Molecular Determinants of BK Channel Functional Diversity and Functioning

2017 ◽  
Vol 97 (1) ◽  
pp. 39-87 ◽  
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.

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

2016 ◽  
Vol 113 (23) ◽  
pp. E3231-E3239 ◽  
Author(s):  
Juan P. Castillo ◽  
Jorge E. Sánchez-Rodríguez ◽  
H. Clark Hyde ◽  
Cristian A. Zaelzer ◽  
Daniel Aguayo ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Channel Structure ◽  
Physiological Processes ◽  
Extracellular Region ◽  
Channel Diversity ◽  
Α Subunits

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.

scholarly journals Calcium-driven regulation of voltage-sensing domains in BK channels

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yenisleidy Lorenzo-Ceballos ◽  
Willy Carrasquel-Ursulaez ◽  
Karen Castillo ◽  
Osvaldo Alvarez ◽  
Ramon Latorre
Keyword(s):  
Binding Sites ◽  
Channel Gating ◽  
Interaction Mechanism ◽  
Bk Channels ◽  
Bk Channel ◽  
Strong Impact ◽  
Gating Currents ◽  
Voltage Sensing ◽  
Α Subunit ◽  
C Terminus

Allosteric interactions between the voltage-sensing domain (VSD), the Ca2+-binding sites, and the pore domain govern the mammalian Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the crosstalk between the Ca2+- and voltage-sensing mechanisms on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation by investigating the effects of internal Ca2+ on BK channel gating currents. Our results indicate that Ca2+ sensor occupancy has a strong impact on VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single α-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between Ca2+ sensors and VSDs.

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 Calcium-driven regulation of voltage-sensing domains in BK channels

2019 ◽  
Author(s):  
Yenisleidy Lorenzo-Ceballos ◽  
Willy Carrasquel-Ursulaez ◽  
Karen Castillo ◽  
Osvaldo Alvarez ◽  
Ramon Latorre
Keyword(s):  
Binding Sites ◽  
Interaction Mechanism ◽  
Bk Channels ◽  
Bk Channel ◽  
Strong Impact ◽  
Gating Currents ◽  
Voltage Sensing ◽  
Α Subunit ◽  
C Terminus ◽  
Voltage Dependent

AbstractAllosteric interplays between voltage-sensing domains (VSD), Ca2+-binding sites, and the pore domain govern the Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the Ca2+- and voltage-sensing mechanisms crosstalk on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation measuring and analyzing the effects of internal Ca2+ on BK channels gating currents. Our results indicate that the Ca2+ sensors occupancy has a strong impact on the VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single α-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, appear to contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between Ca2+ sensors and VSDs.

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 Chronic Prenatal Hypoxia Down-Regulated BK Channel Β1 Subunits in Mesenteric Artery Smooth Muscle Cells of the Offspring

2018 ◽  
Vol 45 (4) ◽  
pp. 1603-1616 ◽  
Author(s):  
Bailin Liu ◽  
Yanping Liu ◽  
Ruixiu Shi ◽  
Xueqin Feng ◽  
Xiang Li ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Bk Channels ◽  
Bk Channel ◽  
Mesenteric Arteries ◽  
Prenatal Hypoxia ◽  
Pregnant Rats ◽  
Α Subunit

Background/Aims: Chronic hypoxia in utero could impair vascular functions in the offspring, underlying mechanisms are unclear. This study investigated functional alteration in large-conductance Ca2+-activated K+ (BK) channels in offspring mesenteric arteries following prenatal hypoxia. Methods: Pregnant rats were exposed to normoxic control (21% O2, Con) or hypoxic (10.5% O2, Hy) conditions from gestational day 5 to 21, their 7-month-old adult male offspring were tested for blood pressure, vascular BK channel functions and expression using patch clamp and wire myograh technique, western blotting, and qRT-PCR. Results: Prenatal hypoxia increased pressor responses and vasoconstrictions to phenylephrine in the offspring. Whole-cell currents density of BK channels and amplitude of spontaneous transient outward currents (STOCs), not the frequency, were significantly reduced in Hy vascular myocytes. The sensitivity of BK channels to voltage, Ca2+, and tamoxifen were reduced in Hy myocytes, whereas the number of channels per patch and the single-channel conductance were unchanged. Prenatal hypoxia impaired NS1102- and tamoxifen-mediated relaxation in mesenteric arteries precontracted with phenylephrine in the presence of Nω-nitro-L-arginine methyl ester. The mRNA and protein expression of BK channel β1, not the α-subunit, was decreased in Hy mesenteric arteries. Conclusions: Impaired BK channel β1-subunits in vascular smooth muscle cells contributed to vascular dysfunction in the offspring exposed to prenatal hypoxia.

scholarly journals Effect of aldosterone on BK channel expression in mammalian cortical collecting duct

2008 ◽  
Vol 295 (3) ◽  
pp. F780-F788 ◽  
Author(s):  
Genevieve Estilo ◽  
Wen Liu ◽  
Nuria Pastor-Soler ◽  
Phillip Mitchell ◽  
Marcelo D. Carattino ◽  
...  
Keyword(s):  
Splice Variants ◽  
Collecting Duct ◽  
Bk Channels ◽  
Bk Channel ◽  
Mrna Abundance ◽  
Cortical Collecting Duct ◽  
Α Subunit ◽  
Channel Expression ◽  
Tubular Flow ◽  
Circulating Levels

Apical large-conductance Ca2+-activated K+ (BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated K+ secretion. Dietary K+ loading for 10–14 days leads to an increase in BK channel mRNA abundance, enhanced flow-stimulated K+ secretion in microperfused CCDs, and a redistribution of immunodetectable channels from an intracellular pool to the apical membrane (Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Am J Physiol Renal Physiol 289: F922–F932, 2005). To test whether this adaptation was mediated by a K+-induced increase in aldosterone, New Zealand White rabbits were fed a low-Na+ (LS) or high-Na+ (HS) diet for 7–10 days to alter circulating levels of aldosterone but not serum K+ concentration. Single CCDs were isolated for quantitation of BK channel subunit (total, α-splice variants, β-isoforms) mRNA abundance by real-time PCR and measurement of net transepithelial Na+ (JNa) and K+ (JK) transport by microperfusion; kidneys were processed for immunolocalization of BK α-subunit by immunofluorescence microscopy. At the time of death, LS rabbits excreted no urinary Na+ and had higher circulating levels of aldosterone than HS animals. The relative abundance of BK α-, β2-, and β4-subunit mRNA and localization of immunodetectable α-subunit were similar in CCDs from LS and HS animals. In response to an increase in tubular flow rate from ∼1 to 5 nl·min−1·mm−1, the increase in JNa was greater in LS vs. HS rabbits, yet the flow-stimulated increase in JK was similar in both groups. These data suggest that aldosterone does not contribute to the regulation of BK channel expression/activity in response to dietary K+ loading.

scholarly journals Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

2010 ◽  
Vol 298 (6) ◽  
pp. F1416-F1423 ◽  
Author(s):  
Shaohua Chang ◽  
Cristiano Mendes Gomes ◽  
Joseph A. Hypolite ◽  
James Marx ◽  
Jaber Alanzi ◽  
...  
Keyword(s):  
Detrusor Overactivity ◽  
Bladder Outlet Obstruction ◽  
Rabbit Model ◽  
Outlet Obstruction ◽  
Bk Channels ◽  
Bk Channel ◽  
Β Subunit ◽  
Α Subunit ◽  
Translational Study ◽  
Channel Expression

Large-conductance voltage- and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstruction-induced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC20) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel α- and β-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BKβ mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK β-subunit was greater than that of the BK α-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK β-subunit was employed to study the effect of BK depletion on MLC20 phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC20 phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC20 phosphorylation.

scholarly journals Position and Role of the BK Channel α Subunit S0 Helix Inferred from Disulfide Crosslinking

2008 ◽  
Vol 131 (6) ◽  
pp. 537-548 ◽  
Author(s):  
Guoxia Liu ◽  
Sergey I. Zakharov ◽  
Lin Yang ◽  
Shi-Xian Deng ◽  
Donald W. Landry ◽  
...  
Keyword(s):  
Bk Channels ◽  
Bk Channel ◽  
Electrostatic Energy ◽  
Extracellular Loop ◽  
Membrane Domain ◽  
Α Subunit ◽  
Disulfide Crosslinking ◽  
Gating Charge ◽  
Sensor Activation

The position and role of the unique N-terminal transmembrane (TM) helix, S0, in large-conductance, voltage- and calcium-activated potassium (BK) channels are undetermined. From the extents of intra-subunit, endogenous disulfide bond formation between cysteines substituted for the residues just outside the membrane domain, we infer that the extracellular flank of S0 is surrounded on three sides by the extracellular flanks of TM helices S1 and S2 and the four-residue extracellular loop between S3 and S4. Eight different double cysteine–substituted alphas, each with one cysteine in the S0 flank and one in the S3–S4 loop, were at least 90% disulfide cross-linked. Two of these alphas formed channels in which 90% cross-linking had no effect on the V50 or on the activation and deactivation rate constants. This implies that the extracellular ends of S0, S3, and S4 are close in the resting state and move in concert during voltage sensor activation. The association of S0 with the gating charge bearing S3 and S4 could contribute to the considerably larger electrostatic energy required to activate the BK channel compared with typical voltage-gated potassium channels with six TM helices.

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

2006 ◽  
Vol 127 (3) ◽  
pp. 309-328 ◽  
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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