Phe-315 may interact in the closed state, forming a major energy barrier that the channel has to overcome to open. Homology modeling and molecular dynamic simulations further support that the side chain of Leu-312 can couple strongly with the aromatic ring of Phe-315 in neighboring subunits (L-F coupling) to maintain the channel closed. Additionally, single-channel recordings indicate that the calcium-insensitive mutants, whose kinetics can be approximately characterized by a two-state closed-open (C-O) model, exhibit nearly 100% open probability under physiological conditions without alterations in single-channel conductance. These findings provide a basis for understanding the structure and gating of the BK channel pore.

Identification of Large Conductance Calcium Activated Potassium Channel Accessory β4 Subunit in Rat and Mouse Bladder Smooth Muscle

The BK (large conductance voltage and Ca(2+) activated K(+)) channel is a key regulator of bladder smooth muscle contractility. To our knowledge in bladder smooth muscle the BK channel pore forming alpha subunit BKalpha associates in homotetramers with 4 regulatory smooth muscle specific beta1 subunits. We challenged this concept in identify whether other regulatory BKbeta subunits exist in mouse and rat bladder smooth muscle. We used a novel approach with single cell reverse transcriptase-polymerase chain reaction combined with immunocytochemical studies in freshly isolated mouse and rat bladder smooth muscle cells. Western blot was also performed. Reverse transcriptase-polymerase chain reaction identified the mRNA expression of various BK channel subunits in freshly isolated bladder smooth muscle cells. Our data indicate that, in addition to BKalpha and BKbeta1, neuronal specific BKbeta4 is expressed in mouse and rat bladder smooth muscle cells. BKbeta4 expression was also revealed by Western blot. Immunocytochemistry was further applied to confirm the specific expression of BKbeta4 protein directly in freshly isolated mouse and rat bladder smooth muscle cells. To our knowledge we performed the first comprehensive examination of the expression of BKalpha and BKbeta subunits in bladder smooth muscle. We identified that the bladder smooth muscle BK channel has a distinctive architecture involving pore forming BKalpha and regulatory BKbeta1/beta4. Further studies of the functional roles of BKalpha, BKbeta1 and BKbeta4 directly in human bladder smooth muscle may help the development of alternative therapeutic strategies to control bladder dysfunction. New drugs targeting specific BK channel subunits in human bladder smooth muscle may prove useful for overactive bladder.

Intrinsic Electrostatic Potential in the BK Channel Pore: Role in Determining Single Channel Conductance and Block

The internal vestibule of large-conductance Ca2+ voltage-activated K+ (BK) channels contains a ring of eight negative charges not present in K+ channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017–9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K+ was 18% lower than the control. Current–voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K+ (30 mM) and vanished at high K+ (1 M K+). We determine the electrostatic potential change, Δφ, caused by the charge neutralization using TEA+ block for the extracellular charges and Ba2+ for intracellular charges. We measured 13 ± 2 mV for Δφ at the TEA+ site when turning off the extracellular charges, and 17 ± 2 mV for the Δφ at the Ba2+ site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Δφ using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.

Unique inner pore properties of BK channels revealed by quaternary ammonium block.

Potassium channels have a very wide distribution of single-channel conductance, with BK type Ca2+-activated K+ channels having by far the largest. Even though crystallographic views of K+ channel pores have become available, the structural basis underlying BK channels' large conductance has not been completely understood. In this study we use intracellularly applied quaternary ammonium compounds to probe the pore of BK channels. We show that molecules as large as decyltriethylammonium (C10) and tetrabutylammonium (TBA) have much faster block and unblock rates in BK channels when compared with any other tested K+ channel types. Additionally, our results suggest that at repolarization large QA molecules may be trapped inside blocked BK channels without slowing the overall process of deactivation. Based on these findings we propose that BK channels may differ from other K+ channels in its geometrical design at the inner mouth, with an enlarged cavity and inner pore providing less spatially restricted access to the cytoplasmic solution. These features could potentially contribute to the large conductance of BK channels.

Intracellular Mg(2+) enhances the function of BK-type Ca(2+)-activated K(+) channels.

BK channels modulate neurotransmitter release due to their activation by voltage and Ca(2+). Intracellular Mg(2+) also modulates BK channels in multiple ways with opposite effects on channel function. Previous single-channel studies have shown that Mg(2+) blocks the pore of BK channels in a voltage-dependent manner. We have confirmed this result by studying macroscopic currents of the mslo1 channel. We find that Mg(2+) activates mslo1 BK channels independently of Ca(2+) and voltage by preferentially binding to their open conformation. The mslo3 channel, which lacks Ca(2+) binding sites in the tail, is not activated by Mg(2+). However, coexpression of the mslo1 core and mslo3 tail produces channels with Mg(2+) sensitivity similar to mslo1 channels, indicating that Mg(2+) sites differ from Ca(2+) sites. We discovered that Mg(2+) also binds to Ca(2+) sites and competitively inhibits Ca(2+)-dependent activation. Quantitative computation of these effects reveals that the overall effect of Mg(2+) under physiological conditions is to enhance BK channel function.