Mechanism of Increased Open Probability by a Mutation of the BK Channel

2006 ◽  
Vol 96 (3) ◽  
pp. 1507-1516 ◽  
Author(s):  
Ana Díez-Sampedro ◽  
William R. Silverman ◽  
Jocelyn F. Bautista ◽  
George B. Richerson
Keyword(s):  
Bk Channel ◽  
Single Channels ◽  
Voltage Sensitivity ◽  
Wild Type ◽  
Open Probability ◽  
Mutant Channel ◽  
Α Subunit ◽  
Brain Excitability ◽  
The Difference ◽  
Channel Properties

A missense mutation (D434G) has recently been identified in the α subunit of the human large-conductance calcium-activated potassium (BK) channel. Interestingly, although the mutation causes an increase in open probability, individuals that carry the mutation have epilepsy and/or paroxysmal dyskinesia, disorders of increased brain excitability. To define the mechanisms of the mutation, we have used recordings from single channels and measurement of macroscopic conductances to examine the gating of the α subunit, modulation by the regulatory β4 subunit, and the effect of Mg2+ on channel properties. Although there was relatively little difference in open dwell times for the mutant and wild-type α subunits, the mutant channel spent less time in a long-lived closed state. Co-expression of the β4 subunit caused the wild-type channel to be less sensitive to calcium at low Ca2+ concentrations but had little effect on the mutant channel, further accentuating the difference between the wild-type and the mutant channels. In the absence of Ca2+, there was no difference in Mg2+ or voltage sensitivity of the mutant and wild-type channels, whereas in 2 mM Ca2+, the mutant channel had greater open probability at every Mg2+ concentration tested. We conclude that the D434G mutation modifies Ca2+-dependent activation, but we find no evidence of a direct effect on activation by Mg2+ or voltage. The resulting enhancement of BK channel function leads to an increase in brain excitability, possibly due to more rapid repolarization of action potentials.

scholarly journals BK in Double-Membrane Organelles: A Biophysical, Pharmacological, and Functional Survey

2021 ◽  
Vol 12 ◽  
Author(s):  
Naileth González-Sanabria ◽  
Felipe Echeverría ◽  
Ignacio Segura ◽  
Rosangelina Alvarado-Sánchez ◽  
Ramon Latorre
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Potassium Channel ◽  
Lipid Bilayers ◽  
Bk Channels ◽  
Bk Channel ◽  
Inner Mitochondrial Membrane ◽  
Modular Architecture ◽  
Open Probability ◽  
Α Subunit

In the 1970s, calcium-activated potassium currents were recorded for the first time. In 10years, this Ca2+-activated potassium channel was identified in rat skeletal muscle, chromaffin cells and characterized in skeletal muscle membranes reconstituted in lipid bilayers. This calcium- and voltage-activated potassium channel, dubbed BK for “Big K” due to its large ionic conductance between 130 and 300 pS in symmetric K+. The BK channel is a tetramer where the pore-forming α subunit contains seven transmembrane segments. It has a modular architecture containing a pore domain with a highly potassium-selective filter, a voltage-sensor domain and two intracellular Ca2+ binding sites in the C-terminus. BK is found in the plasma membrane of different cell types, the inner mitochondrial membrane (mitoBK) and the nuclear envelope’s outer membrane (nBK). Like BK channels in the plasma membrane (pmBK), the open probability of mitoBK and nBK channels are regulated by Ca2+ and voltage and modulated by auxiliary subunits. BK channels share common pharmacology to toxins such as iberiotoxin, charybdotoxin, paxilline, and agonists of the benzimidazole family. However, the precise role of mitoBK and nBK remains largely unknown. To date, mitoBK has been reported to play a role in protecting the heart from ischemic injury. At the same time, pharmacology suggests that nBK has a role in regulating nuclear Ca2+, membrane potential and expression of eNOS. Here, we will discuss at the biophysical level the properties and differences of mitoBK and nBK compared to those of pmBK and their pharmacology and function.

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 Differential Effects of β1 and β2 Subunits on BK Channel Activity

2005 ◽  
Vol 125 (4) ◽  
pp. 395-411 ◽  
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.

scholarly journals Structural Determinants for Functional Coupling Between the β and α Subunits in the Ca2+-activated K+ (BK) Channel

2006 ◽  
Vol 127 (2) ◽  
pp. 191-204 ◽  
Author(s):  
Patricio Orio ◽  
Yolima Torres ◽  
Patricio Rojas ◽  
Ingrid Carvacho ◽  
Maria L. Garcia ◽  
...  
Keyword(s):  
Calcium Sensitivity ◽  
Bk Channels ◽  
Bk Channel ◽  
Fine Tuning ◽  
Functional Coupling ◽  
Xenopus Laevis Oocytes ◽  
Voltage Sensitivity ◽  
Patch Clamp Technique ◽  
Α Subunit ◽  
Intracellular Domains

High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (β) subunits. The most remarkable effects of β1 and β2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by α and β1 or β2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of β1 but not β2. Here we reveal the molecular regions in these β subunits that determine their differential functional coupling with the pore-forming α-subunit. We made chimeric constructs between β1 and β2 subunits, and BK channels formed by α and chimeric β subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the β1 and β2 subunits demonstrates that the NH3 and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these β subunits. Moreover, the intracellular domains of β1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the α-subunit to be the target of the modulation by the β1-subunit.

scholarly journals Enhanced large conductance K+ channel activity contributes to the impaired myogenic response in the cerebral vasculature of Fawn Hooded Hypertensive rats

2014 ◽  
Vol 306 (7) ◽  
pp. H989-H1000 ◽  
Author(s):  
Mallikarjuna R. Pabbidi ◽  
Olga Mazur ◽  
Fan Fan ◽  
Jerry M. Farley ◽  
Debebe Gebremedhin ◽  
...  
Keyword(s):  
Congenic Strain ◽  
Cerebral Arteries ◽  
Bk Channel ◽  
Chromosome 1 ◽  
Brown Norway ◽  
K Channel ◽  
Myogenic Response ◽  
Voltage Sensitivity ◽  
Channel Activity ◽  
Open Probability

Recent studies have indicated that the myogenic response (MR) in cerebral arteries is impaired in Fawn Hooded Hypertensive (FHH) rats and that transfer of a 2.4 megabase pair region of chromosome 1 (RNO1) containing 15 genes from the Brown Norway rat into the FHH genetic background restores MR in a FHH.1BN congenic strain. However, the mechanisms involved remain to be determined. The present study examined the role of the large conductance calcium-activated potassium (BK) channel in impairing the MR in FHH rats. Whole-cell patch-clamp studies of cerebral vascular smooth muscle cells (VSMCs) revealed that iberiotoxin (IBTX; BK inhibitor)-sensitive outward potassium (K+) channel current densities are four- to fivefold greater in FHH than in FHH.1BN congenic strain. Inside-out patches indicated that the BK channel open probability ( NP o) is 10-fold higher and IBTX reduced NP o to a greater extent in VSMCs isolated from FHH than in FHH.1BN rats. Voltage sensitivity of the BK channel is enhanced in FHH as compared with FHH.1BN rats. The frequency and amplitude of spontaneous transient outward currents are significantly greater in VSMCs isolated from FHH than in FHH.1BN rats. However, the expression of the BK-α and -β-subunit proteins in cerebral vessels as determined by Western blot is similar between the two groups. Middle cerebral arteries (MCAs) isolated from FHH rats exhibited an impaired MR, and administration of IBTX restored this response. These results indicate that there is a gene on RNO1 that impairs MR in the MCAs of FHH rats by enhancing BK channel activity.

scholarly journals The β Subunit Increases the Ca2+ Sensitivity of Large Conductance Ca2+-activated Potassium Channels by Retaining the Gating in the Bursting States

1999 ◽  
Vol 113 (3) ◽  
pp. 425-440 ◽  
Author(s):  
Crina M. Nimigean ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Β Subunit ◽  
Open Probability ◽  
Α Subunit ◽  
Closed Intervals ◽  
Single Channel Analysis ◽  
Channel Analysis ◽  
The Mean

Coexpression of the β subunit (KV,Caβ) with the α subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the β subunit increased open probability (Po) by increasing burst duration 20–100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the β subunit does not act by increasing all the Ca2+ binding rates proportionally. The β subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the β subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.

scholarly journals Single-channel Properties of Human NaV1.1 and Mechanism of Channel Dysfunction in SCN1A-associated Epilepsy

2005 ◽  
Vol 127 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Carlos G. Vanoye ◽  
Christoph Lossin ◽  
Thomas H. Rhodes ◽  
Alfred L. George
Keyword(s):  
Sodium Channel ◽  
Single Channel ◽  
Sodium Current ◽  
Febrile Seizures ◽  
Persistent Sodium Current ◽  
Open Probability ◽  
Α Subunit ◽  
Whole Cell ◽  
Open Time ◽  
Channel Properties

Mutations in genes encoding neuronal voltage-gated sodium channel subunits have been linked to inherited forms of epilepsy. The majority of mutations (>100) associated with generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI) occur in SCN1A encoding the NaV1.1 neuronal sodium channel α-subunit. Previous studies demonstrated functional heterogeneity among mutant SCN1A channels, revealing a complex relationship between clinical and biophysical phenotypes. To further understand the mechanisms responsible for mutant SCN1A behavior, we performed a comprehensive analysis of the single-channel properties of heterologously expressed recombinant WT-SCN1A channels. Based on these data, we then determined the mechanisms for dysfunction of two GEFS+-associated mutations (R1648H, R1657C) both affecting the S4 segment of domain 4. WT-SCN1A has a slope conductance (17 pS) similar to channels found in native mammalian neurons. The mean open time is ∼0.3 ms in the −30 to −10 mV range. The R1648H mutant, previously shown to display persistent sodium current in whole-cell recordings, exhibited similar slope conductance but had an increased probability of late reopening and a subfraction of channels with prolonged open times. We did not observe bursting behavior and found no evidence for a gating mode shift to explain the increased persistent current caused by R1648H. Cells expressing R1657C exhibited conductance, open probability, mean open time, and latency to first opening similar to WT channels but reduced whole-cell current density, suggesting decreased number of functional channels at the plasma membrane. In summary, our findings define single-channel properties for WT-SCN1A, detail the functional phenotypes for two human epilepsy-associated sodium channel mutants, and clarify the mechanism for increased persistent sodium current induced by the R1648H allele.

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 An S6 Mutation in BK Channels Reveals β1 Subunit Effects on Intrinsic and Voltage-dependent Gating

2006 ◽  
Vol 128 (6) ◽  
pp. 731-744 ◽  
Author(s):  
Bin Wang ◽  
Robert Brenner
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Open Probability ◽  
Α Subunit ◽  
Channel Opening ◽  
Sensor Activation ◽  
Intracellular Domains

Large conductance, Ca2+- and voltage-activated K+ (BK) channels are exquisitely regulated to suit their diverse roles in a large variety of physiological processes. BK channels are composed of pore-forming α subunits and a family of tissue-specific accessory β subunits. The smooth muscle–specific β1 subunit has an essential role in regulating smooth muscle contraction and modulates BK channel steady-state open probability and gating kinetics. Effects of β1 on channel's gating energetics are not completely understood. One of the difficulties is that it has not yet been possible to measure the effects of β1 on channel's intrinsic closed-to-open transition (in the absence of voltage sensor activation and Ca2+ binding) due to the very low open probability in the presence of β1. In this study, we used a mutation of the α subunit (F315Y) that increases channel openings by greater than four orders of magnitude to directly compare channels' intrinsic open probabilities in the presence and absence of the β1 subunit. Effects of β1 on steady-state open probabilities of both wild-type α and the F315Y mutation were analyzed using the dual allosteric HA model. We found that mouse β1 has two major effects on channel's gating energetics. β1 reduces the intrinsic closed-to-open equilibrium that underlies the inhibition of BK channel opening seen in submicromolar Ca2+. Further, PO measurements at limiting slope allow us to infer that β1 shifts open channel voltage sensor activation to negative membrane potentials, which contributes to enhanced channel opening seen at micromolar Ca2+ concentrations. Using the F315Y α subunit with deletion mutants of β1, we also demonstrate that the small N- and C-terminal intracellular domains of β1 play important roles in altering channel's intrinsic opening and voltage sensor activation. In summary, these results demonstrate that β1 has distinct effects on BK channel intrinsic gating and voltage sensor activation that can be functionally uncoupled by mutations in the intracellular domains.

scholarly journals NFATc3 regulates BK channel function in murine urinary bladder smooth muscle

2008 ◽  
Vol 295 (3) ◽  
pp. C611-C623 ◽  
Author(s):  
JJ Layne ◽  
ME Werner ◽  
DC Hill-Eubanks ◽  
MT Nelson
Keyword(s):  
Transcription Factor ◽  
Smooth Muscle ◽  
Urinary Bladder ◽  
Bk Channels ◽  
Bk Channel ◽  
Wild Type ◽  
Bladder Smooth Muscle ◽  
Activated T Cells ◽  
Α Subunit ◽  
Urinary Bladder Smooth Muscle

The nuclear factor of activated T-cells (NFAT) is a Ca2+-dependent transcription factor that has been reported to regulate the expression of smooth muscle contractile proteins and ion channels. Here we report that large conductance Ca2+-sensitive potassium (BK) channels and voltage-gated K+ (KV) channels may be regulatory targets of NFATc3 in urinary bladder smooth muscle (UBSM). UBSM myocytes from NFATc3-null mice displayed a reduction in iberiotoxin (IBTX)-sensitive BK currents, a decrease in mRNA for the pore-forming α-subunit of the BK channel, and a reduction in BK channel density compared with myocytes from wild-type mice. Tetraethylammonium chloride-sensitive KV currents were elevated in UBSM myocytes from NFATc3-null mice, as was mRNA for the Shab family member KV2.1. Despite KV current upregulation, bladder strips from NFATc3-null mice displayed an elevated contractile response to electrical field stimulation relative to strips from wild-type mice, but this difference was abrogated in the presence of the BK channel blocker IBTX. These results support a role for the transcription factor NFATc3 in regulating UBSM contractility, primarily through an NFATc3-dependent increase in BK channel activity.

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