Sergey Zakharov - Academia.edu (original) (raw)

Papers by Sergey Zakharov

Proceedings of the National Academy of Sciences of the United States of America, Mar 20, 2006

Proceedings of the National Academy of Sciences of the United States of America, Aug 5, 2008

Fig. 1. Mouse BK ␣and ␤1-subunits. (A) Scheme of the threading of BK ␣ through the membrane. The ... more Fig. 1. Mouse BK ␣and ␤1-subunits. (A) Scheme of the threading of BK ␣ through the membrane. The extracellular regions flanking S0-S6, in which Cys were substituted, are indicated by thick lines. (B) Scheme of the threading of BK ␤1 through the membrane. The extracellular regions flanking TM1 and TM2, in which Cys were substituted, are indicated by thick lines. Two disulfide bonds within the extracellular loop are shown.

Nature Cardiovascular Research

Fight-or-flight responses involve β-adrenergic-induced increases in heart rate and contractile fo... more 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 lev...

Nature, 2020

Increased cardiac contractility during fight-or-flight response is caused by β-adrenergic augment... more Increased cardiac contractility during fight-or-flight response is caused by β-adrenergic augmentation of Ca V 1.2 channels 1-4. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant Ca V 1.2 α 1C and β subunits, this regulation persists, implying involvement of extra-channel factors. Here, we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated Ca 2+ channels. We expressed α 1C or β 2B subunits conjugated to ascorbate-peroxidase 5 in mouse hearts and used multiplexed, quantitative proteomics 6,7 to track hundreds of proteins in proximity of Ca V 1.2. We observed that the Ca 2+ channel inhibitor Rad 8,9 , a monomeric G-protein, is enriched in the Ca V 1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific Ser residues on Rad decreases its affinity for auxiliary β-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Journal of Clinical Investigation, 2018

Statistics. Results are mean ± SEM. For multiple group comparisons, 1-way ANOVA followed by multi... more Statistics. Results are mean ± SEM. For multiple group comparisons, 1-way ANOVA followed by multiple comparison testing was performed. For comparisons between 2 groups, an unpaired Student's t test was used. Statistical analyses were performed using Prism 6 (Graphpad Software). Differences were considered statistically significant at P values less than 0.05. Data availability. The data and study materials will be made available to other researchers for purposes of reproducing the results or replicating the procedure. Study approval. The Institutional Animal Care and Use Committee at Columbia University approved all animal experiments.

The Journal of Physiology, 1997

1. Acetylcholine (ACh)‐induced rebound stimulation of the cAMP‐regulated Cl‐ current was studied ... more 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. Expos...

Proceedings of the National Academy of Sciences, 2017

Significance Calcium influx through the cardiac voltage-dependent L-type calcium channel (Ca V 1.... more Significance Calcium influx through the cardiac voltage-dependent L-type calcium channel (Ca V 1.2) increases during “fight or flight” through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. None of the previously identified sites in the α 1C subunit, each painstakingly and singly investigated, were shown to be required for adrenergic modulation of Ca V 1.2. Our approach allowed an unprecedented and massive increase in throughput, because we mutated many potential PKA phosphorylation sites throughout α 1C . By creating transgenic mice expressing either α 1C with alanines substituted for all conserved consensus PKA phosphorylation sites or, separately, α 1C without C-terminal proteolytic cleavage, our paradigm-shifting results show that acute β-adrenergic regulation of Ca V 1.2 does not require phosphorylation of any conserved Ser/Thr of α 1C or the proteolytic cleavage of the C terminus of α 1C .

EP Europace, 2016

Introduction: Atrial fibrillation (AF) occurs and perpetuates for unclear reasons. Women have a d... more Introduction: Atrial fibrillation (AF) occurs and perpetuates for unclear reasons. Women have a decreased incidence of AF compared to men. Previous studies have shown both gain-of-function and loss-of-function mutations in SCN5A are associated with an increased incidence of AF in humans. In a transgenic mouse line expressing human NaV1.5 with a mutation in the local anesthetic binding site (F1759A), spontaneous and prolonged episodes of AF were observed, and enabled the investigation of substrates responsible for AF in mice and gender differences in AF burden. Methods: Whole cell patch and calcium imaging were performed on cardiomyocytes of control and F1759A-Tg mice. Echocardiography, histology, transmission electron microscopy (TEM), and optical mapping were performed in vivo and ex vivo in male (n ¼ 5) and female mice (n ¼ 3). Explanted hearts from Tg and non-Tg mice from 3 to 8 months of age were Langendorff-perfused and a voltage-mapping performed using high-resolution CMOS camera. Action potential duration (APD) regional maps of the atria were analyzed in normal sinus rhythm (NSR) and spontaneous AF. Results: Atrial and ventricular cardiomyocytes isolated from the F1759A-Tg mice had increased persistent Naþ current, which caused reduced cardiac contractility, increased atrial size, atrial hypertrophy, increased atrial fibrosis, myofibril disarray, increased mitochondrial dysfunction with decreased cristae density, and increased in heteregeneity of mitochondrial area. Spontaneous and prolonged episodes of paroxysmal AF was observed during optical mapping in eight F1759A-Tg mice. There was increased APD dispersion in F1759A-Tg atria compared to non-Tgmice. Compared to male mice, female mice had significant decrease in AF burden in vivo and ex vivo, and decreased atrial fibrosis. Conclusions: Incomplete Naþ channel inactivation is sufficient to initiate the structural and arrhythmogenic substrates required to initiate and perpetuate AF in mice. These mice offer a new approach to understand and treat AF, and lead to further understanding of the role of gender and mitochondrial injury in AF

PloS one, 2013

The large-conductance potassium channel (BK) α subunit contains a transmembrane (TM) helix S0 pre... more 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...

Journal of Neuroscience, 2009

Large-conductance, voltage-and Ca 2ϩ-gated potassium (BK) channels control excitability in a numb... more Large-conductance, voltage-and Ca 2ϩ-gated potassium (BK) channels control excitability in a number of cell types. BK channels are composed of ␣ subunits, which contain the voltage-sensor domains and the Ca 2ϩ-sensor domains and form the pore, and often one of four types of ␤ subunits, which modulate the channel in a cell-specific manner. ␤4 is expressed in neurons throughout the brain. Deletion of ␤4 in mice causes temporal lobe epilepsy. Compared with channels composed of ␣ alone, channels composed of ␣ and ␤4 activate and deactivate more slowly. We inferred the locations of the two ␤4 transmembrane (TM) helices TM1 and TM2 relative to the seven ␣ 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 ␤4 TM2 is close to ␣ S0 and that ␤4 TM1 is close to both ␣ 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. Materials and Methods Constructs. Mutants of the BK ␣ subunit (mSlo1, KCNMA1; GenBank accession number NM_010610; 1169 residues; molecular weight 131,700) and human BK ␤4 subunit (KCNMB4, Open Biosystems clone/

The FASEB Journal, 2013

Excessively increased peripheral vasoconstriction is a hallmark of heart failure (HF). Here, we s... more Excessively increased peripheral vasoconstriction is a hallmark of heart failure (HF). Here, we show that in mice with systolic HF post-myocardial infarction, the myogenic tone of third-order mesenteric resistance vessels is increased, the vascular smooth muscle (VSM) membrane potential is depolarized by ϳ20 mV, and vessel wall intracellular [Ca 2؉ ] is elevated relative to that in sham-operated control mice. Despite the increased [Ca 2؉ ], the frequency and amplitude of spontaneous transient outward currents (STOCs), mediated by large conductance, Ca 2؉-activated BK channels, were reduced by nearly 80% (P<0.01) and 25% (P<0.05), respectively, in HF. The expression of the BK ␣ and ␤1 subunits was reduced in HF mice compared to controls (65 and 82% lower, respectively, P<0.01). Consistent with the importance of a reduction in BK channel expression and function in mediating the HF-induced increase in myogenic tone are two further findings: a blunting of paxilline-induced increase in myogenic tone in HF mice compared to controls (0.9 vs. 10.9%, respectively), and that HF does not alter the increased myogenic tone of BK ␤1-null mice. These findings identify electrical dysregulation within VSM, specifically the reduction of BK currents, as a key molecular mechanism sensitizing resistance vessels to pressure-induced vasoconstriction in systolic HF.

Nature Cell Biology, 2004

Activation of store-operated channels (SOCs) and capacitative calcium influx are triggered by dep... more 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.

The Journal of General Physiology, 2008

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

Journal of General Physiology, 2012

Large-conductance voltage- and Ca2+-gated K+ channels are negative-feedback regulators of excitab... more Large-conductance voltage- and Ca2+-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 addit...

Journal of General Physiology, 2010

Large-conductance voltage- and calcium-activated potassium (BK) channels contain four pore-formin... more Large-conductance voltage- and calcium-activated potassium (BK) channels contain four pore-forming α subunits and four modulatory β 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 α, 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 β1 TM helices, TM2 is next to S0, and TM1 is next to TM2. Coexpression of α with two substituted Cys’s, one in S0 and one in S2, and β1 also with two substituted Cys’s, one in TM1 and one in TM2, resulted in two αs cross-linked by one β. Thus, each β lies between and can interact with the voltage-sensing domains of two adjacent α subunits.

Journal of Biological Chemistry, 2004

Voltage-dependent Ca 2؉ channel (Ca v 1.2, L-type Ca 2؉ channel) function is highly regulated by ... more 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 ␣ 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 ␣ 1c subunit.

Journal of Biological Chemistry, 2003

Store-operated cation (SOC) channels and capacitative Ca 2؉ entry (CCE) play very important role ... more 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؉-releaseactivated 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.

Journal of Biological Chemistry, 2006

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