Interaction between residues in the Mg2+-binding site regulates BK channel activation

As a unique member of the voltage-gated potassium channel family, a large conductance, voltage- and Ca2+-activated K+ (BK) channel has a large cytosolic domain that serves as the Ca2+ sensor, in addition to a membrane-spanning domain that contains the voltage-sensing (VSD) and pore-gate domains. The conformational changes of the cytosolic domain induced by Ca2+ binding and the conformational changes of the VSD induced by membrane voltage changes trigger the opening of the pore-gate domain. Although some structural information of these individual functional domains is available, how the interactions among these domains, especially the noncovalent interactions, control the dynamic gating process of BK channels is still not clear. Previous studies discovered that intracellular Mg2+ binds to an interdomain binding site consisting of D99 and N172 from the membrane-spanning domain and E374 and E399 from the cytosolic domain. The bound Mg2+ at this narrow interdomain interface activates the BK channel through an electrostatic interaction with a positively charged residue in the VSD. In this study, we investigated the potential interdomain interactions between the Mg2+-coordination residues and their effects on channel gating. By introducing different charges to these residues, we discovered a native interdomain interaction between D99 and E374 that can affect BK channel activation. To understand the underlying mechanism of the interdomain interactions between the Mg2+-coordination residues, we introduced artificial electrostatic interactions between residues 172 and 399 from two different domains. We found that the interdomain interactions between these two positions not only alter the local conformations near the Mg2+-binding site but also change distant conformations including the pore-gate domain, thereby affecting the voltage- and Ca2+-dependent activation of the BK channel. These results illustrate the importance of interdomain interactions to the allosteric gating mechanisms of BK channels.

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WS05.1 AVX-770 binding site within CFTR membrane spanning domain 2 enables ATP-independent channel activation

Journal of Cystic Fibrosis ◽

10.1016/s1569-1993(20)30189-2 ◽

2020 ◽

Vol 19 ◽

pp. S8

Author(s):

N. Baatallah ◽

A. Elbahnsi ◽

J.-P. Mornon ◽

B. Chevalier ◽

I. Pranke ◽

...

Keyword(s):

Binding Site ◽

Channel Activation ◽

Membrane Spanning

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β-Subunit–Dependent Modulation of hSlo BK Current by Arachidonic Acid

Journal of Neurophysiology ◽

10.1152/jn.00700.2006 ◽

2007 ◽

Vol 97 (1) ◽

pp. 62-69 ◽

Cited By ~ 28

Author(s):

X. Sun ◽

D. Zhou ◽

P. Zhang ◽

E. G. Moczydlowski ◽

G. G. Haddad

Keyword(s):

Fatty Acids ◽

Arachidonic Acid ◽

Conformational Changes ◽

Unsaturated Fatty Acids ◽

Saturated Fatty Acids ◽

Nordihydroguaiaretic Acid ◽

Bk Channels ◽

Bk Channel ◽

Β Subunit ◽

Α Subunit

In this study, we examined the effect of arachidonic acid (AA) on the BK α-subunit with or without β-subunits expressed in Xenopus oocytes. In excised patches, AA potentiated the hSlo-α current and slowed inactivation only when β2/3 subunit was co-expressed. The β2-subunit–dependent modulation by AA persisted in the presence of either superoxide dismutase or inhibitors of AA metabolism such as nordihydroguaiaretic acid and eicosatetraynoic acid, suggesting that AA acts directly rather than through its metabolites. Other cis unsaturated fatty acids (docosahexaenoic and oleic acid) also enhanced hSlo-α + β2 currents and slowed inactivation, whereas saturated fatty acids (palmitic, stearic, and caprylic acid) were without effect. Pretreatment with trypsin to remove the cytosolic inactivation domain largely occluded AA action. Intracellularly applied free synthetic β2-ball peptide induced inactivation of the hSlo-α current, and AA failed to enhance this current and slow the inactivation. These results suggest that AA removes inactivation by interacting, possibly through conformational changes, with β2 to prevent the inactivation ball from reaching its receptor. Our data reveal a novel mechanism of β-subunit–dependent modulation of BK channels by AA. In freshly dissociated mouse neocortical neurons, AA eliminated a transient component of whole cell K+ currents. BK channel inactivation may be a specific mechanism by which AA and other unsaturated fatty acids influence neuronal death/survival in neuropathological conditions.

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BK Channel Gating Mechanisms: Progresses Toward a Better Understanding of Variants Linked Neurological Diseases

Frontiers in Physiology ◽

10.3389/fphys.2021.762175 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jianmin Cui

Keyword(s):

Neurological Disorders ◽

Recent Progress ◽

Molecular Mechanisms ◽

Neurological Diseases ◽

Bk Channels ◽

Bk Channel ◽

Channel Activation ◽

Gating Mechanisms ◽

Intracellular Ca ◽

Atomistic Structure

The large conductance Ca2+-activated potassium (BK) channel is activated by both membrane potential depolarization and intracellular Ca2+ with distinct mechanisms. Neural physiology is sensitive to the function of BK channels, which is shown by the discoveries of neurological disorders that are associated with BK channel mutations. This article reviews the molecular mechanisms of BK channel activation in response to voltage and Ca2+ binding, including the recent progress since the publication of the atomistic structure of the whole BK channel protein, and the neurological disorders associated with BK channel mutations. These results demonstrate the unique mechanisms of BK channel activation and that these mechanisms are important factors in linking BK channel mutations to neurological disorders.

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The BK channel gating ring is strongly coupled to the voltage sensor

10.1101/381319 ◽

2018 ◽

Author(s):

Pablo Miranda ◽

Miguel Holmgren ◽

Teresa Giraldez

Keyword(s):

Conformational Changes ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

Voltage Sensitivity ◽

Divalent Ions ◽

Gating Currents ◽

Open Probability ◽

Voltage Dependent ◽

Tightly Coupled

ABSTRACTThe open probability of large conductance voltage- and calcium-dependent potassium (BK) channels is regulated allosterically by changes in the transmembrane voltage and intracellular concentration of divalent ions (Ca2+ and Mg2+). The divalent cation sensors reside within the gating ring formed by eight Regulator of Conductance of Potassium (RCK) domains, two from each of the four channel subunits. Overall, the gating ring contains 12 sites that can bind Ca2+ with different affinities. Using patch-clamp fluorometry, we have shown robust changes in FRET signals within the gating ring in response to divalent ions and voltage, which do not directly track open probability. Only the conformational changes triggered through the RCK1 binding site are voltage-dependent in presence of Ca2+. Because the gating ring is outside the electric field, it must gain voltage sensitivity from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of voltage sensor dynamics known to shift gating currents produce a cognate shift in the gating ring voltage dependence, whereas changing BK channels’ relative probability of opening had little effect. These results strongly suggest that the conformational changes of the RCK1 domain of the gating ring are tightly coupled to the voltage sensor function, and this interaction is central to the allosteric modulation of BK channels.

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Molecular mechanism of BK channel activation by the smooth muscle relaxant NS11021

The Journal of General Physiology ◽

10.1085/jgp.201912506 ◽

2020 ◽

Vol 152 (6) ◽

Cited By ~ 1

Author(s):

Michael E. Rockman ◽

Alexandre G. Vouga ◽

Brad S. Rothberg

Keyword(s):

Smooth Muscle ◽

Muscle Relaxant ◽

Time Course ◽

Bk Channels ◽

Bk Channel ◽

Neurological Deficits ◽

Open Probability ◽

Channel Activation ◽

Voltage Sensors ◽

Smooth Muscle Relaxant

Large-conductance Ca2+-activated K+ channels (BK channels) are activated by cytosolic calcium and depolarized membrane potential under physiological conditions. Thus, these channels control electrical excitability in neurons and smooth muscle by gating K+ efflux and hyperpolarizing the membrane in response to Ca2+ signaling. Altered BK channel function has been linked to epilepsy, dyskinesia, and other neurological deficits in humans, making these channels a key target for drug therapies. To gain insight into mechanisms underlying pharmacological modulation of BK channel gating, here we studied mechanisms underlying activation of BK channels by the biarylthiourea derivative, NS11021, which acts as a smooth muscle relaxant. We observe that increasing NS11021 shifts the half-maximal activation voltage for BK channels toward more hyperpolarized voltages, in both the presence and nominal absence of Ca2+, suggesting that NS11021 facilitates BK channel activation primarily by a mechanism that is distinct from Ca2+ activation. 30 µM NS11021 slows the time course of BK channel deactivation at −200 mV by ∼10-fold compared with 0 µM NS11021, while having little effect on the time course of activation. This action is most pronounced at negative voltages, at which the BK channel voltage sensors are at rest. Single-channel kinetic analysis further shows that 30 µM NS11021 increases open probability by 62-fold and increases mean open time from 0.15 to 0.52 ms in the nominal absence of Ca2+ at voltages less than −60 mV, conditions in which BK voltage sensors are largely in the resting state. We could therefore account for the major activating effects of NS11021 by a scheme in which the drug primarily shifts the pore-gate equilibrium toward the open state.

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Spontaneous Transient Outward Currents Arise from Microdomains Where BK Channels Are Exposed to a Mean Ca2+ Concentration on the Order of 10 μM during a Ca2+ Spark

The Journal of General Physiology ◽

10.1085/jgp.20028571 ◽

2002 ◽

Vol 120 (1) ◽

pp. 15-27 ◽

Cited By ~ 81

Author(s):

Ronghua ZhuGe ◽

Kevin E. Fogarty ◽

Richard A. Tuft ◽

John V. Walsh

Keyword(s):

Voltage Dependence ◽

Ryanodine Receptors ◽

Bk Channels ◽

Bk Channel ◽

Channel Activation ◽

Membrane Area ◽

Outward Currents ◽

Cell Recording ◽

Excised Patches ◽

Spontaneous Transient Outward Currents

Ca2+ sparks are small, localized cytosolic Ca2+ transients due to Ca2+ release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca2+ sparks activate large conductance Ca2+-activated K+ channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca2+ to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g(STOC)), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca2+]s. The Ca2+ sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g(STOC) remained roughly constant from 20 to −40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca2+] on the order of 10 μM during a Ca2+ spark. The membrane area over which a concentration ≥10 μM is reached has an estimated radius of 150–300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca2+ current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

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Closed state-coupled C-type inactivation in BK channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1607584113 ◽

2016 ◽

Vol 113 (25) ◽

pp. 6991-6996 ◽

Cited By ~ 2

Author(s):

Jiusheng Yan ◽

Qin Li ◽

Richard W. Aldrich

Keyword(s):

Conformational Changes ◽

Bk Channels ◽

Bk Channel ◽

Membrane Potentials ◽

Selectivity Filter ◽

Open Probability ◽

Closed State ◽

State Dependent ◽

Channel Opening ◽

Activation Gate

Ion channels regulate ion flow by opening and closing their pore gates. K+ channels commonly possess two pore gates, one at the intracellular end for fast channel activation/deactivation and the other at the selectivity filter for slow C-type inactivation/recovery. The large-conductance calcium-activated potassium (BK) channel lacks a classic intracellular bundle-crossing activation gate and normally show no C-type inactivation. We hypothesized that the BK channel’s activation gate may spatially overlap or coexist with the C-type inactivation gate at or near the selectivity filter. We induced C-type inactivation in BK channels and studied the relationship between activation/deactivation and C-type inactivation/recovery. We observed prominent slow C-type inactivation/recovery in BK channels by an extreme low concentration of extracellular K+ together with a Y294E/K/Q/S or Y279F mutation whose equivalent in Shaker channels (T449E/K/D/Q/S or W434F) caused a greatly accelerated rate of C-type inactivation or constitutive C-inactivation. C-type inactivation in most K+ channels occurs upon sustained membrane depolarization or channel opening and then recovers during hyperpolarized membrane potentials or channel closure. However, we found that the BK channel C-type inactivation occurred during hyperpolarized membrane potentials or with decreased intracellular calcium ([Ca2+]i) and recovered with depolarized membrane potentials or elevated [Ca2+]i. Constitutively open mutation prevented BK channels from C-type inactivation. We concluded that BK channel C-type inactivation is closed state-dependent and that its extents and rates inversely correlate with channel-open probability. Because C-type inactivation can involve multiple conformational changes at the selectivity filter, we propose that the BK channel’s normal closing may represent an early conformational stage of C-type inactivation.

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Differential Effects of β1 and β2 Subunits on BK Channel Activity

The Journal of General Physiology ◽

10.1085/jgp.200409236 ◽

2005 ◽

Vol 125 (4) ◽

pp. 395-411 ◽

Cited By ~ 97

Author(s):

Patricio Orio ◽

Ramon Latorre

Keyword(s):

Steady State ◽

Calcium Binding ◽

Voltage Dependence ◽

Calcium Sensitivity ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensitivity ◽

Channel Activation ◽

Α Subunit ◽

Voltage Sensors

High conductance, calcium- and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (β) subunits. β1 and β2 subunits increase apparent channel calcium sensitivity. The β1 subunit also decreases the voltage sensitivity of the channel and the β2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the β1 and β2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a β2 subunit without its N-type inactivation domain (β2IR). The results indicate that the β2IR subunit, like the β1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the β1 subunit, the β2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied β subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the β1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both β subunits reduce the intrinsic equilibrium constant for channel opening (L0). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the β1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the α subunit is coexpressed with the β2IR subunit.

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Cadmium–cysteine coordination in the BK inner pore region and its structural and functional implications

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1500953112 ◽

2015 ◽

Vol 112 (16) ◽

pp. 5237-5242 ◽

Cited By ~ 11

Author(s):

Yu Zhou ◽

Xiao-Ming Xia ◽

Christopher J. Lingle

Keyword(s):

Conformational Changes ◽

State Dependence ◽

Bk Channels ◽

Bk Channel ◽

Pore Region ◽

Kv Channel ◽

Kv Channels ◽

Voltage Dependent ◽

Channel Structures ◽

Inner Pore

To probe structure and gating-associated conformational changes in BK-type potassium (BK) channels, we examined consequences of Cd2+ coordination with cysteines introduced at two positions in the BK inner pore. At V319C, the equivalent of valine in the conserved Kv proline-valine-proline (PVP) motif, Cd2+ forms intrasubunit coordination with a native glutamate E321, which would place the side chains of V319C and E321 much closer together than observed in voltage-dependent K+ (Kv) channel structures, requiring that the proline between V319C and E321 introduces a kink in the BK S6 inner helix sharper than that observed in Kv channel structures. At inner pore position A316C, Cd2+ binds with modest state dependence, suggesting the absence of an ion permeation gate at the cytosolic side of BK channel. These results highlight fundamental structural differences between BK and Kv channels in their inner pore region, which likely underlie differences in voltage-dependent gating between these channels.

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Recognition of histone H3 methylation states by the PHD1 domain of histone demethylase KDM5A

10.1101/715474 ◽

2019 ◽

Author(s):

James E Longbotham ◽

Mark J S Kelly ◽

Danica Galonić Fujimori

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Solution Structure ◽

Structural Information ◽

Protein Complexes ◽

Histone H3 ◽

Histone Demethylase ◽

Helical Conformation ◽

Regulatory Function ◽

Small Molecule Modulators

AbstractPHD reader domains are chromatin binding modules often responsible for the recruitment of large protein complexes that contain histone modifying enzymes, chromatin remodelers and DNA repair machinery. A majority of PHD domains recognize N–terminal residues of histone H3 and are sensitive to the methylation state of Lys4 in histone H3 (H3K4). Histone demethylase KDM5A, an epigenetic eraser enzyme that contains three PHD domains, is often overexpressed in various cancers and its demethylation activity is allosterically enhanced when its PHD1 domain is bound to the H3 tail. The allosteric regulatory function of PHD1 expands roles of reader domains, suggesting unique features of this chromatin interacting module. Our previous studies determined the H3 binding site of PHD1, although it remains unclear how the H3 tail interacts with the N–terminal residues of PHD1 and how PHD1 discriminates against H3 tails with varying degrees of H3K4 methylation. Here we have determined the solution structure of apo and H3 bound PHD1. We observe conformational changes occurring in PHD1 in order to accommodate H3, which interestingly binds in a helical conformation. We also observe differential interactions of binding residues with differently methylated H3K4 peptides (me0, me1, me2 or me3), providing a rational for this PHD1 domain’s preference for lower methylation states of H3K4. We further assessed the contributions of various H3 interacting residues in the PHD1 domain to the binding of H3 peptides. The structural information of the H3 binding site could provide useful information to aid development of allosteric small molecule modulators of KDM5A.

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