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 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 Elimination of the BKCa Channel's High-Affinity Ca2+ Sensitivity

2002 ◽  
Vol 120 (2) ◽  
pp. 173-189 ◽  
Author(s):  
Lin Bao ◽  
Anne M. Rapin ◽  
Ericka C. Holmstrand ◽  
Daniel H. Cox
Keyword(s):  
Binding Sites ◽  
K Channel ◽  
Bkca Channel ◽  
Binding Characteristics ◽  
High Affinity ◽  
Channel Opening ◽  
High Concentrations

We report here a combination of site-directed mutations that eliminate the high-affinity Ca2+ response of the large-conductance Ca2+-activated K+ channel (BKCa), leaving only a low-affinity response blocked by high concentrations of Mg2+. Mutations at two sites are required, the “Ca2+ bowl,” which has been implicated previously in Ca2+ binding, and M513, at the end of the channel's seventh hydrophobic segment. Energetic analyses of mutations at these positions, alone and in combination, argue that the BKCa channel contains three types of Ca2+ binding sites, one of low affinity that is Mg2+ sensitive (as has been suggested previously) and two of higher affinity that have similar binding characteristics and contribute approximately equally to the power of Ca2+ to influence channel opening. Estimates of the binding characteristics of the BKCa channel's high-affinity Ca2+-binding sites are provided.

scholarly journals Mg2+ Enhances Voltage Sensor/Gate Coupling in BK Channels

2007 ◽  
Vol 131 (1) ◽  
pp. 13-32 ◽  
Author(s):  
Frank T. Horrigan ◽  
Zhongming Ma
Keyword(s):  
Resting State ◽  
Binding Sites ◽  
Bk Channels ◽  
Voltage Sensor ◽  
Detectable Effect ◽  
Open Probability ◽  
Independent Manner ◽  
Voltage Sensors ◽  
Channel Opening ◽  
Sensor Activation

BK (Slo1) potassium channels are activated by millimolar intracellular Mg2+ as well as micromolar Ca2+ and membrane depolarization. Mg2+ and Ca2+ act in an approximately additive manner at different binding sites to shift the conductance–voltage (GK-V) relation, suggesting that these ligands might work through functionally similar but independent mechanisms. However, we find that the mechanism of Mg2+ action is highly dependent on voltage sensor activation and therefore differs fundamentally from that of Ca2+. Evidence that Ca2+ acts independently of voltage sensor activation includes an ability to increase open probability (PO) at extreme negative voltages where voltage sensors are in the resting state; 2 μM Ca2+ increases PO more than 15-fold at −120 mV. However 10 mM Mg2+, which has an effect on the GK-V relation similar to 2 μM Ca2+, has no detectable effect on PO when voltage sensors are in the resting state. Gating currents are only slightly altered by Mg2+ when channels are closed, indicating that Mg2+ does not act merely to promote voltage sensor activation. Indeed, channel opening is facilitated in a voltage-independent manner by Mg2+ in a mutant (R210C) whose voltage sensors are constitutively activated. Thus, 10 mM Mg2+ increases PO only when voltage sensors are activated, effectively strengthening the allosteric coupling of voltage sensor activation to channel opening. Increasing Mg2+ from 10 to 100 mM, to occupy very low affinity binding sites, has additional effects on gating that more closely resemble those of Ca2+. The effects of Mg2+ on steady-state activation and IK kinetics are discussed in terms of an allosteric gating scheme and the state-dependent interactions between Mg2+ and voltage sensor that may underlie this mechanism.

scholarly journals The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating

2005 ◽  
Vol 126 (3) ◽  
pp. 227-241 ◽  
Author(s):  
Gayathri Krishnamoorthy ◽  
Jingyi Shi ◽  
David Sept ◽  
Jianmin Cui
Keyword(s):  
Conformational Changes ◽  
K Channel ◽  
Bkca Channel ◽  
Bkca Channels ◽  
Allosteric Coupling ◽  
Channel Opening ◽  
Activation Gate ◽  
Rck Domain ◽  
Dual Activation ◽  
Α Subunits

Large conductance, voltage- and Ca2+-activated K+ (BKCa) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four α subunits of BKCa may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BKCa gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BKCa activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BKCa channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.

scholarly journals Role of the β1 Subunit in Large-Conductance Ca2+-Activated K+ Channel Gating Energetics

2000 ◽  
Vol 116 (3) ◽  
pp. 411-432 ◽  
Author(s):  
D.H. Cox ◽  
R.W. Aldrich
Keyword(s):  
Steady State ◽  
Channel Gating ◽  
K Channel ◽  
Bkca Channel ◽  
Voltage Sensitivity ◽  
Specific Expression ◽  
Α Subunit ◽  
Gating Charge ◽  
Channel Opening ◽  
Wide Range

Over the past few years, it has become clear that an important mechanism by which large-conductance Ca2+-activated K+ channel (BKCa) activity is regulated is the tissue-specific expression of auxiliary β subunits. The first of these to be identified, β1, is expressed predominately in smooth muscle and causes dramatic effects, increasing the apparent affinity of the channel for Ca2+ 10-fold at 0 mV, and shifting the range of voltages over which the channel activates −80 mV at 9.1 μM Ca2+. With this study, we address the question: which aspects of BKCa gating are altered by β1 to bring about these effects: Ca2+ binding, voltage sensing, or the intrinsic energetics of channel opening? The approach we have taken is to express the β1 subunit together with the BKCa α subunit in Xenopus oocytes, and then to compare β1's steady state effects over a wide range of Ca2+ concentrations and membrane voltages to those predicted by allosteric models whose parameters have been altered to mimic changes in the aspects of gating listed above. The results of our analysis suggest that much of β1's steady state effects can be accounted for by a reduction in the intrinsic energy the channel must overcome to open and a decrease in its voltage sensitivity, with little change in the affinity of the channel for Ca2+ when it is either open or closed. Interestingly, however, the small changes in Ca2+ binding affinity suggested by our analysis (Kc 7.4 μM → 9.6 μM; Ko = 0.80 μM → 0.65 μM) do appear to be functionally important. We also show that β1 affects the mSlo conductance–voltage relation in the essential absence of Ca2+, shifting it +20 mV and reducing its apparent gating charge 38%, and we develop methods for distinguishing between alterations in Ca2+ binding and other aspects of BKCa channel gating that may be of general use.

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.

scholarly journals Tissue-Restricted Expression of the Cardiac α-Myosin Heavy Chain Gene Is Controlled by a Downstream Repressor Element Containing a Palindrome of Two Ets-Binding Sites

1998 ◽  
Vol 18 (12) ◽  
pp. 7243-7258 ◽  
Author(s):  
Madhu Gupta ◽  
Radovan Zak ◽  
Towia A. Libermann ◽  
Mahesh P. Gupta
Keyword(s):  
Gene Regulation ◽  
Myosin Heavy Chain ◽  
Cardiac Myocytes ◽  
Heavy Chain ◽  
Binding Sites ◽  
Cardiac Myocyte ◽  
Specific Expression ◽  
Tissue Specific Expression ◽  
Nonmuscle Cells ◽  
Muscle Gene

ABSTRACT The expression of the α-myosin heavy chain (MHC) gene is restricted primarily to cardiac myocytes. To date, several positive regulatory elements and their binding factors involved in α-MHC gene regulation have been identified; however, the mechanism restricting the expression of this gene to cardiac myocytes has yet to be elucidated. In this study, we have identified by using sequential deletion mutants of the rat cardiac α-MHC gene a 30-bp purine-rich negative regulatory (PNR) element located in the first intronic region that appeared to be essential for the tissue-specific expression of the α-MHC gene. Removal of this element alone elevated (20- to 30-fold) the expression of the α-MHC gene in cardiac myocyte cultures and in heart muscle directly injected with plasmid DNA. Surprisingly, this deletion also allowed a significant expression of the α-MHC gene in HeLa and other nonmuscle cells, where it is normally inactive. The PNR element required upstream sequences of the α-MHC gene for negative gene regulation. By DNase I footprint analysis of the PNR element, a palindrome of two high-affinity Ets-binding sites (CTTCCCTGGAAG) was identified. Furthermore, by analyses of site-specific base-pair mutation, mobility gel shift competition, and UV cross-linking, two different Ets-like proteins from cardiac and HeLa cell nuclear extracts were found to bind to the PNR motif. Moreover, the activity of the PNR-binding factor was found to be increased two- to threefold in adult rat hearts subjected to pressure overload hypertrophy, where the α-MHC gene is usually suppressed. These data demonstrate that the PNR element plays a dual role, both downregulating the expression of the α-MHC gene in cardiac myocytes and silencing the muscle gene activity in nonmuscle cells. Similar palindromic Ets-binding motifs are found conserved in the α-MHC genes from different species and in other cardiac myocyte-restricted genes. These results are the first to reveal a role of the Ets class of proteins in controlling the tissue-specific expression of a cardiac muscle gene.

scholarly journals cAMP-dependent regulation of IKs single-channel kinetics

2017 ◽  
Vol 149 (8) ◽  
pp. 781-798 ◽  
Author(s):  
Emely Thompson ◽  
Jodene Eldstrom ◽  
Maartje Westhoff ◽  
Donald McAfee ◽  
Elise Balse ◽  
...  
Keyword(s):  
Action Potential ◽  
Single Channel ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Surface Expression ◽  
Voltage Sensor ◽  
Single Channels ◽  
Adrenergic Stimulation ◽  
Channel Opening ◽  
Sensor Activation

The delayed potassium rectifier current, IKs, is composed of KCNQ1 and KCNE1 subunits and plays an important role in cardiac action potential repolarization. During β-adrenergic stimulation, 3′-5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylates KCNQ1, producing an increase in IKs current and a shortening of the action potential. Here, using cell-attached macropatches and single-channel recordings, we investigate the microscopic mechanisms underlying the cAMP-dependent increase in IKs current. A membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), causes a marked leftward shift of the conductance–voltage relation in macropatches, with or without an increase in current size. Single channels exhibit fewer silent sweeps, reduced first latency to opening (control, 1.61 ± 0.13 s; cAMP, 1.06 ± 0.11 s), and increased higher-subconductance-level occupancy in the presence of cAMP. The E160R/R237E and S209F KCNQ1 mutants, which show fixed and enhanced voltage sensor activation, respectively, largely abolish the effect of cAMP. The phosphomimetic KCNQ1 mutations, S27D and S27D/S92D, are much less and not at all responsive, respectively, to the effects of PKA phosphorylation (first latency of S27D + KCNE1 channels: control, 1.81 ± 0.1 s; 8-CPT-cAMP, 1.44 ± 0.1 s, P < 0.05; latency of S27D/S92D + KCNE1: control, 1.62 ± 0.1 s; cAMP, 1.43 ± 0.1 s, nonsignificant). Using total internal reflection fluorescence microscopy, we find no overall increase in surface expression of the channel during exposure to 8-CPT-cAMP. Our data suggest that the cAMP-dependent increase in IKs current is caused by an increase in the likelihood of channel opening, combined with faster openings and greater occupancy of higher subconductance levels, and is mediated by enhanced voltage sensor activation.

scholarly journals Two-Step Regulation of Ad4BP/SF-1 Gene Transcription during Fetal Adrenal Development: Initiation by a Hox-Pbx1-Prep1 Complex and Maintenance via Autoregulation by Ad4BP/SF-1

2006 ◽  
Vol 26 (11) ◽  
pp. 4111-4121 ◽  
Author(s):  
Mohamad Zubair ◽  
Satoru Ishihara ◽  
Sanae Oka ◽  
Katsuzumi Okumura ◽  
Ken-ichirou Morohashi
Keyword(s):  
Adrenal Cortex ◽  
Binding Sites ◽  
Orphan Nuclear Receptor ◽  
Specific Expression ◽  
Steroidogenic Factor 1 ◽  
Tissue Specific ◽  
Adrenal Cells ◽  
Tissue Specific Expression ◽  
And Function ◽  
Intron 4

ABSTRACT The orphan nuclear receptor Ad4BP/SF-1 (adrenal 4 binding protein/steroidogenic factor 1) is essential for the proper development and function of reproductive and steroidogenic tissues. Although the expression of Ad4BP/SF-1 is specific for those tissues, the mechanisms underlying this tissue-specific expression remain unknown. In this study, we used transgenic mouse assays to examine the regulation of the tissue-specific expression of Ad4BP/SF-1. An investigation of the entire Ad4BP/SF-1 gene locus revealed a fetal adrenal enhancer (FAdE) in intron 4 containing highly conserved binding sites for Pbx-Prep, Pbx-Hox, and Ad4BP/SF-1. Transgenic assays revealed that the Ad4 sites, together with Ad4BP/SF-1, develop an autoregulatory loop and thereby maintain transcription, while the Pbx/Prep and Pbx/Hox sites initiate transcription prior to the establishment of the autoregulatory loop. Indeed, a limited number of Hox family members were found to be expressed in the adrenal primordia. Whether a true fetal-type adrenal cortex is present in mice remained controversial, and this argument was complicated by the postnatal development of the so-called X zone. Using transgenic mice with lacZ driven by the FAdE, we clearly identified a fetal adrenal cortex in mice, and the X zone is the fetal adrenal cells accumulated at the juxtamedullary region after birth.

The Dynamic Response of a Simple Elastic System to Antisymmetric Forcing Functions Characteristic of Airplanes in Unsymmetric Landing Impact

1949 ◽  
Vol 16 (3) ◽  
pp. 310-316
Author(s):  
Joseph B. Woodson
Keyword(s):  
Dynamic Response ◽  
Sine Wave ◽  
Response Curves ◽  
Response Factors ◽  
Natural Period ◽  
Wave Pulse ◽  
Pulse Disturbance ◽  
Landing Impact ◽  
Forcing Functions ◽  
The Difference

Abstract This paper presents an analysis of the dynamic response of an undamped mechanical system with one degree of freedom subjected to disturbances which are described by antisymmetric forcing functions. The analysis was undertaken to throw light on the effect on the vibration of the wings caused by unsymmetric landing impact of an airplane. Two types of disturbances are considered; a full-sine-wave pulse, and a pulse which is the difference between two overlapping half sine waves. The results are presented in the form of dynamic-response curves and dynamic-response-factor curves. The numerically greatest dynamic-response factors, approximately 3.24 and −3.26, resulted for a full-sine-wave pulse disturbance with a ratio of duration of impact to natural period, Ti/T ≅ 1.11. When Ti/T is in the neighborhood of 1, the first positive peak of dynamic response is numerically less than the negative and positive peaks which follow it. For much of the range, the positive and negative dynamic-response factors are numerically approximately equal. The analysis was confined to values of Ti/T between 0.33 and 12. As Ti/T increases without limit, the positive and negative dynamic-response factors tend to 1 and −1, respectively.

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