β1-subunit–induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel

Large-conductance Ca2+- and voltage-activated K+ (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit–induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.

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Regulatory γ1 subunits defy symmetry in functional modulation of BK channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1804560115 ◽

2018 ◽

Vol 115 (40) ◽

pp. 9923-9928 ◽

Cited By ~ 3

Author(s):

Vivian Gonzalez-Perez ◽

Manu Ben Johny ◽

Xiao-Ming Xia ◽

Christopher J. Lingle

Keyword(s):

Single Channel ◽

Regulatory Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Bk Channels ◽

Bk Channel ◽

Single Type ◽

Regulatory Subunits ◽

And Function

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, β and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four β subunits per channel, with each β subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+channel.

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An improved open-channel structure of MscL determined from FRET confocal microscopy and simulation

The Journal of General Physiology ◽

10.1085/jgp.200910376 ◽

2010 ◽

Vol 136 (4) ◽

pp. 483-494 ◽

Cited By ~ 64

Author(s):

Ben Corry ◽

Annette C. Hurst ◽

Prithwish Pal ◽

Takeshi Nomura ◽

Paul Rigby ◽

...

Keyword(s):

Patch Clamp ◽

Conformational Changes ◽

Brownian Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Channel Structure ◽

Paramagnetic Resonance ◽

Physiological Processes ◽

Lipid Environment ◽

Dynamics Simulations

Mechanosensitive channels act as molecular transducers of mechanical force exerted on the membrane of living cells by opening in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing, blood pressure regulation, and osmoregulation. Here, we determine the likely structure of the open state of the mechanosensitive channel of large conductance using a combination of patch clamp, fluorescence resonance energy transfer (FRET) spectroscopy, data from previous electron paramagnetic resonance experiments, and molecular and Brownian dynamics simulations. We show that structural rearrangements of the protein can be measured in similar conditions as patch clamp recordings while controlling the state of the pore in its natural lipid environment by modifying the lateral pressure distribution via the lipid bilayer. Transition to the open state is less dramatic than previously proposed, while the N terminus remains anchored at the surface of the membrane where it can either guide the tilt of or directly translate membrane tension to the conformation of the pore-lining helix. Combining FRET data obtained in physiological conditions with simulations is likely to be of great value for studying conformational changes in a range of multimeric membrane proteins.

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Molecular Determinants of BK Channel Functional Diversity and Functioning

Physiological Reviews ◽

10.1152/physrev.00001.2016 ◽

2017 ◽

Vol 97 (1) ◽

pp. 39-87 ◽

Cited By ~ 78

Author(s):

Ramon Latorre ◽

Karen Castillo ◽

Willy Carrasquel-Ursulaez ◽

Romina V. Sepulveda ◽

Fernando Gonzalez-Nilo ◽

...

Keyword(s):

Binding Sites ◽

Muscle Tone ◽

Gene Diversity ◽

Single Gene ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

Channel Structure ◽

Α Subunit ◽

Physiological Importance

Large-conductance Ca2+- and voltage-activated K+ (BK) channels play many physiological roles ranging from the maintenance of smooth muscle tone to hearing and neurosecretion. BK channels are tetramers in which the pore-forming α subunit is coded by a single gene ( Slowpoke, KCNMA1). In this review, we first highlight the physiological importance of this ubiquitous channel, emphasizing the role that BK channels play in different channelopathies. We next discuss the modular nature of BK channel-forming protein, in which the different modules (the voltage sensor and the Ca2+ binding sites) communicate with the pore gates allosterically. In this regard, we review in detail the allosteric models proposed to explain channel activation and how the models are related to channel structure. Considering their extremely large conductance and unique selectivity to K+, we also offer an account of how these two apparently paradoxical characteristics can be understood consistently in unison, and what we have learned about the conduction system and the activation gates using ions, blockers, and toxins. Attention is paid here to the molecular nature of the voltage sensor and the Ca2+ binding sites that are located in a gating ring of known crystal structure and constituted by four COOH termini. Despite the fact that BK channels are coded by a single gene, diversity is obtained by means of alternative splicing and modulatory β and γ subunits. We finish this review by describing how the association of the α subunit with β or with γ subunits can change the BK channel phenotype and pharmacology.

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Role of Charged Residues in the S1–S4 Voltage Sensor of BK Channels

The Journal of General Physiology ◽

10.1085/jgp.200509421 ◽

2006 ◽

Vol 127 (3) ◽

pp. 309-328 ◽

Cited By ~ 109

Author(s):

Zhongming Ma ◽

Xing Jian Lou ◽

Frank T. Horrigan

Keyword(s):

Voltage Dependence ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

Voltage Gating ◽

Voltage Sensing ◽

Kv Channels ◽

Gating Charge ◽

Charged Residues ◽

Sensor Activation

The activation of large conductance Ca2+-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K+ (KV) channels. Yet BK and KV channels share many conserved charged residues in transmembrane segments S1–S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (Po) and IK kinetics (τ(IK)) over an extended voltage range in 0–50 μM [Ca2+]i. mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of PO. The voltage dependence of PO and τ(IK) at extreme negative potentials was also reduced, implying that the closed–open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and KV channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to KV channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1–S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3–7 kcal mol−1, indicating a strong contribution of non–voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.

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Heme Regulates Allosteric Activation of the Slo1 BK Channel

The Journal of General Physiology ◽

10.1085/jgp.200509262 ◽

2005 ◽

Vol 126 (1) ◽

pp. 7-21 ◽

Cited By ~ 66

Author(s):

Frank T. Horrigan ◽

Stefan H. Heinemann ◽

Toshinori Hoshi

Keyword(s):

Divalent Cations ◽

Ionic Currents ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

Gating Currents ◽

Calcium Dependent ◽

Channel Opening ◽

Activation Gate ◽

Voltage Dependent

Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531–535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G–V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G–V simulated by weakening the coupling of both Ca2+ binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca2+- and voltage-dependent gating as well as intrinsic stability of the open state.

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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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Mechanism of β4 Subunit Modulation of BK Channels

The Journal of General Physiology ◽

10.1085/jgp.200509436 ◽

2006 ◽

Vol 127 (4) ◽

pp. 449-465 ◽

Cited By ~ 80

Author(s):

Bin Wang ◽

Brad S. Rothberg ◽

Robert Brenner

Keyword(s):

Calcium Binding ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

Type Ii ◽

Allosteric Coupling ◽

Channel Opening ◽

Sensor Activation ◽

Compensatory Effect ◽

Allosteric Model

Large-conductance (BK-type) Ca2+-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca2+. BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (β1–β4). Biophysical characterization has shown that the β4 subunit confers properties of the so-called “type II” BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the β4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca2+ sensitivity. Specifically, channel activity at low Ca2+ is inhibited, while at high Ca2+, activity is enhanced. The goal of this study is to understand the mechanism underlying β4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that β4's most profound effect is a decrease in Po (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, β4 promotes channel opening by increasing voltage dependence of Po-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of β4 on BK channels. β4 reduces channel opening by decreasing the intrinsic gating equilibrium (L0), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, β4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vho) to more negative membrane potentials. The consequence is that β4 causes a net positive shift of the G-V relationship (relative to α subunit alone) at low calcium. At higher calcium, the contribution by Vho and an increase in allosteric coupling to Ca2+ binding (C) promotes a negative G-V shift of α+β4 channels as compared to α subunits alone. This manner of modulation predicts that type II BK channels are downregulated by β4 at resting voltages through effects on L0. However, β4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.

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Molecular structure and function of big calcium-activated potassium channels in skeletal muscle: pharmacological perspectives

Physiological Genomics ◽

10.1152/physiolgenomics.00121.2016 ◽

2017 ◽

Vol 49 (6) ◽

pp. 306-317 ◽

Cited By ~ 6

Author(s):

Fatima Maqoud ◽

Michela Cetrone ◽

Antonietta Mele ◽

Domenico Tricarico

Keyword(s):

Skeletal Muscle ◽

Alternative Splicing ◽

Mammalian Cells ◽

Electrical Potential ◽

Smooth Muscles ◽

Periodic Paralysis ◽

Bk Channels ◽

Bk Channel ◽

Α Subunit ◽

Channel Diversity

The large-conductance Ca2+-activated K+ (BK) channel is broadly expressed in various mammalian cells and tissues such as neurons, skeletal muscles (sarco-BK), and smooth muscles. These channels are activated by changes in membrane electrical potential and by increases in the concentration of intracellular calcium ion (Ca2+). The BK channel is subjected to many mechanisms that add diversity to the BK channel α-subunit gene. These channels are indeed subject to alternative splicing, auxiliary subunits modulation, posttranslational modifications, and protein-protein interactions. BK channels can be modulated by diverse molecules that may induce either an increase or decrease in channel activity. The linkage of these channels to many intracellular metabolites and pathways, as well as their modulation by extracellular natural agents, have been found to be relevant in many physiological processes. BK channel diversity is obtained by means of alternative splicing and modulatory β- and γ-subunits. The association of the α-subunit with β- or with γ-subunits can change the BK channel phenotype, functional diversity, and pharmacological properties in different tissues. In the case of the skeletal muscle BK channel (sarco-BK channel), we established that the main mechanism regulating BK channel diversity is the alternative splicing of the KCNMA1/slo1 gene encoding for the α-subunit generating different splicing isoform in the muscle phenotypes. This finding helps to design molecules selectively targeting the skeletal muscle subtypes. The use of drugs selectively targeting the skeletal muscle BK channels is a promising strategy in the treatment of familial disorders affecting muscular skeletal apparatus including hyperkalemia and hypokalemia periodic paralysis.

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Akt1 mediates purinergic-dependent NOS3 activation in thick ascending limbs

AJP Renal Physiology ◽

10.1152/ajprenal.00270.2009 ◽

2009 ◽

Vol 297 (3) ◽

pp. F646-F652 ◽

Cited By ~ 16

Author(s):

Guillermo B. Silva ◽

Jeffrey L. Garvin

Keyword(s):

Kinase Inhibitor ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Pi3 Kinase ◽

Dominant Negative ◽

Wild Type ◽

Akt Isoforms ◽

Physiological Processes ◽

Nephron Segment

Extracellular ATP regulates many physiological processes via release of nitric oxide (NO). ATP stimulates NO in thick ascending limbs (TALs), but the signaling cascade involved in the cells of this nephron segment, as well as many other types of cells, is poorly understood. We hypothesized that ATP enhances NO synthase (NOS) activity by stimulating PI3 kinase and Akt. We measured 1) NO in TALs using the NO-sensitive dye DAF-2 DA and 2) Akt activity by fluorescence resonance energy transfer and phosphorylation of Akt isoforms. ATP (100 μM) stimulated NO in wild-type mice [26 ± 4 arbitrary units (AU)], but not in NOS3 −/− mice (2 ± 2 AU; P < 0.04). In the presence of the NOS1- and NOS2-selective inhibitors 7-NI and 1400W, ATP stimulated NO by 30 ± 2 and 33 ± 3 AU, respectively (not significant vs. control). In the presence of the PI3 kinase inhibitor LY294002, ATP-increased NO was reduced by 85% (5 ± 2 vs. 28 ± 4 AU; P < 0.02). ATP alone increased Akt activity and this effect was significantly blocked by suramin, a P2 receptor antagonist. In the presence of an Akt-selective inhibitor, ATP-induced NO was blocked by 90 ± 4%. ATP significantly stimulated Akt1 phosphorylation at Ser473 by 91 ± 13%, whereas Akt2 phosphorylation remained unchanged and Akt3 phosphorylation decreased. In vivo transduction of TALs with a dominant-negative Akt1 significantly decreased ATP-induced NO by 88 ± 6%. We concluded that ATP increases NOS3-derived NO via Akt1 activation in the TAL.

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Coupling and cooperativity in voltage activation of a limited-state BK channel gating in saturating Ca2+

The Journal of General Physiology ◽

10.1085/jgp.200910331 ◽

2010 ◽

Vol 135 (5) ◽

pp. 461-480 ◽

Cited By ~ 19

Author(s):

Christopher Shelley ◽

Xiaowei Niu ◽

Yanyan Geng ◽

Karl L. Magleby

Keyword(s):

Single Channel ◽

Coupling Factor ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

Channel Kinetics ◽

Voltage Sensors ◽

Gating Mechanisms ◽

Voltage Dependent ◽

Single Channel Currents

Voltage-dependent gating mechanisms of large conductance Ca2+ and voltage-activated (BK) channels were investigated using two-dimensional maximum likelihood analysis of single-channel open and closed intervals. To obtain sufficient data at negative as well as positive voltages, single-channel currents were recorded at saturating Ca2+ from BK channels mutated to remove the RCK1 Ca2+ and Mg2+ sensors. The saturating Ca2+ acting on the Ca2+ bowl sensors of the resulting BKB channels increased channel activity while driving the gating into a reduced number of states, simplifying the model. Five highly constrained idealized gating mechanisms based on extensions of the Monod-Wyman-Changeux model for allosteric proteins were examined. A 10-state model without coupling between the voltage sensors and the opening/closing transitions partially described the voltage dependence of Po but not the single-channel kinetics. With allowed coupling, the model gave improved descriptions of Po and approximated the single-channel kinetics; each activated voltage sensor increased the opening rate approximately an additional 23-fold while having little effect on the closing rate. Allowing cooperativity among voltage sensors further improved the description of the data: each activated voltage sensor increased the activation rate of the remaining voltage sensors approximately fourfold, with little effect on the deactivation rate. The coupling factor was decreased in models with cooperativity from ∼23 to ∼18. Whether the apparent cooperativity among voltage sensors arises from imposing highly idealized models or from actual cooperativity will require additional studies to resolve. For both cooperative and noncooperative models, allowing transitions to five additional brief (flicker) closed states further improved the description of the data. These observations show that the voltage-dependent single-channel kinetics of BKB channels can be approximated by highly idealized allosteric models in which voltage sensor movement increases Po mainly through an increase in channel opening rates, with limited effects on closing rates.

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