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The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on β-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavβ subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVβ, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVβ, is sufficient for sympathetic upregulation of Ca2+ currents.

Fight-or-flight responses involve β-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart's innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for β-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of β-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel β-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility., Competing Interests: Competing interests Columbia University, Harvard University and NY Presbyterian Hospital have filed a patent (WO/2021/003389), which is published and pending review, reporting a FRET-based method for screening small molecules that increase contractility for the treatment of heart failure. Inventors on this patent application are S.O.M., H.M.C., M.K., S.I.Z., A.N.K., M.B.J. and G.L. The FRET-based assay was utilized in this manuscript for assessing the effects of calyculin and whether 3DA-β2B and 2DA-β2B Ca2+ channel subunits bind to Rad.

Rationale: Changing activity of cardiac Ca V 1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac Ca V 1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic., Objective: The primary goal of this study was to determine how various factors converging at the Ca V 1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure., Methods and Results: We generated transgenic mice with expression of Ca V 1.2 α 1C subunits with (1) mutations ablating interaction between α 1C and β-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α 1C ), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α 1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of Ca V β to α 1C I-II loop and eliminated β-adrenergic agonist stimulation of Ca V 1.2 current. In contrast, introduction of the exon 9* splice variant in the α 1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β 2B and Rad or transgenically expressed in cardiomyocytes., Conclusions: Ca 2+ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α 1C I-II loop. Ca V β binding to α 1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca 2+ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β-binding to α 1C .

The Ca2+-binding protein calmodulin has emerged as a pivotal player in tuning Na+ channel function, although its impact in vivo remains to be resolved. Here, we identify the role of calmodulin and the NaV1.5 interactome in regulating late Na+ current in cardiomyocytes. We created transgenic mice with cardiac-specific expression of human NaV1.5 channels with alanine substitutions for the IQ motif (IQ/AA). The mutations rendered the channels incapable of binding calmodulin to the C-terminus. The IQ/AA transgenic mice exhibited normal ventricular repolarization without arrhythmias and an absence of increased late Na+ current. In comparison, transgenic mice expressing a lidocaine-resistant (F1759A) human NaV1.5 demonstrated increased late Na+ current and prolonged repolarization in cardiomyocytes, with spontaneous arrhythmias. To determine regulatory factors that prevent late Na+ current for the IQ/AA mutant channel, we considered fibroblast growth factor homologous factors (FHFs), which are within the NaV1.5 proteomic subdomain shown by proximity labeling in transgenic mice expressing NaV1.5 conjugated to ascorbate peroxidase. We found that FGF13 diminished late current of the IQ/AA but not F1759A mutant cardiomyocytes, suggesting that endogenous FHFs may serve to prevent late Na+ current in mouse cardiomyocytes. Leveraging endogenous mechanisms may furnish an alternative avenue for developing novel pharmacology that selectively blunts late Na+ current.

Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of Ca V 1.2 voltage-gated calcium channels 1-4 . However, this augmentation persists in transgenic murine hearts expressing mutant Ca V 1.2 α 1C and β subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of β-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated calcium channels. We express α 1C or β 2B subunits conjugated to ascorbate peroxidase 5 in mouse hearts, and use multiplexed quantitative proteomics 6,7 to track hundreds of proteins in the proximity of Ca V 1.2. We observe that the calcium-channel inhibitor Rad 8,9 , a monomeric G protein, is enriched in the Ca V 1.2 microenvironment but is depleted during β-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for β subunits and relieves constitutive inhibition of Ca V 1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of Ca V 1.3 and Ca V 2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

Ca2+ channel β-subunit interactions with pore-forming α-subunits are long-thought to be obligatory for channel trafficking to the cell surface and for tuning of basal biophysical properties in many tissues. Unexpectedly, we demonstrate that transgenic expression of mutant α1C subunits lacking capacity to bind CaVβ can traffic to the sarcolemma in adult cardiomyocytes in vivo and sustain normal excitation-contraction coupling. However, these β-less Ca2+ channels cannot be stimulated by β-adrenergic pathway agonists, and thus adrenergic augmentation of contractility is markedly impaired in isolated cardiomyocytes and in hearts. Similarly, viral-mediated expression of a β-subunit-sequestering peptide sharply curtailed β-adrenergic stimulation of WT Ca2+ channels, identifying an approach to specifically modulate β-adrenergic regulation of cardiac contractility. Our data demonstrate that β subunits are required for β-adrenergic regulation of CaV1.2 channels and positive inotropy in the heart, but are dispensable for CaV1.2 trafficking to the adult cardiomyocyte cell surface, and for basal function and excitation-contraction coupling.

Calcium influx through the voltage-dependent L-type calcium channel (Ca V 1.2) rapidly increases in the heart during "fight or flight" through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac Ca V 1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α 1C and C-terminal proteolytic cleavage of the α 1C subunit. We generated transgenic mice expressing an α 1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for β-adrenergic regulation of Ca V 1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute β-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α 1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α 1C Prevention of C-terminal cleavage did not alter β-adrenergic stimulation of Ca V 1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α 1C in β-adrenergic stimulation of Ca V 1.2, and show that phosphoregulatory sites on α 1C are not redundant and do not each fractionally contribute to the net stimulatory effect of β-adrenergic stimulation. Further, proteolytic cleavage of α 1C is not required for β-adrenergic stimulation of Ca V 1.2., Competing Interests: The authors declare no conflict of interest.

Increased sodium influx via incomplete inactivation of the major cardiac sodium channel Na(V)1.5 is correlated with an increased incidence of atrial fibrillation (AF) in humans. Here, we sought to determine whether increased sodium entry is sufficient to cause the structural and electrophysiological perturbations that are required to initiate and sustain AF. We used mice expressing a human Na(V)1.5 variant with a mutation in the anesthetic-binding site (F1759A-Na(V)1.5) and demonstrated that incomplete Na+ channel inactivation is sufficient to drive structural alterations, including atrial and ventricular enlargement, myofibril disarray, fibrosis and mitochondrial injury, and electrophysiological dysfunctions that together lead to spontaneous and prolonged episodes of AF in these mice. Using this model, we determined that the increase in a persistent sodium current causes heterogeneously prolonged action potential duration and rotors, as well as wave and wavelets in the atria, and thereby mimics mechanistic theories that have been proposed for AF in humans. Acute inhibition of the sodium-calcium exchanger, which targets the downstream effects of enhanced sodium entry, markedly reduced the burden of AF and ventricular arrhythmias in this model, suggesting a potential therapeutic approach for AF. Together, our results indicate that these mice will be important for assessing the cellular mechanisms and potential effectiveness of antiarrhythmic therapies.

The large-conductance, voltage- and Ca(2+)-gated K(+) (BK) channel consists of four α subunits, which form a voltage- and Ca(2+)-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance-voltage (G-V) curve to the left at [Ca(2+)] > 2 µM. In addition to the six transmembrane (TM) helices, S1-S6, conserved in all voltage-dependent K(+) channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2-S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2-S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2-S3) has no effect on V50 for activation, implying that the cytoplasmic end of S0 and the S2-S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V50., (© 2015 Liu et al.)

Large conductance voltage- and calcium-activated potassium (BK) channels are highly expressed in airway smooth muscle (ASM). Utilizing the ovalbumin (OVA) and house dust mite (HDM) models of asthma in C57BL/6 mice, we demonstrate that systemic administration of the BK channel agonist rottlerin (5 μg/g) during the challenge period reduced methacholine-induced airway hyperreactivity (AHR) in OVA- and HDM-sensitized mice (47% decrease in peak airway resistance in OVA-asthma animals, P<0.01; 54% decrease in HDM-asthma animals, P<0.01) with a 35-40% reduction in inflammatory cells and 20-35% reduction in Th2 cytokines in bronchoalveolar lavage fluid. Intravenous rottlerin (5 μg/g) reduced AHR within 5 min in the OVA-asthma mice by 45% (P<0.01). With the use of an ex vivo lung slice technique, rottlerin relaxed acetylcholine-stimulated murine airway lumen area to 87 ± 4% of the precontracted area (P<0.01 vs. DMSO control). Rottlerin increased BK channel activity in human ASM cells (V50 shifted by 73.5±13.5 and 71.8±14.6 mV in control and asthmatic cells, respectively, both P<0.05 as compared with pretreatment) and reduced the frequency of acetylcholine-induced Ca(2+) oscillations in murine ex vivo lung slices. These findings suggest that rottlerin, with both anti-inflammatory and ASM relaxation properties, may have benefit in treating asthma.

Large-conductance voltage- and Ca(2+)-gated K(+) channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory β subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in β2 and β3, as in β1 and β4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse β3 (mβ3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0-αS6. We identified by elimination mβ3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in β1. These are conserved in β2-β4, which have four additional Cys (eight in total), except that mβ3 has one fewer. In β1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mβ3, the free Cys is at position 7; position 2 lacks a Cys present in all other β2-β4; and the disulfide pattern is 1-8, 3-4, and 5-6. Presumably, Cys 2 cross-links to Cys 7 in all other β2-β4. Cross-linking of mβ3 Cys152 to Cys substituted in the flanks of αS0-S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mβ3 perturbed. Although the mβ3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.

The large-conductance potassium channel (BK) α subunit contains a transmembrane (TM) helix S0 preceding the canonical TM helices S1 through S6. S0 lies between S4 and the TM2 helix of the regulatory β1 subunit. Pairs of Cys were substituted in the first helical turns in the membrane of BK α S0 and S4 and in β1 TM2. One such pair, W22C in S0 and W203C in S4, was 95% crosslinked endogenously. Under voltage-clamp conditions in outside-out patches, this crosslink was reduced by DTT and reoxidized by a membrane-impermeant bis-quaternary ammonium derivative of diamide. The rate constants for this reoxidation were not significantly different in the open and closed states of the channel. Thus, these two residues are approximately equally close in the two states. In addition, 90% crosslinking of a second pair, R20C in S0 and W203C in S4, had no effect on the V50 for opening. Taken together, these findings indicate that separation between residues at the extracellular ends of S0 and S4 is not required for voltage-sensor activation. On the contrary, even though W22C and W203C were equally likely to form a disulfide in the activated and deactivated states, relative immobilization by crosslinking of these two residues favored the activated state. Furthermore, the efficiency of recrosslinking of W22C and W203C on the cell surface was greater in the presence of the β1 subunit than in its absence, consistent with β1 acting through S0 to stabilize its immobilization relative to α S4.

Large-conductance voltage- and calcium-activated potassium (BK) channels contain four pore-forming alpha subunits and four modulatory beta subunits. From the extents of disulfide cross-linking in channels on the cell surface between cysteine (Cys) substituted for residues in the first turns in the membrane of the S0 transmembrane (TM) helix, unique to BK alpha, and of the voltage-sensing domain TM helices S1-S4, we infer that S0 is next to S3 and S4, but not to S1 and S2. Furthermore, of the two beta1 TM helices, TM2 is next to S0, and TM1 is next to TM2. Coexpression of alpha with two substituted Cys's, one in S0 and one in S2, and beta1 also with two substituted Cys's, one in TM1 and one in TM2, resulted in two alphas cross-linked by one beta. Thus, each beta lies between and can interact with the voltage-sensing domains of two adjacent alpha subunits.

Large-conductance, voltage- and Ca(2+)-gated potassium (BK) channels control excitability in a number of cell types. BK channels are composed of alpha subunits, which contain the voltage-sensor domains and the Ca(2+)- sensor domains and form the pore, and often one of four types of beta subunits, which modulate the channel in a cell-specific manner. beta 4 is expressed in neurons throughout the brain. Deletion of beta 4 in mice causes temporal lobe epilepsy. Compared with channels composed of alpha alone, channels composed of alpha and beta 4 activate and deactivate more slowly. We inferred the locations of the two beta 4 transmembrane (TM) helices TM1 and TM2 relative to the seven alpha TM helices, S0-S6, from the extent of disulfide bond formation between cysteines substituted in the extracellular flanks of these TM helices. We found that beta 4 TM2 is close to alpha S0 and that beta 4 TM1 is close to both alpha S1 and S2. At least at their extracellular ends, TM1 and TM2 are not close to S3-S6. In six of eight of the most highly crosslinked cysteine pairs, four crosslinks from TM2 to S0 and one each from TM1 to S1 and S2 had small effects on the V(50) and on the rates of activation and deactivation. That disulfide crosslinking caused only small functional perturbations is consistent with the proximity of the extracellular ends of TM2 to S0 and of TM1 to S1 and to S2, in both the open and closed states.

BK channels are composed of alpha-subunits, which form a voltage- and Ca(2+)-gated potassium channel, and of modulatory beta-subunits. The beta1-subunit is expressed in smooth muscle, where it renders the BK channel sensitive to [Ca(2+)](i) in a voltage range near the smooth-muscle resting potential and slows activation and deactivation. BK channel acts thereby as a damped feedback regulator of voltage-dependent Ca(2+) channels and of smooth muscle tone. We explored the contacts between alpha and beta1 by determining the extent of endogenous disulfide bond formation between cysteines substituted just extracellular to the two beta1 transmembrane (TM) helices, TM1 and TM2, and to the seven alpha TM helices, consisting of S1-S6, conserved in all voltage-dependent potassium channels, and the unique S0 helix, which we previously concluded was partly surrounded by S1-S4. We now find that the extracellular ends of beta1 TM2 and alpha S0 are in contact and that beta1 TM1 is close to both S1 and S2. The extracellular ends of TM1 and TM2 are not close to S3-S6. In almost all cases, cross-linking of TM2 to S0 or of TM1 to S1 or S2 shifted the conductance-voltage curves toward more positive potentials, slowed activation, and speeded deactivation, and in general favored the closed state. TM1 and TM2 are in position to contribute, in concert with the extracellular loop and the intracellular N- and C-terminal tails of beta1, to the modulation of BK channel function.

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 V(50) 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.

Here we tested the role of calcium influx factor (CIF) and calcium-independent phospholipase A2 (iPLA2) in activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry in rat basophilic leukemia (RBL-2H3) cells. We demonstrate that 1) endogenous CIF production may be triggered by Ca2+ release (net loss) as well as by simple buffering of free Ca2+ within the stores, 2) a specific 82-kDa variant of iPLA2beta and its corresponding activity are present in membrane fraction of RBL cells, 3) exogenous CIF (extracted from other species) mimics the effects of endogenous CIF and activates iPLA2beta when applied to cell homogenates but not intact cells, 4) activation of ICRAC can be triggered in resting RBL cells by dialysis with exogenous CIF, 5) molecular or functional inhibition of iPLA2beta prevents activation of ICRAC, which could be rescued by cell dialysis with a human recombinant iPLA2beta, 6) dependence of ICRAC on intracellular pH strictly follows pH dependence of iPLA2beta activity, and 7) (S)-BEL, a chiral enantiomer of suicidal substrate specific for iPLA2beta, could be effectively used for pharmacological inhibition of ICRAC and store-operated Ca2+ entry. These findings validate and significantly advance our understanding of the CIF-iPLA2-dependent mechanism of activation of ICRAC and store-operated Ca2+ entry.

In a wide variety of cell types, including neurons and smooth muscle cells, activation of the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels causes transient membrane hyperpolarization, thereby regulating cellular excitability. Similar to other voltage-gated ion channels, BK channels, a tetramer of alpha-subunits, associate with auxiliary beta-subunits in a tissue-specific manner, modifying the channel's gating properties. The BK beta1-subunit, which is expressed in smooth muscle, increases the apparent Ca(2+) sensitivity (marked by a hyperpolarizing shift in the conductance-voltage relationship at a given Ca(2+) concentration), slows macroscopic activation and deactivation, and is required for channel activation by 17beta-estradiol. The beta1-subunit is essential for normal regulation of vascular smooth muscle contractility and blood pressure. Little is known, however, about the molecular mechanisms of beta1-subunit modulation of alpha-subunits. Here we show that the beta1-subunit's modulation of the Ca(2+) and 17beta-estradiol sensitivities can be dissociated from its effects on gating kinetics by truncation of the alpha-subunit's extracellular N-terminal residues. The BK alpha-subunit N terminus interacts uniquely with the beta1-subunit: beta2 regulation of the alpha-subunit is unaltered by truncation of the N terminus. Although the functional interaction of alpha and beta1 requires the N-terminal tail of alpha, the physical association requires the S1, S2, and S3 transmembrane helices of alpha.

Pharmacologic approaches to activate K+ channels represent an emerging strategy to regulate membrane excitability. Here we report the identification and characterization of a lipid soluble toxin, mallotoxin (rottlerin), which potently activates the large conductance voltage and Ca2+-activated K+ channel (BK) expressed in a heterologous expression system and human vascular smooth muscle cells, shifting the conductance/voltage relationship by >100 mV. Probing the mechanism of action, we discover that the BK channel can be activated in the absence of divalent cations (Ca2+, Mg2+), suggesting that the mallotoxin mechanism of action involves the voltage-dependent gating of the channel. Mallotoxin-activated channels remain incrementally sensitive to Ca2+ and beta subunits. In comparison to other small hydrophobic poisons, anesthetic agents, and protein toxins that inhibit ion channel activity, mallotoxin potently activates channel activity. In certain respects, mallotoxin acts as a BK channel beta1 subunit mimetic, preserving BK channel Ca2+ sensitivity yet adjusting the set-point for BK channel activation to a more hyperpolarized membrane potential.

Voltage-dependent Ca(2+) channel (Ca(v)1.2, L-type Ca(2+) channel) function is highly regulated by hormones and neurotransmitters in large part through the activation of kinases and phosphatases. Regulation of Ca(v)1.2 by protein kinase C (PKC) is of significant physiologic importance, mediating, in part, the cardiac response to hormonal regulation. Although PKC has been reported to mediate activation and/or inhibition of Ca(v)1.2 function, the molecular mechanisms mediating the response have not been definitively elucidated. We show that PKC forms a macromolecular complex with the alpha(1c) subunit of Ca(v)1.2 through direct interaction with the C terminus. This interaction leads to phosphorylation of the channel in response to activators of PKC. We identify Ser(1928) as the residue that is phosphorylated by PKC in vitro and in vivo. Ser(1928) has been identified previously as the site mediating, in part, the protein kinase A up-regulation of channel activity. Thus, the protein kinase A and PKC signaling pathways converge on the Ca(v)1.2 complex at Ser(1928) to increase channel activity. Our results identify two mechanisms leading to regulation of Ca(v)1.2 activity by PKC: pre-association of the channel with PKC isoforms and phosphorylation of specific sites within the alpha(1c) subunit.

Cardiac tissue expresses several TRP proteins as well as a Mg2+ -inhibited, non-selective cation current (IMIC) that bears many characteristics of TRP channel currents. We used the whole-cell voltage clamp technique in pig and rat ventricular myocytes to characterize the permeation, blockage properties and regulation of the cardiac IMIC channels in order to compare them with TRP channels, in particular with Mg2+ -sensitive TRPM6 and TRPM7. We show that removing extracellular divalent cations unmasks large inward and outward monovalent currents, which can be inhibited by intracellular Mg2+. Inward currents are suppressed upon replacing extracellular Na+ by NMDG+. Divalent cations block monovalent IMIC and, at 10-20 mm, carry measurable currents. Their efficacy sequence in decreasing outward IMIC (Ni2+ = Mg2+ > Ca2+ > Ba2+) and in inducing inward IMIC (Ni2+ >> Mg2+ = Ca2+ approximately Ba2+), and their permeabilities calculated from reversal potentials are similar to those of TRPM6 and TRPM7 channels. The trivalent cations Gd3+ and Dy3+ also block IMIC in a voltage-dependent manner (delta = 0.4-0.5). In addition they inhibit the inward current carried by divalent cations. IMIC is regulated by pH. Decreasing or increasing extracellular pH decreased and increased IMIC, respectively (pH0.5 = 6.9, nH = 0.98). Qualitatively similar results were obtained on IMIC in rat basophilic leukaemia cells. These effects in cardiac myocytes were absent in the presence of high intracellular buffering by 40 mm Hepes. Our results suggest that IMIC in cardiac cells is due to TRPM channels, most probably to TRPM6 or TRPM7 channels or to their heteromultimeres.

We have recently found that diethylstilbestrol (DES), a synthetic estrogen agonist, inhibits thrombin-induced Ca(2+) influx in human platelets, but it remains unclear to what extend this effect might be related to the store-operated Ca(2+) influx pathway. To study the effect of DES on store-operated channels and capacitative Ca(2+) influx, we used rat basophilic leukemia (RBL) cells, vascular smooth muscle cells (SMC), and human platelets, and recorded whole-cell Ca(2+) release-activated Ca(2+) (CRAC) currents and thapsigargin (TG)-induced capacitative Ca(2+) influx. In this study, we demonstrate that extracellular DES produces a dose-dependent and reversible inhibition of CRAC currents in RBL cells (IC(50), approximately 0.5 microM), whereas intracellular DES (25 microM) has no effect. Extracellular DES (up to 30 microM) inhibited only CRAC but did not affect a whole-cell monovalent cation current mediated by TRPM7 channels. DES effectively inhibited TG-induced capacitative Ca(2+) influx in a dose-dependent manner with an IC(50) values of approximately 0.1 microM in RBL cells, <0.1 microM in SMC, and approximately 1 microM in human platelets. It is noteworthy that trans-stilbene, a close structural analog of DES that lacks hydroxyl and ethyl groups, had no effect on CRAC current and on store-operated Ca(2+) influx. Thus, we found DES to be a very effective inhibitor of store-operated channels and Ca(2+) influx in a variety of cell types.

Activation of store-operated channels (SOCs) and capacitative calcium influx are triggered by depletion of intracellular calcium stores. However, the exact molecular mechanism of such communication remains unclear. Recently, we demonstrated that native SOC channels can be activated by calcium influx factor (CIF) that is produced upon depletion of calcium stores, and showed that Ca(2+)-independent phospholipase A(2) (iPLA(2)) has an important role in the store-operated calcium influx pathway. Here, we identify the key plasma-membrane-delimited events that result in activation of SOC channels. We also propose a novel molecular mechanism in which CIF displaces inhibitory calmodulin (CaM) from iPLA(2), resulting in activation of iPLA(2) and generation of lysophospholipids that in turn activate soc channels and capacitative calcium influx. Upon refilling of the stores and termination of CIF production, CaM rebinds to iPLA(2), inhibits it, and the activity of SOC channels and capacitative calcium influx is terminated.

Store-operated cation (SOC) channels and capacitative Ca(2+) entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca(2+) signaling. Here, we present the first evidence that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is a crucial molecular determinant in activation of SOC channels and store-operated Ca(2+) entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA(2) leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca(2+)-release-activated Ca(2+) (CRAC) channels. Transfection of vascular smooth muscle cells (SMC) with antisense, but not sense, oligonucleotides for iPLA(2) impaired thapsigargin (TG)-induced activation of iPLA(2) and TG-induced Ca(2+) and Mn(2+) influx. Identical inhibition of TG-induced Ca(2+) and Mn(2+) influx (but not Ca(2+) release) was observed in SMC, human platelets, and Jurkat T-lymphocytes when functional activity of iPLA(2) was inhibited by its mechanism-based suicidal substrate, bromoenol lactone (BEL). Moreover, irreversible inhibition of iPLA(2) impaired TG-induced activation of single nonselective SOC channels in SMC and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-induced activation of whole-cell CRAC current in rat basophilic leukemia cells. Thus, functional iPLA(2) is required for activation of store-operated channels and capacitative Ca(2+) influx in wide variety of cell types.

1 Previously we have described a monovalent cation (MC) current that could be unmasked by the removal of extracellular divalent cations in vascular smooth muscle cells (SMC) and cardiac myocytes, but specific and potent inhibitors of MC current have not been found, and the mechanism of its intracellular regulation remains obscure. 2 Here we show that small MC current is present in intact cells and could be dramatically up-regulated during cell dialysis. MC current in dialyzed cells strongly resembled monovalent cation current attributed to Ca(2+) release-activated Ca(2+)-selective (CRAC) channels, but its activation did not require depletion of Ca(2+) stores, and was observed when the cells were dialyzed with, or without BAPTA. 3 Intracellular free Mg(2+) inhibits MC current with K(d)=250 microM. 4 Extracellular (but not intracellular) spermine effectively blocked MC current with K(d) =3-10 microM, while store-operated cations (SOC) channels and capacitative Ca(2+) influx were not affected. 5 Spermine effectively inhibited MC current-induced SMC depolarization, and prevented Ca(2+) paradox-induced vascular contracture. 6 Both, MC and SOC currents were inhibited by 2-aminoethoxydiphenyl borate (2-APB). 7 It is concluded that MC current could be regulated by intracellular Mg(2+), and low concentrations of extracellular spermine could be used to discriminate it from SOC current, and to assess its role in cellular function.

1. The effects of removal of extracellular divalent cations (experimental calcium paradox conditions) were studied on the whole-cell current in freshly isolated smooth muscle cells (SMCs), and on contraction in rabbit aortic rings. 2. Aortic rings treated for 30-60 min with extracellular Ca2+- and Mg2+-free solution contracted following readmission of extracellular Ca2+, even in the presence of nifedipine. 3. In isolated SMCs, the removal of extracellular Ca2+ and Mg2+ induced a non-inactivating whole-cell inward current and membrane depolarization. This current was a monovalent cation (MC) current which reversed at around 0 mV and conducted K+ >= Cs+ > Na+ > Li+. Extracellular divalent cations (Ca2+, Mg2+, Ba2+, Mn2+ and Ni2+) inhibited MC current. 4. Using noise analysis of the whole-cell MC current, the single MC channel conductance was estimated to be < 450 fS. 5. MC current was insensitive to nifedipine, TEA, 4-aminopyridine, SK&F 96365 and S-nitroso-N-acetyl-penicillamine (SNAP), but was decreased by amiloride and low pH. 6. When EGTA was present in Ca2+- and Mg2+-free solution, a significant nifedipine-sensitive Na+ current through L-type Ca2+ channels developed in addition to MC current. 7. It is concluded that upon the removal of extracellular Ca2+ and Mg2+ from resting SMCs, an inward MC current develops allowing Na+ influx and causing SMC depolarization which could be the important steps leading to vessel contraction upon Ca2+ readmission. Addition of EGTA to Ca2+- and Mg2+-free solution greatly potentiates Na+ influx and vessel contraction by allowing additional Na+ influx through L-type Ca2+ channels which are activated presumably by MC current-induced depolarization.

1. Acetylcholine (ACh)-induced rebound stimulation of the cAMP-regulated Cl- current was studied in isolated guinea-pig ventricular myocytes using dialysing and dialysis-limiting configurations of the whole-cell patch-clamp technique. 2. Exposure to and subsequent washout of ACh produced a transient rebound stimulation of the Cl- current. However, this rebound response was only observed in the presence of submaximally stimulating concentrations of the cAMP-producing agonists isoprenaline (Iso) or histamine. ACh-induced rebound stimulation was not observed in the presence of maximally stimulating concentrations of Iso, nor was it observed in the absence of Iso. 3. To prevent saturation of responses during rebound, the effects of ACh were studied in the presence of a subthreshold concentration of Iso (0.001 microM). Varying the duration of exposure to ACh before washout demonstrated that the stimulatory effect of 1 microM ACh approaches steady state with a time constant of 34 s. Exposing myocytes to varying concentrations of ACh for 90 s demonstrated that the EC50 for the stimulatory effect of ACh was 0.15 microM with a maximum response equal to 67% of that obtained by a maximally stimulating concentration of Iso alone. 4. Rebound stimulation of the Cl- current could also be elicited by washing in 2 microM atropine during exposure to ACh, instead of washing out ACh. Furthermore, ACh-induced rebound was blocked by the M2 muscarinic receptor antagonist methoctramine but not by the M1 receptor antagonist pirenzepine. Rebound was also blocked in pertussis toxin (PTX)-treated myocytes. 5. ACh-induced rebound stimulation was not blocked by: (a) L-NMMA, an inhibitor of nitric oxide synthase activity; (b) Methylene Blue, LY-83583, and ODQ, inhibitors of cGMP production; or (c) milrinone, an inhibitor of cGMP-dependent phosphodiesterase activity. 6. These results indicate that ACh can stimulate cAMP-regulated ion channel activity in cardiac ventricular myocytes by facilitating beta-adrenergic and histaminergic responses. This is opposite to the inhibitory actions more typically associated with muscarinic receptor stimulation in ventricular myocardium. This stimulatory effect of ACh is mediated through M2 muscarinic receptors and a PTX-sensitive G-protein, but it does not appear to involve the production of nitric oxide or cGMP.

It has recently been demonstrated that NO plays an obligatory role in muscarinic inhibition of beta-adrenergically stimulated ion channels in cardiac sinoatrial node cells (J Gen Physiol. 1995;106:45-65). We looked for evidence that NO might play a similar role in ventricular cells by using histochemical staining for NO synthase (NOS) activity and whole-cell patch-clamp recording of cAMP-regulated Cl- currents. Myocytes isolated from guinea pig hearts stained positively for NADPH-diaphorase activity, suggesting that these cells do express NOS. Acetylcholine (ACh) inhibition of the R(-)-isoproterenol bitartrate (Iso)-activated Cl- current was also reversed by the cGMP-lowering agents LY-83583 and methylene blue, consistent with idea that NO activation of guanylate cyclase may contribute to muscarinic responses. However, LY-83583 and methylene blue activated the Cl- current in the presence of subthreshold concentrations of Iso alone, suggesting that their effects may not be due to antagonism of an NO/cGMP-dependent response. Furthermore, ACh inhibition of Iso-activated Cl- currents could not be mimicked by the NO donors sodium nitroprusside,3-morpholinosydnonimine, and spermine-NO. Similarly, ACh inhibition of the Iso-activated Cl- current could not be blocked by the NOS inhibitor NG-monomethyl-L-arginine. These results indicate that even though ventricular myocytes possess NOS activity, NO production does not play an important role in muscarinic inhibition of beta-adrenergically regulated Cl- channels in these cells.

Autonomic regulation of the cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current was studied in isolated guinea pig ventricular myocytes using various configurations of the whole cell patch-clamp technique. When currents were recorded using the conventional patch-clamp technique, it was possible to continue to activate the Cl- current on repeated exposure to isoproterenol (Iso) for up to 60 min after initiating dialysis. However, there was significant rundown of the magnitude of the Cl- current response to the maximally stimulating concentrations of Iso. In addition, the concentration of Iso that produced half-maximal activation of the Cl- current (K1/2) increased with time. Conversely, the K1/2 for acetylcholine inhibition of the Iso-activated current decreased with time. When currents were recorded using the perforated patch-clamp technique, the sensitivity to both beta-adrenergic- and muscarinic-receptor stimulation was stable. Immediately after initiation of dialysis with the conventional patch-clamp technique, the sensitivity to Iso was nearly identical to that determined using the perforated patch-clamp technique. However, the initial sensitivity to muscarinic-receptor activation was significantly greater. These results indicate that cell dialysis associated with conventional patch-clamp techniques not only results in a time-dependent rundown of current amplitude, but it also significantly alters the concentration dependence of beta-adrenergic and muscarinic-receptor regulation of ion channel function.

In guinea pig ventricle, the protein kinase A-regulated Cl- current (ICl) is conducted by an alternatively spliced isoform of the cystic fibrosis transmembrane conductance regulator. We studied muscarinic regulation of this current using the whole-cell configuration of the patch-clamp technique. Acetylcholine (ACh) antagonized activation of ICl activated by 1 microM isoproterenol (ISO) in a concentration-dependent manner. The concentration of ACh that produced a half-maximal effect (K1/2) was 36 nM, the slope factor was 1.1, and the relative magnitude of the Cl- conductance at maximally effective concentrations of ACh (Gmin) was 21% of that observed in the presence of ISO alone. In the presence of 100 nM atropine, a competitive antagonist at the muscarinic receptor, the K1/2 value for ACh inhibition of ICl was increased to 4.3 microM, but the slope factor and Gmin were not affected, which indicated that the dissociation constant (KB) for atropine was < 1 nM. ACh-induced inhibition of the ISO-activated ICl was also blocked by the quaternary ammonium compound tetraethylammonium (TEA). Like atropine, TEA increased the K1/2 value for ACh inhibition of ICl without affecting the slope factor or Gmin. Schild analysis confirmed that TEA is also a competitive antagonist at the muscarinic receptor, with a KB value of 137 microM. However, tetramethylammonium (TMA), a structurally related compound, acted as an agonist at the muscarinic receptor. TMA inhibited ICl activated by 1 microM ISO with a K1/2 value of 342 microM, a slope factor of 0.87 and a Gmin value of 17%. Increasing the concentration of ISO shifted the K1/2 value for both ACh and TMA inhibition of ICl to higher concentrations and increased Gmin, without significantly affecting the slope factor. These results indicate that muscarinic regulation of ICl depends on the level of beta adrenergic stimulation in a functionally uncompetitive manner. They also suggest that TMA acts like ACh, a full agonist at the muscarinic receptor. Furthermore, we conclude that quaternary ammonium compounds, which are often used as ion substitutes and direct ion channel blockers, should be used with caution because of the significant and diverse effects they exert at muscarinic receptors.

Replacement of extracellular Na+ with tetramethylammonium (TMA) reduces the magnitude of the Cl- current activated by beta-adrenergic receptor stimulation in guinea pig ventricular myocytes. However, the effects of replacing Na+ appear to be associated with the presence of TMA, rather than the absence of Na+. Direct addition of TMA to extracellular solutions, without changing the Na+ concentration, was able to inhibit the Cl- current activated by isoproterenol (Iso) in a concentration-dependent manner. The concentration of TMA that caused half-maximal inhibition was 327 microM when the Cl- current was activated by 1 microM Iso and 29 microM when the Cl- current was activated by 0.03 microM Iso. The effect of TMA was also blocked by atropine, suggesting that TMA exerts its effect through stimulation of the muscarinic receptors. Furthermore, TMA inhibited the Iso-activated Ca2+ current, as would be expected for an effect involving muscarinic receptor stimulation. The response to complete Na+ replacement with TMA could not be overcome by increasing the concentration of Iso 1,000-fold, and direct addition of TMA was able to antagonize the Cl- current activated independently of the beta-adrenergic receptor, using forskolin and histamine. These results are consistent with the hypothesis that TMA does not exert its effects through a competitive mechanism at the beta-adrenergic receptor. It is concluded that TMA is able to antagonize adenosine 3',5'-cyclic monophosphate-dependent activation of ion channels in the heart through activation of muscarinic receptors.

In experiments on isolated rat hearts the effects of focused continuous and impulse ultrasound (543 Hz, with intensity up to 7.8 W/cm2 at a focal region) on a pressure developed by left ventricle and electrograms were studied. In all experiments ultrasound induced extra-excitations of the heart, which appeared when intensity was 1.35 +/- 0.21 W/cm2 (n = 9). Simultaneously with the extra-excitations the cavitation bursts were recorded at intensity of 1.52 +/- 0.18 W/cm2 (n = 6). Acoustic cavitation (after 30 sec of exposure) resulted in a significant decrease of the developed pressure (from 100.8 +/- 3.8 mm Hg to 95.1 +/- 4.3 mm Hg, p 0.001), measured in 2 min after the end of the exposure. In the absence of cavitation the ultrasound was found to have no effects on cardiac performance. Electrograms recorded during acoustic pacing show that a pattern of the heart excitation changed from stimulus to stimulus.

The effects of ryanodine on ventricular arrhythmias in guinea-pigs in vivo, on delayed after potentials and after contractions, and on spontaneous oscillations of the membrane potential (SOP) and of resting tension (SOT) of guinea-pig papillary muscle under ouabain intoxication were studied. After addition of ouabain (1 microM) the after potentials, after contractions, and SOP and SOT amplitude were significantly increased. The power spectra of SOT and SOP under these conditions had a resonance harmonic with the frequency of about 5 Hz. Three to 5 mins after the addition of ryanodine (0.1-0.5 microM), the after potentials, after contractions, and SOP and SOT were abolished, suggesting a close relationship between these oscillations and the oscillatory activity of sarcoplasmic reticulum. In in vivo experiments, ouabain-induced (75-115 micrograms/kg) ventricular arrhythmias were terminated 4 to 5 min after intravenous injection of ryanodine (15 micrograms/kg); within 8-10 min, sinus rhythm was completely restored. We attribute the antiarrhythmic effect of ryanodine to a cellular effect and alteration of SR function, rather than to effects that are secondary to this.

Acoustic cavitation induced by continuous focused ultrasound (1.4 W/cm2, 543 Hz) was found to result in reversible membrane depolarization (by 54 mV), loss of excitability and contracture in the rat papillary muscles. The same intensities of impulse ultrasound had positive inotropic effects.