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

2015 ◽  
Vol 112 (16) ◽  
pp. 5237-5242 ◽  
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.

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

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.

scholarly journals Voltage-dependent dynamics of the BK channel cytosolic gating ring are coupled to the membrane-embedded voltage sensor

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Pablo Miranda ◽  
Miguel Holmgren ◽  
Teresa Giraldez
Keyword(s):  
Conformational Changes ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Cation Binding ◽  
Voltage Sensitivity ◽  
Open Probability ◽  
Calcium Dependent ◽  
Transmembrane Voltage ◽  
Voltage Dependent

In humans, large conductance voltage- and calcium-dependent potassium (BK) channels are regulated allosterically by transmembrane voltage and intracellular Ca2+. Divalent cation binding sites reside within the gating ring formed by two Regulator of Conductance of Potassium (RCK) domains per subunit. Using patch-clamp fluorometry, we show that Ca2+ binding to the RCK1 domain triggers gating ring rearrangements that depend on transmembrane voltage. Because the gating ring is outside the electric field, this voltage sensitivity must originate from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of the voltage sensor, either by mutagenesis or regulation by auxiliary subunits, are paralleled by changes in the voltage dependence of the gating ring movements, whereas modifications of the relative open probability are not. These results strongly suggest that conformational changes of RCK1 domains are specifically coupled to the voltage sensor function during allosteric modulation of BK channels.

scholarly journals The functionally relevant site for paxilline inhibition of BK channels

2019 ◽  
Vol 117 (2) ◽  
pp. 1021-1026
Author(s):  
Yu Zhou ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Computational Analysis ◽  
Open Channel ◽  
Specific Inhibitor ◽  
State Dependence ◽  
Functional Results ◽  
Bk Channels ◽  
Bk Channel ◽  
Central Cavity ◽  
Type K ◽  
Channel Structures

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.

scholarly journals Charge substitution for a deep-pore residue reveals structural dynamics during BK channel gating

2011 ◽  
Vol 138 (2) ◽  
pp. 137-154 ◽  
Author(s):  
Xixi Chen ◽  
Richard W. Aldrich
Keyword(s):  
Amino Acids ◽  
Structural Dynamics ◽  
Conformational Changes ◽  
Channel Gating ◽  
Bk Channels ◽  
Bk Channel ◽  
Pore Region ◽  
Channel Opening ◽  
Essential Components ◽  
Ion Channel Proteins

The pore-lining amino acids of ion channel proteins reside on the interface between a polar (the pore) and a nonpolar environment (the rest of the protein). The structural dynamics of this region, which physically controls ionic flow, are essential components of channel gating. Using large-conductance, Ca2+-dependent K+ (BK) channels, we devised a systematic charge–substitution method to probe conformational changes in the pore region during channel gating. We identified a deep-pore residue (314 in hSlo1) as a marker of structural dynamics. We manipulated the charge states of this residue by substituting amino acids with different valence and pKa, and by adjusting intracellular pH. We found that the charged states of the 314 residues stabilized an open state of the BK channel. With models based on known structures of related channels, we postulate a dynamic rearrangement of the deep-pore region during BK channel opening/closing, which involves a change of the degree of pore exposure for 314.

scholarly journals The liverwort Marchantia polymorpha operates a depolarization-activated Slowpoke (SLO) K+ channel that recognises pH changes in the environment

2021 ◽  
Author(s):  
Frances C. Sussmilch ◽  
Jennifer Boehm ◽  
Guido Gessner ◽  
Tobias Maierhofer ◽  
Thomas D. Mueller ◽  
...  
Keyword(s):  
Critical Role ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Marchantia Polymorpha ◽  
Terrestrial Environment ◽  
Ph Changes ◽  
Kv Channels ◽  
Voltage Dependent ◽  
Genes Encoding

Voltage-dependent ion channels are a prerequisite for cellular excitability and electrical communication - important traits for multicellular organisms to thrive in a changeable terrestrial environment. Based on their presence in extant embryophytes and closely-related green algae, the first plants to survive on land likely possessed genes encoding channels with homology to large-conductance calcium-activated K+ channels (BK channels from the Slo family) in addition to primary voltage-gated potassium channels from the plant VG-type family (Shaker or Kv channels). While the function and gating of Shaker channels has been characterised in flowering plants, so far knowledge of BK channels has been limited to animal models. In humans, BK-mediated K+ efflux has a critical role in sperm motility and membrane polarisation to enable fertilisation. In the liverwort Marchantia polymorpha, the MpBK2a channel gene is most highly expressed in male reproductive tissue, suggesting that these channels may function in sexual reproduction. We characterised MpBK2a channels and found them to be strongly K+-selective, outward-rectifying, 80-pS channels capable of repolarising the membrane after stimulus-dependent depolarisation. In contrast to its animal counterpart, MpBK2a is insensitive to cytoplasmic Ca2+ variations but effectively gated by pH changes. Given that this plant BK channel is active even in the presence of trace amounts of external K+ and at low pH, the liverwort channel could have stabilised the membrane potential under stressful pre-historic conditions including nutrient-depleted and acid environments as early plant pioneers conquered land.

scholarly journals BK channel inhibition by strong extracellular acidification

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Yu Zhou ◽  
Xiao-Ming Xia ◽  
Christopher J Lingle
Keyword(s):  
Bk Channels ◽  
Bk Channel ◽  
Extracellular Acidification ◽  
Channel Inhibition ◽  
Wide Range ◽  
The Family ◽  
Voltage Dependent ◽  
Intracellular Organelles ◽  
Extracellular Side ◽  
Key Residues

Mammalian BK-type voltage- and Ca2+-dependent K+ channels are found in a wide range of cells and intracellular organelles. Among different loci, the composition of the extracellular microenvironment, including pH, may differ substantially. For example, it has been reported that BK channels are expressed in lysosomes with their extracellular side facing the strongly acidified lysosomal lumen (pH ~4.5). Here we show that BK activation is strongly and reversibly inhibited by extracellular H+, with its conductance-voltage relationship shifted by more than +100 mV at pHO 4. Our results reveal that this inhibition is mainly caused by H+ inhibition of BK voltage-sensor (VSD) activation through three acidic residues on the extracellular side of BK VSD. Given that these key residues (D133, D147, D153) are highly conserved among members in the voltage-dependent cation channel superfamily, the mechanism underlying BK inhibition by extracellular acidification might also be applicable to other members in the family.

scholarly journals LRRC52 regulates BK channel function and localization in mouse cochlear inner hair cells

2019 ◽  
Vol 116 (37) ◽  
pp. 18397-18403 ◽  
Author(s):  
Christopher J. Lingle ◽  
Pedro L. Martinez-Espinosa ◽  
Aizhen Yang-Hood ◽  
Luis E. Boero ◽  
Shelby Payne ◽  
...  
Keyword(s):  
Hair Cells ◽  
Glutamate Release ◽  
Nerve Fibers ◽  
Bk Channels ◽  
Bk Channel ◽  
Membrane Potentials ◽  
Macromolecular Complex ◽  
Inner Hair Cells ◽  
Channel Function ◽  
Voltage Dependent

The perception of sound relies on sensory hair cells in the cochlea that convert the mechanical energy of sound into release of glutamate onto postsynaptic auditory nerve fibers. The hair cell receptor potential regulates the strength of synaptic transmission and is shaped by a variety of voltage-dependent conductances. Among these conductances, the Ca2+- and voltage-activated large conductance Ca2+-activated K+channel (BK) current is prominent, and in mammalian inner hair cells (IHCs) displays unusual properties. First, BK currents activate at unprecedentedly negative membrane potentials (−60 mV) even in the absence of intracellular Ca2+elevations. Second, BK channels are positioned in clusters away from the voltage-dependent Ca2+channels that mediate glutamate release from IHCs. Here, we test the contributions of two recently identified leucine-rich-repeat–containing (LRRC) regulatory γ subunits, LRRC26 and LRRC52, to BK channel function and localization in mouse IHCs. Whereas BK currents and channel localization were unaltered in IHCs fromLrrc26knockout (KO) mice, BK current activation was shifted more than +200 mV in IHCs fromLrrc52KO mice. Furthermore, the absence of LRRC52 disrupted BK channel localization in the IHCs. Given that heterologous coexpression of LRRC52 with BK α subunits shifts BK current gating about −90 mV, to account for the profound change in BK activation range caused by removal of LRRC52, we suggest that additional factors may help define the IHC BK gating range. LRRC52, through stabilization of a macromolecular complex, may help retain some other components essential both for activation of BK currents at negative membrane potentials and for appropriate BK channel positioning.

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

2007 ◽  
Vol 97 (1) ◽  
pp. 62-69 ◽  
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.

scholarly journals Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels

2014 ◽  
Vol 144 (5) ◽  
pp. 457-467 ◽  
Author(s):  
Sandipan Chowdhury ◽  
Benjamin M. Haehnel ◽  
Baron Chanda
Keyword(s):  
Potassium Channels ◽  
Conformational Changes ◽  
Electromechanical Coupling ◽  
Structural Integrity ◽  
Voltage Sensor ◽  
Amino Acid Residues ◽  
Kv Channel ◽  
Electromechanical Transduction ◽  
Voltage Dependent ◽  
Graph Theoretical Approach

Voltage-dependent potassium channels play a crucial role in electrical excitability and cellular signaling by regulating potassium ion flux across membranes. Movement of charged residues in the voltage-sensing domain leads to a series of conformational changes that culminate in channel opening in response to changes in membrane potential. However, the molecular machinery that relays these conformational changes from voltage sensor to the pore is not well understood. Here we use generalized interaction-energy analysis (GIA) to estimate the strength of site-specific interactions between amino acid residues putatively involved in the electromechanical coupling of the voltage sensor and pore in the outwardly rectifying KV channel. We identified candidate interactors at the interface between the S4–S5 linker and the pore domain using a structure-guided graph theoretical approach that revealed clusters of conserved and closely packed residues. One such cluster, located at the intracellular intersubunit interface, comprises three residues (arginine 394, glutamate 395, and tyrosine 485) that interact with each other. The calculated interaction energies were 3–5 kcal, which is especially notable given that the net free-energy change during activation of the Shaker KV channel is ∼14 kcal. We find that this triad is delicately maintained by balance of interactions that are responsible for structural integrity of the intersubunit interface while maintaining sufficient flexibility at a critical gating hinge for optimal transmission of force to the pore gate.

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