L-Type Ca2+ Channel Regulation by Calmodulin and CaBP1

L-type voltage-gated Ca2+ channels (CaV1.2 and CaV1.3, called CaV) interact with the Ca2+ sensor proteins, calmodulin (CaM) and Ca2+ binding Protein 1 (CaBP1), that oppositely control Ca2+-dependent channel activity. CaM and CaBP1 can each bind to the IQ-motif within the C-terminal cytosolic domain of CaV, which promotes increased channel open probability under basal conditions. At elevated cytosolic Ca2+ levels (caused by CaV channel opening), Ca2+-bound CaM binding to CaV is essential for promoting rapid Ca2+-dependent channel inactivation (CDI). By contrast, CaV binding to CaBP1 prevents CDI and promotes Ca2+-induced channel opening (called CDF). In this review, I provide an overview of the known structures of CaM and CaBP1 and their structural interactions with the IQ-motif to help understand how CaM promotes CDI, whereas CaBP1 prevents CDI and instead promotes CDF. Previous electrophysiology studies suggest that Ca2+-free forms of CaM and CaBP1 may pre-associate with CaV under basal conditions. However, previous Ca2+ binding data suggest that CaM and CaBP1 are both calculated to bind to Ca2+ with an apparent dissociation constant of ~100 nM when CaM or CaBP1 is bound to the IQ-motif. Since the neuronal basal cytosolic Ca2+ concentration is ~100 nM, nearly half of the neuronal CaV channels are suggested to be bound to Ca2+-bound forms of either CaM or CaBP1 under basal conditions. The pre-association of CaV with calcified forms of CaM or CaBP1 are predicted here to have functional implications. The Ca2+-bound form of CaBP1 is proposed to bind to CaV under basal conditions to block CaV binding to CaM, which could explain how CaBP1 might prevent CDI.

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α-Actinin-1 promotes activity of the L-type Ca2+ Channel CaV1.2

10.1101/664102 ◽

2019 ◽

Cited By ~ 1

Author(s):

Matthew Turner ◽

David E. Anderson ◽

Madeline Nieves-Cintron ◽

Peter Bartels ◽

Andrea M. Coleman ◽

...

Keyword(s):

Single Channel ◽

Cardiac Contraction ◽

Charge Movement ◽

Open Probability ◽

Iq Motif ◽

Nmr Structures ◽

Gating Charge ◽

Channel Opening ◽

Target Sites ◽

Surface Localization

ABSTRACTThe L-type Ca2+ channel CaV1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1646-1664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV1.2 to defined surface regions and simultaneously boosts its open probability so that CaV1.2 is mostly active when appropriately localized.

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Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00325.2013 ◽

2014 ◽

Vol 306 (5) ◽

pp. C460-C470 ◽

Cited By ~ 18

Author(s):

Kiril L. Hristov ◽

Amy C. Smith ◽

Shankar P. Parajuli ◽

John Malysz ◽

Georgi V. Petkov

Keyword(s):

Smooth Muscle ◽

Membrane Potential ◽

Guinea Pig ◽

Bk Channels ◽

Bk Channel ◽

K Channel ◽

Channel Activity ◽

Open Probability ◽

Channel Regulation ◽

Pkc Activation

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility.

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Irreversible temperature gating in trpv1 sheds light on channel activation

eLife ◽

10.7554/elife.36372 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 18

Author(s):

Ana Sánchez-Moreno ◽

Eduardo Guevara-Hernández ◽

Ricardo Contreras-Cervera ◽

Gisela Rangel-Yescas ◽

Ernesto Ladrón-de-Guevara ◽

...

Keyword(s):

Trp Channels ◽

Structural Information ◽

Heat Absorption ◽

Absorption Process ◽

Open Probability ◽

Temperature Changes ◽

Trpv1 Channels ◽

Channel Opening ◽

Temperature Activation ◽

Thermal Stimuli

Temperature-activated TRP channels or thermoTRPs are among the only proteins that can directly convert temperature changes into changes in channel open probability. In spite of a wealth of functional and structural information, the mechanism of temperature activation remains unknown. We have carefully characterized the repeated activation of TRPV1 by thermal stimuli and discovered a previously unknown inactivation process, which is irreversible. We propose that this form of gating in TRPV1 channels is a consequence of the heat absorption process that leads to channel opening.

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Activation of Ca2+-activated K+(maxi-K+) channel by angiotensin II in myocytes of the guinea pig ileum

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.4.c983 ◽

1998 ◽

Vol 274 (4) ◽

pp. C983-C991 ◽

Cited By ~ 35

Author(s):

Fernando Romero ◽

Bagnólia A. Silva ◽

Viviane L. A. Nouailhetas ◽

Jeannine Aboulafia

Keyword(s):

Angiotensin Ii ◽

K Channel ◽

Intermediate Step ◽

Synthetic Analog ◽

Ang Ii ◽

Open Probability ◽

Channel Opening ◽

Intracellular Ca ◽

Patch Configuration ◽

Pharmacomechanical Coupling

We investigated the regulation of the Ca2+-activated K+(maxi-K+) channel by angiotensin II (ANG II) and its synthetic analog, [Lys2]ANG II, in freshly dispersed intestinal myocytes. We identified a maxi-K+ channel population in the inside-out patch configuration on the basis of its conductance (257 ± 4 pS in symmetrical 150 mM KCl solution), voltage and Ca2+ dependence of channel opening, low Na+-to-K+and Cl−-to-K+permeability ratios, and blockade by external Cs+ and tetraethylammonium chloride. ANG II and [Lys2]ANG II caused an indirect, reversible, Ca2+- and dose-dependent activation of maxi-K+ channels in cell-attached experiments when cells were bathed in high-K+ solution. This effect was reversibly blocked by DUP-753, being that it is mediated by the AT1 receptor. Evidences that activation of the maxi-K+ channel by ANG II requires a rise in intracellular Ca2+concentration ([Ca2+]i) as an intermediate step were the shift of the open probability of the channel-membrane potential relationship to less positive membrane potentials and the sustained increase in [Ca2+]iin fura 2-loaded myocytes. The preservation of the pharmacomechanical coupling of ANG II in these cells provides a good model for the study of transmembrane signaling responses to ANG II and analogs in this tissue.

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Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.6.c1769 ◽

2001 ◽

Vol 281 (6) ◽

pp. C1769-C1775 ◽

Cited By ~ 155

Author(s):

Guillermo J. Pérez ◽

Adrian D. Bonev ◽

Mark T. Nelson

Keyword(s):

Smooth Muscle ◽

Laser Scanning ◽

Cerebral Arteries ◽

Bk Channels ◽

Bk Channel ◽

Hill Coefficient ◽

Channel Activity ◽

Acetoxymethyl Ester ◽

Open Probability ◽

Apparent Dissociation Constant

The goal of the present study was to test the hypothesis that local Ca2+ release events (Ca2+ sparks) deliver high local Ca2+concentration to activate nearby Ca2+-sensitive K+ (BK) channels in the cell membrane of arterial smooth muscle cells. Ca2+ sparks and BK channels were examined in isolated myocytes from rat cerebral arteries with laser scanning confocal microscopy and patch-clamp techniques. BK channels had an apparent dissociation constant for Ca2+ of 19 μM and a Hill coefficient of 2.9 at −40 mV. At near-physiological intracellular Ca2+ concentration ([Ca2+]i; 100 nM) and membrane potential (−40 mV), the open probability of a single BK channel was low (1.2 × 10−6). A Ca2+spark increased BK channel activity to 18. Assuming that 1–100% of the BK channels are activated by a single Ca2+ spark, BK channel activity increases 6 × 105-fold to 6 × 103-fold, which corresponds to ∼30 μM to 4 μM spark Ca2+ concentration. 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid acetoxymethyl ester caused the disappearance of all Ca2+sparks while leaving the transient BK currents unchanged. Our results support the idea that Ca2+ spark sites are in close proximity to the BK channels and that local [Ca2+]i reaches micromolar levels to activate BK channels.

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Effects of Inhalational Anesthetics on L-type Ca2+Currents in Human Atrial Cardiomyocytes during β-Adrenergic Stimulation

Anesthesiology ◽

10.1097/00000542-200307000-00017 ◽

2003 ◽

Vol 99 (1) ◽

pp. 90-96 ◽

Cited By ~ 14

Author(s):

Jens Fassl ◽

Christian R. Halaszovich ◽

Rocco Hüneke ◽

Eberhard Jüngling ◽

Rolf Rossaint ◽

...

Keyword(s):

Minimum Alveolar Concentration ◽

Direct Effects ◽

Adrenergic Stimulation ◽

Patch Clamp Technique ◽

Dual Effect ◽

Cardiac Side Effects ◽

Atrial Cardiomyocytes ◽

Channel Regulation ◽

Ca2 Channels ◽

Peak Value

Background Anesthetics may cause cardiac side effects by their action on L-type Ca2+ channels. Direct effects on the channels have not yet been discriminated from an interference with the beta-adrenergic channel regulation. The authors therefore studied the effects of halothane, sevoflurane, and xenon on human cardiac Ca2+ currents during stimulation with isoproterenol. Methods Currents through L-type Ca2+ channels were measured with the patch clamp technique in atrial cardiomyocytes obtained from patients undergoing cardiac surgery. Cells were superfused with solutions equilibrated with anesthetics at the desired concentrations. Ca2+ currents during pulses to 10 mV were evaluated with respect to their peak value (I(max)) and to the total moved charge (Q). Results In the absence and in the presence of isoproterenol (1 microm), sevoflurane (0.29 mm, 1 minimum alveolar concentration [MAC]) significantly depressed Q by 37.8 +/- 7.2% (mean +/- SD) and 40.8 +/- 10.3%, respectively. I(max) was not significantly affected in comparison with control cells never exposed to an anesthetic. Xenon (65%, 1 MAC) did not evoke significant effects. Exposure to halothane (0.39 mm, 1 MAC) during stimulation with isoproterenol significantly reduced Q by 31.3 +/- 23.3% (but not I(max)). After washout of halothane, Q was increased above the level prior to the application of halothane. Moreover, whereas Q promptly declined to baseline levels after washout of isoproterenol in controls, the previous exposure to halothane markedly delayed this decline, leaving Q significantly elevated for several minutes. Conclusions Halothane exerts a dual effect on Ca2+ currents. The long-lasting stimulatory effect may contribute to the proarrhythmic potency of the drug that exceeds that of sevoflurane, which only depressed Ca2+ currents.

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Regulation of Cloned Atp-Sensitive K Channels by Adenine Nucleotides and Sulfonylureas

The Journal of General Physiology ◽

10.1085/jgp.118.4.391 ◽

2001 ◽

Vol 118 (4) ◽

pp. 391-406 ◽

Cited By ~ 21

Author(s):

Scott A. John ◽

James N. Weiss ◽

Bernard Ribalet

Keyword(s):

Katp Channel ◽

Adenine Nucleotides ◽

Katp Channels ◽

Functional Coupling ◽

Sulfonylurea Receptor ◽

K Channels ◽

Channel Regulation ◽

Channel Opening ◽

Insight Into ◽

Positively Charged

KATP channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 “PIP2-insensitive” mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the KATP channel. In contrast, the R301C and R314C mutants, which are also “PIP2-insensitive,” remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the KATP channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since KATP channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.

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Activation of L-type Ca2+ channels after purinoceptor stimulation by ATP in an alveolar epithelial cell (L2)

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.269.6.l873 ◽

1995 ◽

Vol 269 (6) ◽

pp. L873-L883 ◽

Cited By ~ 2

Author(s):

P. Dietl ◽

T. Haller ◽

B. Wirleitner ◽

H. Volkl ◽

F. Friedrich ◽

...

Keyword(s):

Epithelial Cell ◽

Alveolar Epithelial Cell ◽

Alveolar Epithelium ◽

Bay K 8644 ◽

Epithelial Cell Line ◽

Apparent Dissociation Constant ◽

Whole Cell ◽

Fura 2 ◽

Alveolar Epithelial ◽

Ca2 Channels

In the alveolar epithelium, ATP increases the intracellular Ca2+ concentration ([Ca2+]i) and stimulates the secretion of surfactant. We investigated the effects of extracellular ATP on the membrane potential (Vm), the whole cell current, and [Ca2+]i in a cloned rat alveolar epithelial cell line (L2). In microelectrode experiments, ATP caused a sustained depolarization of Vm, resulting from the activation of cation and Cl- conductances, as revealed by ion replacements. The depolarizing phase of the Vm shift was superimposed by Ca(2+)-dependent depolarizing spikes. Spikes were also induced by depolarizing Vm with charybdotoxin or maitotoxin. Replacement of bath Ca2+ with Ba2+ or Sr2+ also evoked repetitive spikes. Ca2+ (Ba2+, Sr2+)-induced spikes were unaffected by pretreatment with ionomycin or thapsigargin. They were, however, completely abolished by (+)-isradipine (100 nM) and stimulated by BAY K 8644 (100 nM). Whole cell L-type Ca2+ (Ba2+, Sr2+) currents were similarly abolished by (+)-isradipine and enhanced by BAY K 8644. L-type Ca2+ channels were further confirmed by demonstrating high-affinity dihydropyridine receptors stereoselectively labeled by (+)-[3H]-isradipine, apparent dissociation constant < 1 nM. In fura 2 experiments, ATP evoked a transient elevation of [Ca2+]i in the absence of Ca2+ and a biphasic sustained elevation in the presence of Ca2+, indicating intracellular Ca2+ release and Ca2+ entry. The ATP-induced fura 2 signals were unaffected by (+)-isradipine. We conclude that in L2 cells, L-type Ca2+ channels are activated after purinoceptor stimulation by ATP. The overall [Ca2+]i response is, however, mediated by Ca2+ entry through and (+)-isradipine-insensitive mechanism and by intracellular Ca2+ release.

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Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength.

The Journal of General Physiology ◽

10.1085/jgp.91.3.317 ◽

1988 ◽

Vol 91 (3) ◽

pp. 317-333 ◽

Cited By ~ 174

Author(s):

C S Anderson ◽

R MacKinnon ◽

C Smith ◽

C Miller

Keyword(s):

Ionic Strength ◽

Plasma Membranes ◽

Scorpion Venom ◽

Dissociation Rate ◽

K Channel ◽

Single Channels ◽

Association Rate ◽

Open Probability ◽

Apparent Dissociation Constant ◽

K Channels

Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K+ channels. The interaction of CTX with Ca2+-activated K+ channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K+ conduction by binding to the external side of the channel, with an apparent dissociation constant of approximately 10 nM at physiological ionic strength. The dwell-time distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K+, Na+, or arginine+) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K+ channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site.

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Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels.

The Journal of General Physiology ◽

10.1085/jgp.108.3.143 ◽

1996 ◽

Vol 108 (3) ◽

pp. 143-155 ◽

Cited By ~ 86

Author(s):

F Noceti ◽

P Baldelli ◽

X Wei ◽

N Qin ◽

L Toro ◽

...

Keyword(s):

Ionic Current ◽

Variance Analysis ◽

Beta Subunit ◽

Charge Movement ◽

Gating Currents ◽

Open Probability ◽

K Channels ◽

Voltage Dependent ◽

Pore Opening ◽

Ca2 Channels

In voltage-dependent ion channels, the gating of the channels is determined by the movement of the voltage sensor. This movement reflects the rearrangement of the protein in response to a voltage stimulus, and it can be thought of as a net displacement of elementary charges (e0) through the membrane (z: effective number of elementary charges). In this paper, we measured z in Shaker IR (inactivation removed) K+ channels, neuronal alpha 1E and alpha 1A, and cardiac alpha 1C Ca2+ channels using two methods: (a) limiting slope analysis of the conductance-voltage relationship and (b) variance analysis, to evaluate the number of active channels in a patch, combined with the measurement of charge movement in the same patch. We found that in Shaker IR K+ channels the two methods agreed with a z congruent to 13. This suggests that all the channels that gate can open and that all the measured charge is coupled to pore opening in a strictly sequential kinetic model. For all Ca2+ channels the limiting slope method gave consistent results regardless of the presence or type of beta subunit tested (z = 8.6). However, as seen with alpha 1E, the variance analysis gave different results depending on the beta subunit used. alpha 1E and alpha 1E beta 1a gave higher z values (z = 14.77 and z = 15.13 respectively) than alpha 1E beta 2a (z = 9.50, which is similar to the limiting slope results). Both the beta 1a and beta 2a subunits, coexpressed with alpha 1E Ca2+ channels facilitated channel opening by shifting the activation curve to more negative potentials, but only the beta 2a subunit increased the maximum open probability. The higher z using variance analysis in alpha 1E and alpha 1E beta 1a can be explained by a set of charges not coupled to pore opening. This set of charges moves in transitions leading to nulls thus not contributing to the ionic current fluctuations but eliciting gating currents. Coexpression of the beta 2a subunit would minimize the fraction of nulls leading to the correct estimation of the number of channels and z.

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