scholarly journals Gating and Ionic Currents Reveal How the BKCa Channel's Ca2+ Sensitivity Is Enhanced by its β1 Subunit

2005 ◽  
Vol 126 (4) ◽  
pp. 393-412 ◽  
Author(s):  
Lin Bao ◽  
Daniel H. Cox
Keyword(s):  
Smooth Muscle ◽  
Muscle Tone ◽  
Ionic Current ◽  
Ionic Currents ◽  
Voltage Sensor ◽  
Bkca Channel ◽  
Bkca Channels ◽  
Specific Expression ◽  
Gating Charge ◽  
Sensor Activation

Large-conductance Ca2+-activated K+ channels (BKCa channels) are regulated by the tissue-specific expression of auxiliary β subunits. β1 is predominately expressed in smooth muscle, where it greatly enhances the BKCa channel's Ca2+ sensitivity, an effect that is required for proper regulation of smooth muscle tone. Here, using gating current recordings, macroscopic ionic current recordings, and unitary ionic current recordings at very low open probabilities, we have investigated the mechanism that underlies this effect. Our results may be summarized as follows. The β1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BKCa channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with β1 present. Furthermore, β1 stabilizes the active voltage sensor more when the channel is closed than when it is open, and this reduces the factor D by which voltage sensor activation promotes opening by ∼24% (16.8→12.8). The effects of β1 on voltage sensing enhance the BKCa channel's Ca2+ sensitivity by decreasing at most voltages the work that Ca2+ binding must do to open the channel. In addition, however, in order to fully account for the increase in efficacy and apparent Ca2+ affinity brought about by β1 at negative voltages, our studies suggest that β1 also decreases the true Ca2+ affinity of the closed channel, increasing its Ca2+ dissociation constant from ∼3.7 μM to between 4.7 and 7.1 μM, depending on how many binding sites are affected.

scholarly journals Measuring the Influence of the BKCa β1 Subunit on Ca2+ Binding to the BKCa Channel

2009 ◽  
Vol 133 (2) ◽  
pp. 139-150 ◽  
Author(s):  
Tara-Beth Sweet ◽  
Daniel H. Cox
Keyword(s):  
Binding Sites ◽  
Bkca Channel ◽  
Response Curves ◽  
Bkca Channels ◽  
Specific Expression ◽  
Auxiliary Subunit ◽  
Channel Opening ◽  
Full Effect ◽  
Sensor Activation ◽  
The Difference

The large-conductance Ca2+-activated potassium (BKCa) channel of smooth muscle is unusually sensitive to Ca2+ as compared with the BKCa channels of brain and skeletal muscle. This is due to the tissue-specific expression of the BKCa auxiliary subunit β1, whose presence dramatically increases both the potency and efficacy of Ca2+ in promoting channel opening. β1 contains no Ca2+ binding sites of its own, and thus the mechanism by which it increases the BKCa channel's Ca2+ sensitivity has been of some interest. Previously, we demonstrated that β1 stabilizes voltage sensor activation, such that activation occurs at more negative voltages with β1 present. This decreases the work that Ca2+ must do to open the channel and thereby increases the channel's apparent Ca2+ affinity without altering the real affinities of the channel's Ca2+ binding sites. To explain the full effect of β1 on the channel's Ca2+ sensitivity, however, we also proposed that there must be effects of β1 on Ca2+ binding. Here, to test this hypothesis, we have used high-resolution Ca2+ dose–response curves together with binding site–specific mutations to measure the effects of β1 on Ca2+ binding. We find that coexpression of β1 alters Ca2+ binding at both of the BKCa channel's two types of high-affinity Ca2+ binding sites, primarily increasing the affinity of the RCK1 sites when the channel is open and decreasing the affinity of the Ca2+ bowl sites when the channel is closed. Both of these modifications increase the difference in affinity between open and closed, such that Ca2+ binding at either site has a larger effect on channel opening when β1 is present.

scholarly journals Effective Activation of BKCa Channels by QO-40 (5-(Chloromethyl)-3-(Naphthalen-1-yl)-2-(Trifluoromethyl)Pyrazolo [1,5-a]pyrimidin-7(4H)-one), Known to Be an Opener of KCNQ2/Q3 Channels

2021 ◽  
Vol 14 (5) ◽  
pp. 388
Author(s):  
Wei-Ting Chang ◽  
Sheng-Nan Wu
Keyword(s):  
Single Channel ◽  
Slow Component ◽  
Ionic Channel ◽  
Gh3 Cells ◽  
Bkca Channel ◽  
Bkca Channels ◽  
Gating Charge ◽  
Ramp Voltage ◽  
K Current ◽  
The Mean

QO-40 (5-(chloromethyl)-3-(naphthalene-1-yl)-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one) is a novel and selective activator of KCNQ2/KCNQ3 K+ channels. However, it remains largely unknown whether this compound can modify any other type of plasmalemmal ionic channel. The effects of QO-40 on ion channels in pituitary GH3 lactotrophs were investigated in this study. QO-40 stimulated Ca2+-activated K+ current (IK(Ca)) with an EC50 value of 2.3 μM in these cells. QO-40-stimulated IK(Ca) was attenuated by the further addition of GAL-021 or paxilline but not by linopirdine or TRAM-34. In inside-out mode, this compound added to the intracellular leaflet of the detached patches stimulated large-conductance Ca2+-activated K+ (BKCa) channels with no change in single-channel conductance; however, there was a decrease in the slow component of the mean closed time of BKCa channels. The KD value required for the QO-40-mediated decrease in the slow component at the mean closure time was 1.96 μM. This compound shifted the steady-state activation curve of BKCa channels to a less positive voltage and decreased the gating charge of the channel. The application of QO-40 also increased the hysteretic strength of BKCa channels elicited by a long-lasting isosceles-triangular ramp voltage. In HEK293T cells expressing α-hSlo, QO-40 stimulated BKCa channel activity. Overall, these findings demonstrate that QO-40 can interact directly with the BKCa channel to increase the amplitude of IK(Ca) in GH3 cells.

EETs relax airway smooth muscle via an EpDHF effect: BKCa channel activation and hyperpolarization

2001 ◽  
Vol 280 (5) ◽  
pp. L965-L973 ◽  
Author(s):  
Catherine Benoit ◽  
Barbara Renaudon ◽  
Dany Salvail ◽  
Eric Rousseau
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Molecular Mechanisms ◽  
Muscle Tone ◽  
Muscle Relaxation ◽  
Smooth Muscle Relaxation ◽  
Epoxyeicosatrienoic Acids ◽  
Bkca Channel ◽  
Channel Activity ◽  
Cytochrome P 450

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K+ conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca2+-activated K+ (BKCa) channel blocker. Moreover, 3 μM 8,9- or 14,15-EET induced hyperpolarizations of −12 ± 3.5 and −16 ± 3 mV, with EC50 values of 0.13 and 0.14 μM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BKCa channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BKCa channels at low free Ca2+ concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.

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.

scholarly journals Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

2010 ◽  
Vol 136 (3) ◽  
pp. 283-291 ◽  
Author(s):  
Guiling Zhao ◽  
Zachary P. Neeb ◽  
M. Dennis Leo ◽  
Judith Pachuau ◽  
Adebowale Adebiyi ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Bkca Channel ◽  
Ip3 Receptors ◽  
Arterial Smooth Muscle ◽  
Bkca Channels ◽  
Channel Activation ◽  
Arterial Smooth Muscle Cells

Plasma membrane large-conductance Ca2+-activated K+ (BKCa) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BKCa channel regulation by IP3 and IP3Rs in rat and mouse cerebral artery smooth muscle cells. IP3 activated BKCa channels both in intact cells and in excised inside-out membrane patches. IP3 caused concentration-dependent BKCa channel activation with an apparent dissociation constant (Kd) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca2+ concentration ([Ca2+]i; 10 µM). IP3 also caused a leftward-shift in BKCa channel apparent Ca2+ sensitivity and reduced the Kd for free [Ca2+]i from ∼20 to 12 µM, but did not alter the slope or maximal Po. BAPTA, a fast Ca2+ buffer, or an elevation in extracellular Ca2+ concentration did not alter IP3-induced BKCa channel activation. Heparin, an IP3R inhibitor, and a monoclonal type 1 IP3R (IP3R1) antibody blocked IP3-induced BKCa channel activation. Adenophostin A, an IP3R agonist, also activated BKCa channels. IP3 activated BKCa channels in inside-out patches from wild-type (IP3R1+/+) mouse arterial smooth muscle cells, but had no effect on BKCa channels of IP3R1-deficient (IP3R1−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP3R1 is located in close spatial proximity to BKCa α subunits. The IP3R1 monoclonal antibody coimmunoprecipitated IP3R1 and BKCa channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP3R1 activation elevates BKCa channel apparent Ca2+ sensitivity through local molecular coupling in arterial smooth muscle cells.

scholarly journals High Efficacy by GAL-021: A Known Intravenous Peripheral Chemoreceptor Modulator that Suppresses BKCa-Channel Activity and Inhibits IK(M) or Ih

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Te-Ling Lu ◽  
Zi-Han Gao ◽  
Shih-Wei Li ◽  
Sheng-Nan Wu
Keyword(s):  
Ionic Currents ◽  
Gh3 Cells ◽  
Delayed Rectifier ◽  
Bkca Channel ◽  
Dependent Manner ◽  
Channel Activity ◽  
Bkca Channels ◽  
Cationic Current ◽  
K Current ◽  
Change In Mean

GAL-021 has recently been developed as a novel breathing control modulator. However, modifications of ionic currents produced by this agent remain uncertain, although its efficacy in suppressing the activity of big-conductance Ca2+-activated K+ (BKCa) channels has been reported. In pituitary tumor (GH3) cells, we found that the presence of GAL-021 decreased the amplitude of macroscopic Ca2+-activated K+ current (IK(Ca)) in a concentration-dependent manner with an effective IC50 of 2.33 μM. GAL-021-mediated reduction of IK(Ca) was reversed by subsequent application of verteporfin or ionomycin; however, it was not by that of diazoxide. In inside-out current recordings, the addition of GAL-021 to the bath markedly decreased the open-state probability of BKCa channels. This agent also resulted in a rightward shift in voltage dependence of the activation curve of BKCa channels; however, neither the gating charge of the curve nor single-channel conductance of the channel was changed. There was an evident lengthening of the mean closed time of BKCa channels in the presence of GAL-021, with no change in mean open time. The GAL-021 addition also suppressed M-type K+ current with an effective IC50 of 3.75 μM; however, its presence did not alter the amplitude of erg-mediated K+ current, or mildly suppressed delayed-rectifier K+ current. GAL-021 at a concentration of 30 μM could also suppress hyperpolarization-activated cationic current. In HEK293T cells expressing α-hSlo, the addition of GAL-021 was also able to suppress the BKCa-channel open probabilities, and GAL-021-mediated suppression of BKCa-channel activity was attenuated by further addition of BMS-191011. Collectively, the GAL-021 effects presented herein do not exclusively act on BKCa channels and these modifications on ionic currents exert significant influence on the functional activities of electrically excitable cells occurring in vivo.

scholarly journals Extracellular Mg2+ Modulates Slow Gating Transitions and the Opening of Drosophila Ether-à-Go-Go Potassium Channels

2000 ◽  
Vol 115 (3) ◽  
pp. 319-338 ◽  
Author(s):  
Chih-Yung Tang ◽  
Francisco Bezanilla ◽  
Diane M. Papazian
Keyword(s):  
Time Course ◽  
Ionic Current ◽  
Ionic Currents ◽  
K Channel ◽  
Gating Currents ◽  
Gating Charge ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Sequential Activation

We have characterized the effects of prepulse hyperpolarization and extracellular Mg2+ on the ionic and gating currents of the Drosophila ether-à-go-go K+ channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg2+ dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg2+ modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg2+ slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg2+ modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg2+. We have also identified mutations in the S3–S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg2+, indicating an important role for this region in the voltage-dependent activation of eag.

scholarly journals Macroscopic Na+ Currents in the “Nonconducting” Shaker Potassium Channel Mutant W434F

1998 ◽  
Vol 112 (1) ◽  
pp. 85-93 ◽  
Author(s):  
John G. Starkus ◽  
Lioba Kuschel ◽  
Martin D. Rayner ◽  
Stefan H. Heinemann
Keyword(s):  
Structural Changes ◽  
Ionic Current ◽  
Outer Region ◽  
Ionic Currents ◽  
Shaker Potassium Channel ◽  
Shaker Channels ◽  
Gating Charge ◽  
Na Currents ◽  
Shaker Potassium Channels ◽  
Kinetics Of

C-type inactivation in Shaker potassium channels inhibits K+ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na+ and Li+ currents in K+-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na+ and Li+ currents (in K+-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na+ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.

scholarly journals Influence of Permeant Ions on Voltage Sensor Function in the Kv2.1 Potassium Channel

2004 ◽  
Vol 123 (4) ◽  
pp. 387-400 ◽  
Author(s):  
Joseph F. Consiglio ◽  
Stephen J. Korn
Keyword(s):  
Potassium Channel ◽  
Ionic Current ◽  
Ionic Currents ◽  
Voltage Sensor ◽  
Selectivity Filter ◽  
Charge Movement ◽  
Channel Activation ◽  
Outer Vestibule ◽  
Specific Selectivity ◽  
Current Activation

We previously demonstrated that the outer vestibule of activated Kv2.1 potassium channels can be in one of two conformations, and that K+occupancy of a specific selectivity filter site determines which conformation the outer vestibule is in. These different outer vestibule conformations result in different sensitivities to internal and external TEA, different inactivation rates, and different macroscopic conductances. The [K+]-dependent switch in outer vestibule conformation is also associated with a change in rate of channel activation. In this paper, we examined the mechanism by which changes in [K+] modulate the rate of channel activation. Elevation of symmetrical [K+] or [Rb+] from 0 to 3 mM doubled the rate of on-gating charge movement (Qon), measured at 0 mV. Cs+produced an identical effect, but required 40-fold higher concentrations. All three permeant ions occupied the selectivity filter over the 0.03–3 mM range, so simple occupancy of the selectivity filter was not sufficient to produce the change in Qon. However, for each of these permeant ions, the speeding of Qonoccurred with the same concentration dependence as the switch between outer vestibule conformations. Neutralization of an amino acid (K356) in the outer vestibule, which abolishes the modulation of channel pharmacology and ionic currents by the K+-dependent reorientation of the outer vestibule, also abolished the K+-dependence of Qon. Together, the data indicate that the K+-dependent reorientation in the outer vestibule was responsible for the change in Qon. Moreover, similar [K+]-dependence and effects of mutagenesis indicate that the K+-dependent change in rate of Qoncan account for the modulation of ionic current activation rate. Simple kinetic analysis suggested that K+reduced an energy barrier for voltage sensor movement. These results provide strong evidence for a direct functional interaction, which is modulated by permeant ions acting at the selectivity filter, between the outer vestibule of the Kv2.1 potassium channel and the voltage sensor.

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