Whole-cell Ca2 Research Articles

Aberrant Splicing Promotes Proteasomal Degradation of L-type CaV1.2 Calcium Channels by Competitive Binding for CaVβ Subunits in Cardiac Hypertrophy.

Decreased expression and activity of CaV1.2 calcium channels has been reported in pressure overload-induced cardiac hypertrophy and heart failure. However, the underlying mechanisms remain unknown. Here we identified in rodents a splice variant of CaV1.2 channel, named CaV1.2e21+22, that contained the pair of mutually exclusive exons 21 and 22. This variant was highly expressed in neonatal hearts. The abundance of this variant was gradually increased by 12.5-folds within 14 days of transverse aortic banding that induced cardiac hypertrophy in adult mouse hearts and was also elevated in left ventricles from patients with dilated cardiomyopathy. Although this variant did not conduct Ca2+ ions, it reduced the cell-surface expression of wild-type CaV1.2 channels and consequently decreased the whole-cell Ca2+ influx via the CaV1.2 channels. In addition, the CaV1.2e21+22 variant interacted with CaVβ subunits significantly more than wild-type CaV1.2 channels, and competition of CaVβ subunits by CaV1.2e21+22 consequently enhanced ubiquitination and subsequent proteasomal degradation of the wild-type CaV1.2 channels. Our findings show that the resurgence of a specific neonatal splice variant of CaV1.2 channels in adult heart under stress may contribute to heart failure.

Dysregulation of Neuronal Ca2+ Channel Linked to Heightened Sympathetic Phenotype in Prohypertensive States.

Hypertension is associated with impaired nitric oxide (NO)-cyclic nucleotide (CN)-coupled intracellular calcium (Ca(2+)) homeostasis that enhances cardiac sympathetic neurotransmission. Because neuronal membrane Ca(2+) currents are reduced by NO-activated S-nitrosylation, we tested whether CNs affect membrane channel conductance directly in neurons isolated from the stellate ganglia of spontaneously hypertensive rats (SHRs) and their normotensive controls. Using voltage-clamp and cAMP-protein kinase A (PKA) FRET sensors, we hypothesized that impaired CN regulation provides a direct link to abnormal signaling of neuronal calcium channels in the SHR and that targeting cGMP can restore the channel phenotype. We found significantly larger whole-cell Ca(2+) currents from diseased neurons that were largely mediated by the N-type Ca(2+) channel (Cav2.2). Elevating cGMP restored the SHR Ca(2+) current to levels seen in normal neurons that were not affected by cGMP. cGMP also decreased cAMP levels and PKA activity in diseased neurons. In contrast, cAMP-PKA activity was increased in normal neurons, suggesting differential switching in phosphodiesterase (PDE) activity. PDE2A inhibition enhanced the Ca(2+) current in normal neurons to a conductance similar to that seen in SHR neurons, whereas the inhibitor slightly decreased the current in diseased neurons. Pharmacological evidence supported a switching from cGMP acting via PDE3 in control neurons to PDE2A in SHR neurons in the modulation of the Ca(2+) current. Our data suggest that a disturbance in the regulation of PDE-coupled CNs linked to N-type Ca(2+) channels is an early hallmark of the prohypertensive phenotype associated with intracellular Ca(2+) impairment underpinning sympathetic dysautonomia. Here, we identify dysregulation of cyclic-nucleotide (CN)-linked neuronal Ca(2+) channel activity that could provide the trigger for the enhanced sympathetic neurotransmission observed in the prohypertensive state. Furthermore, we provide evidence that increasing cGMP rescues the channel phenotype and restores ion channel activity to levels seen in normal neurons. We also observed CN cross-talk in sympathetic neurons that may be related to a differential switching in phosphodiesterase activity. The presence of these early molecular changes in asymptomatic, prohypertensive animals could facilitate the identification of novel therapeutic targets with which to modulate intracellular Ca(2+) Turning down the gain of sympathetic hyperresponsiveness in cardiovascular disease associated with sympathetic dysautonomia would have significant therapeutic utility.

Deduction of a calcium ion circuit affecting rooster sperm in vitro.

Four premises for rooster sperm preservation were outlined previously. Understanding mitochondrial Ca cycling in terms of whole-cell Ca flux was one premise. The present work tested the hypothesis that sperm mitochondria can be damaged by intracellular as well as extracellular Ca. Sperm were washed by centrifugation through 12% (wt/vol) Sperm were washed by centrifugation through 12%(at/vol) Accudenz to procure sperm at a physiological concentration within a chemically-defined suspension. Five solutions were tested. Each solution contained 30 m glucose, and had an osmolality of 320 mmol/kg and a pH of 7.4. Washed sperm were diluted to 2.0 × 10 sperm/mL. Each replicate sperm suspension was cooled to 10°C. Sperm mobility was measured after 1, 2, 4, 8, 12, and 24 h. Data were plotted as a function of time in each experiment. Function type was confirmed by lack of fit analysis. A parabola with a maximum at 3.7 h was observed when sperm were suspended in 205 m taurine buffered with 50 m-tris[hydroxyl-methyl]methyl-2-amino-ethanesulfonic acid (TES). This effect was attributed to a Ca flux from the nuclear envelope into mitochondria. An exponential decay was observed when TES-buffered taurine contained 2 m Ca. This effect was attributed to mitochondrial Ca overload induced by uptake of extracellular Ca. Exponential decay also was observed when TES-buffered taurine contained a Ca chelator. This effect was attributed to a Ca flux from the nuclear envelope through mitochondria and then into an extracellular Ca sink. This possibility was supported by the response of sperm to thapsigargin. Specifically, inhibition of sarcoendoplasmic reticulum Ca-ATPase compromised sperm mobility relative to a buffer control. Finally, a 60 m phosphate buffer containing 2 m citrate yielded a linear relationship in contrast to the TES-buffered solutions tested. Sperm mobility after 24 h of storage in the phosphate buffer was 92% of that observed for prewashed sperm. The linear response was attributed to weak chelators providing resistance within a Ca circuit and thereby preventing mitochondrial Ca overload. Fertility, however, was compromised when hens were inseminated with mobile sperm recovered after either 8 or 24 h of storage at 10°C. In conclusion, sperm cell Ca homeostasis was proven to be critical for maintaining sperm mobility in vitro, but mitochondrial Ca uptake is not the sole phenomenon that compromises sperm function during in vitro storage.

The gain-of-function enhancement of IP3-receptor channel gating by familial Alzheimer's disease-linked presenilin mutants increases the open probability of mitochondrial permeability transition pore

Mutants in presenilins (PS1 or PS2) are the major cause of familial Alzheimer's disease (FAD). They affect intracellular Ca2+ homeostasis by increasing the open probability (Po) of inositol 1,4,5-trisposphate (IP3) receptor (IP3R) Ca2+ release channel located on the endoplasmic reticulum (ER) leading to exaggerated Ca2+ release into a cytoplasmic microdomain formed by neighboring cluster of a few IP3R channels and mitochondrial Ca2+ uniporter (MCU). Ca2+ concentration in the microdomain ([Ca2+]mic) depends on the distance between the cluster and MCU (r); the number of IP3R in the cluster releasing Ca2+ to the cytoplasm (nIP3R), and Po of IP3R. Using experimental whole-cell IP3R-mediated cytosolic Ca2+ data, in conjunction with a computational model of cell bioenergetics, a data-driven Markov chain model for IP3R gating, and a model for the dynamics of the mitochondrial permeability transition pore (PTP), we explore differences in mitochondrial Ca2+ uptake in cells expressing wild type (PS1-WT) and FAD-causing mutant (PS1-M146L) PS. We find that increased mitochondrial [Ca2+]m due to the gain-of-function enhancement of IP3R channels in the cells expressing PS1-M146L leads to the opening of PTP in high conductance state (PTPh), where the latency of opening is inversely correlated with r and proportional to nIP3R. Furthermore, we observe diminished inner mitochondrial membrane potential (ΔΨm), [NADH], [Ca2+]m, and [ATP] when PTP opens. Additionally, we explore how parameters such as the pH gradient, inorganic phosphate concentration, and the rate of the Na+/Ca2+-exchanger affect the latency of PTP to open in PTPh.

Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure.

Abnormal cellular Ca2+ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca2+ transient amplitude due to decreased sarcoplasmic reticulum Ca2+ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca2+ release events which occur when sarcoplasmic reticulum Ca2+ content exceeds a certain threshold level. Such Ca2+ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca2+ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca2+ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca2+ transients and Ca2+ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca2+ content, decreased Ca2+ transient amplitude, and increased frequency of Ca2+ waves. These results are in line with a decreased threshold for diastolic Ca2+ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca2+ release were increased THR286 phosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca2+ content and increased propensity for diastolic Ca2+ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca2+ handling after a myocardial infarction.

Berberine alleviates the cerebrovascular contractility in streptozotocin-induced diabetic rats through modulation of intracellular Ca²⁺ handling in smooth muscle cells.

BackgroundVascular dysfunction is a distinctive phenotype in diabetes mellitus. Current treatments mostly focus on the tight glycemic control and few of these treatments have been designed to directly recover the vascular dysfunction in diabetes. As a classical natural medicine, berberine has been explored as a possible therapy for DM. In addition, it is reported that berberine has an extra-protective effect in diabetic vascular dysfunction. However, little is known whether the berberine treatment could ameliorate the smooth muscle contractility independent of a functional endothelium under hyperglycemia. Furthermore, it remains unknown whether berberine affects the arterial contractility by regulating the intracellular Ca2+ handling in vascular smooth cells (VSMCs) under hyperglycemia.MethodsSprague–Dawley rats were used to establish the diabetic model with a high-fat diet plus injections of streptozotocin (STZ). Berberine (50, 100, and 200 mg/kg/day) were intragastrically administered to control and diabetic rats for 8 weeks since the injection of STZ. The intracellular Ca2+ handling of isolated cerebral VSMCs was investigated by recording the whole-cell L-type Ca2+ channel (CaL) currents, assessing the protein expressions of CaL channel, and measuring the intracellular Ca2+ in response to caffeine. Our results showed that chronic administration of 100 mg/kg/day berberine not only reduced glucose levels, but also inhibited the augmented contractile function of cerebral artery to KCl and 5-hydroxytryptamine (5-HT) in diabetic rats. Furthermore, chronic administration of 100 mg/kg/day berberine significantly inhibited the CaL channel current densities, reduced the α1C-subunit expressions of CaL channel, decreased the resting intracellular Ca2+ ([Ca2+]i) level, and suppressed the Ca2+ releases from RyRs in cerebral VSMCs isolated from diabetic rats. Correspondingly, acute application of 10 μM berberine could directly inhibit the hyperglycemia-induced CaL currents and suppress the hyperglycemia-induced Ca2+ releases from RyRs in cerebral VSMCs isolated from normal control rats.ConclusionsOur study indicated that berberine alleviated the cerebral arterial contractility in the rat model of streptozotocin-induced diabetes via regulating the intracellular Ca2+ handling of smooth muscle cells.

Suppression of Sleep Spindle Rhythmogenesis in Mice with Deletion of CaV3.2 and CaV3.3 T-type Ca2+ Channels

Low-threshold voltage-gated T-type Ca(2+) channels (T-channels or CaV3 channels) sustain oscillatory discharges of thalamocortical (TC) and nucleus Reticularis thalami (nRt) cells. The CaV3.3 subtype dominates nRt rhythmic bursting and mediates a substantial fraction of spindle power in the NREM sleep EEG. CaV3.2 channels are also found in nRt, but whether these contribute to nRt-dependent spindle generation is unexplored. We investigated thalamic rhythmogenesis in mice lacking this subtype in isolation (CaV3.2KO mice) or in concomitance with CaV3.3 deletion (CaV3.double-knockout (DKO) mice). We examined discharge characteristics of thalamic cells and intrathalamic evoked synaptic transmission in brain slices from wild-type, CaV3.2KO and CaV3.DKO mice through patch-clamp recordings. The sleep profile of freely behaving CaV3.2KO and CaV3.DKO mice was assessed by polysomnographic recordings. CaV3.2 channel deficiency left nRt discharge properties largely unaltered, but additional deletion of CaV3.3 channels fully abolished low-threshold whole-cell Ca(2+) currents and bursting, and suppressed burst-mediated inhibitory responses in TC cells. CaV3.DKO mice had more fragmented sleep, with shorter NREM sleep episodes and more frequent microarousals. The NREM sleep EEG power spectrum displayed a relative suppression of the σ frequency band (10-15 Hz), which was accompanied by an increase in the δ band (1-4 Hz). Consistent with previous findings, CaV3.3 channels dominate nRt rhythmogenesis, but the lack of CaV3.2 channels further aggravates neuronal, synaptic, and EEG deficits. Therefore, CaV3.2 channels can boost intrathalamic synaptic transmission, and might play a modulatory role adjusting the relative presence of NREM sleep EEG rhythms.

Modeling the Role of NCX in the Regulation of Cardiomyocyte Microdomain Ca2+ Dynamics

The sodium (Na+)/calcium (Ca2+) exchanger (NCX) is an electrogenic membrane transporter that mainly serves as a Ca2+ removal mechanism during relaxation and maintains Ca2+ homeostasis in cardiomyocytes. The direction of transport reverses at membrane potentials near action the potential plateau, generating an influx of Ca2+ into the cell. Therefore, there has been great interest in the role NCX plays in cardiac Ca2+-induced Ca2+-release (CICR), with some controversial findings questioning whether NCX has any role at all. A recent super-resolution optical imaging study suggested that ∼17% of NCX co-localize within the domain occupied by ryanodine receptor (RyR) clusters and ∼30% of NCX are within 100nm of the dyad boundary. Therefore, NCX may play a critical role in modulating Ca2+ spark activity and CICR as a result of its spatial distribution in the T-tubule. Here we examine the potential role of NCX using computational modeling. We have developed a novel mechanistic and biophysically-detailed model of NCX that describes both NCX transport kinetics and dynamics of Ca2+-dependent allosteric regulation. We incorporated this new NCX model into a previously developed super-resolution Ca2+ spark model to examine effects of local NCX spatial distribution on properties of Ca2+ sparks. Dyadic NCX reduces spark frequency but has minimal effects on spark amplitude and half-width. Furthermore, we incorporate NCX into a previously developed computational model of the cardiac ventricular myocyte that includes a detailed description of CICR simulated with stochastic gating of L-Type Ca2+ channels and RyRs in the dyad and accounts for local Ca2+ gradients via inclusion of peri-dyadic compartments. We use this model to investigate the role of NCX and its localization on whole-cell Ca2+ dynamics, the Ca2+ levels in the junctional sarcoplasmic reticulum, and APs.

Inactivation of Cav1.1 Channels in Adult Skeletal Muscle: Effects of a C-Terminal Pre-IQ Mutation

Calcium-dependent inactivation (CDI) has been documented for the great majority of Cav1 channels but whether native Cav1.1 of adult skeletal muscle exhibits this feature remains controversial. In CDI, Ca2+ ions can bind to specific sites in the C-terminal IQ domain, a process that requires the Ca2+-binding protein calmodulin (CaM). In addition to CaM, other Ca2+-binding proteins could in principle regulate CDI. Here we sought to explore whether S100A1, a Ca2+-binding protein highly expressed in striated muscle that fine tunes SR Ca2+ release, is also involved in the regulation of CDI. The binding of S100A1 to two different Cav1.1 C-terminal regions (pre-IQ and IQ) was determined by isothermal titration calorimetry. Under Ca2+-loaded conditions, S100A1 binds with higher affinity to the pre-IQ region in the skeletal isoform (Cav1.1) than in the cardiac isoform (CaV1.2). Then, to test for a functional role of the pre-IQ region in adult muscle fibers, we expressed EGFP-Cav1.1 wild type (used as control) or EGFP-Cav1.1 pre-IQ (I or W→A) mutant, which should not bind S100A1, via electroporation and used whole-cell voltage clamp Ca2+ current recordings to evaluate Cav1.1 activation and inactivation properties. The pre-IQ mutation did not modify any Cav1.1 activation parameters; however, the fractional inactivation during a 300 ms pulse was significantly increased. Our results highlight a Ca2+/S100A1 binding site in the C-terminal of the Cav1.1 channel that could be critical for tuning CDI in Cav1.1. While not directly critical for excitation-contraction coupling, recent evidence indicates that Ca2+ entry through Cav1.1 is important for the long-term contractile and metabolic function of skeletal muscle; therefore this S100A1 binding site may impact muscle fiber plasticity. Supported by NIH-NIAMS (R37-R055099).

Quercetin Stimulates Insulin Secretion and Reduces the Viability of Rat INS-1 Beta-Cells

Background/Aims: Previously we described insulinotropic effects of Leonurus sibiricus L. plant extracts used for diabetes mellitus treatment in Traditional Mongolian Medicine. The flavonoid quercetin and its glycoside rutin, which exert anti-diabetic properties in vivo by interfering with insulin signaling in peripheral target tissues, are constituents of these extracts. This study was performed to better understand short- and long-term effects of quercetin and rutin on beta-cells. Methods: Cell viability, apoptosis, phospho-protein abundance and insulin release were determined using resazurin, annexin-V binding assays, Western blot and ELISA, respectively. Membrane potentials (V_mem), whole-cell Ca²⁺ (ICa)- and ATP-sensitive K⁺ (IK_ATP) currents were measured by patch clamp. Intracellular Ca²⁺ (Ca_i) levels were measured by time-lapse imaging using the ratiometric Ca²⁺ indicator Fura-2. Results: Rutin, quercetin and the phosphoinositide-3-kinase (PI3K) inhibitor LY294002 caused a dose-dependent reduction in cell viability with IC₅₀ values of ∼75 µM, ∼25 µM and ∼3.5 µM, respectively. Quercetin (50 µM) significantly increased the percentage of Annexin-V+ cells within 48 hrs. The mean cell volume (MCV) of quercetin-treated cells was significantly lower. Within 2 hrs, quercetin significantly decreased basal- and insulin-stimulated Akt(T308) phosphorylation and increased Erk1/2 phosphorylation, without affecting P-Akt(S473) abundance. Basal- and glucose-stimulated insulin release were significantly stimulated by quercetin. Quercetin significantly depolarized V_mem by ∼25 mV which was prevented by the K_ATP-channel opener diazoxide, but not by the L-type ICa inhibitor nifedipine. Quercetin significantly stimulated ICa and caused a 50% inhibition of IK_ATP. The effects on V_mem, ICa and IK_ATP rapidly reached peak values and then gradually diminished to control values within ∼1 minute. With a similar time-response quercetin induced an elevation in Ca_i which was completely abolished in the absence of Ca²⁺ in the bath solution. Rutin (50 µM) did not significantly alter the percentage of Annexin-V+ cells, MCV, Akt or Erk1/2 phosphorylation, insulin secretion, or the electrophysiological behavior of INS-1 cells. Conclusion: We conclude that quercetin acutely stimulates insulin release, presumably by transient K_ATP channel inhibition and ICa stimulation. Long term application of quercetin inhibits cell proliferation and induces apoptosis, most likely by inhibition of PI3K/Akt signaling.

Electrophysiological effect and the gating mechanism of astragaloside IV on l-type Ca2+ channels of guinea-pig ventricular myocytes

Astragaloside IV (AS-IV) is one of the main active ingredients of Astragalus membranaceus. This study is aimed to investigate AS-IV׳s effects on Ca2+ channel activity of single cardiomyocytes and single Ca2+ channels. Whole-cell Ca2+ currents in freshly dissociated cardiomyocytes were measured using the whole-cell patch-clamp technique. Single Ca2+ channel currents were examined in cell-attached patches and inside-out patches. In the whole-cell recording, AS-IV reduced the amplitude of l-type Ca2+ currents (ICaL) in a concentration-dependent manner. Although AS-IV did not alter the steady-state activation curves, the voltage dependence of the current inactivation curves was negatively shifted by AS-IV in a concentration dependent manner. Consistent with the results of the whole-cell recording, in the inside-out configuration the ensemble average of single Ba2+ current via l-type Ca2+ channel was dose-dependently reduced by AS-IV. The reduction of unitary Ba2+ current at 0.1 or 1µM AS-IV was accounted for a decrease in the channel activity (NPo). In addition to the decrease in NPo, there was a reduction of Po without a change in channel number or an apparent change in single channel current. Furthermore, we found that the open-closed kinetics of the channel were affected by AS-IV. AS-IV induced the shift of l-type Ca2+ channels from either brief openings (mode 1) or long-lasting openings (mode 2) to no active opening (mode 0). Our results suggest that AS-IV blocks the currents through Ca2+ channels in guinea-pig ventricular myocytes by affecting the open-closed kinetics of l-type Ca2+ channels to inhibit the channel activities. This study could provide theoretical basis for the drug exploiting of the monomer of Astragalus membranaceus.

Reduced IP3-Mediated Ca2+ Signaling in Autism Spectrum Disorders in the Context of Fragile X and Tuberous Sclerosis Syndromes

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder affecting up to 2% of children and characterized by impaired social skills, delayed or disordered language and communication skills, and repetitive, stereotypic behavior. Growing evidence supports a role of Ca2+ signaling in the pathogenesis of ASD. Inositol trisphosphate (IP3)-mediated Ca2+ release from intracellular stores participates in a variety of functions, from synaptic plasticity and memory, to long-term gene transcription changes and immune response. IP3 is produced upon stimulation of G-protein coupled receptors (GPCR) and binds to IP3 receptor/channel (IP3R) in the membrane of the endoplasmic reticulum (ER), liberating Ca2+ sequestered in the ER lumen into the cytoplasm. Here, we report that human fibroblasts from three genetically distinct monogenic models of ASD – fragile X and tuberous sclerosis TSC1 and TSC2 – uniformly display depressed Ca2+ release through IP3 receptors. We observed defects in whole-cell Ca2+ signals evoked by G-protein-coupled cell surface receptors and by photoreleased IP3, and at the level of local elementary Ca2+ events, suggesting fundamental defects in IP3R channel activity in ASD. Given its ubiquitous functions in the body, malfunctioning of IP3-mediated signaling may account for the heterogeneity of non-neuronal symptoms seen in ASD, such as gastrointestinal tract problems and immunological complications.

Subcellular Ca Channel Distribution and Ca Alternans in Atrial Myocytes

Unlike ventricular myocytes, atrial myocytes have a much less well-developed T-tubular network, especially in rodents. In this study, we carried out computer simulations to investigate the consequences of a T tubular network on the development of spatially discordant Ca alternans in atrial myocytes. Our study was motivated by experimental observations in isolated mouse atrial myocytes, which express both Cav1.2 and Cav1.3 channels. Knocking out Cav1.3, but not Cav1.2, promoted spatially discordant Ca alternans in which Ca amplitude alternates out-of-phase in different regions of the myocyte. Based on observations in sinoatrial myocytes that Cav1.2 channels are mainly distributed on the surface of the cell while Cav1.3 channels are more uniformly distributed throughout the whole cell, in our computer model, we coupled both types of channels to Ca release units on the surface of the cell, but only Cav1.3 to Ca release units in the interior of the cell. The cell model was paced with a clamped voltage waveform at a pacing cycle length of 300 msec. Under control, Ca transient was normal, and no alternans were observed. When Cav1.2 channels were removed, the Ca transient amplitude decreased, but alternans did not occur. When Cav1.3 channels were removed, however, Ca alternans occurred. The magnitude of the whole-cell Ca transient alternans varied over time, exhibiting a modulated pattern. The line scan showed spatially discordant alternans. These simulations show that the subcellular distribution of Ca channels in relation to Ca release units has important effects on the proclivity for spatially discordant Ca alternans. Specifically, a high fraction of orphaned RyR clusters in the center of the myocyte facilitates spatially discordant Ca alternans.

Abstract 011: Identification and Electrophysiological Characterization of a Novel Somatic Mutation (insT149KCNJ5) of the Potassium Channel Kir3.4 (KCNJ5)

Context: Understanding the function of the Kir3.4 (KCNJ5 gene) potassium channel through characterization of occurring novel mutations is key for dissecting the mechanism(s) of autonomous aldosterone secretion in primary aldosteronism. Objective: To identify novel KCNJ5 channel mutations and functionally characterize them in a large database of patients with aldosterone-producing adenomas (APA). Methods: We sequenced the APA and germinal DNA of 195 consecutive patients, diagnosed with the four corners criteria of the PAPY study. Among the 24.6% (48/195) APA patients who showed somatic KCNJ5 mutations we discovered a novel c.446insAAC insertion resulting in the mutant protein KCNJ5-insT149 in a patient with severe drug-resistant hypertension. The mutated cDNA generated by site-directed mutagenesis was transfected along with KCNJ3 cDNA in mammalian cells. 17α-hydroxylase, CYP11B1, and CYP11B2 were immunochemically localized in the excised adrenal gland. Whole-cell patch clamp recordings, CYP11B2 mRNA, aldosterone and intracellular Ca2+ measurement (Fura-2), and molecular modeling were performed to characterize the KCNJ5-insT149 mutation. Results: The patient’s high blood pressure was long-term cured; his LVMI fell from 168 to 106 g/m2 after 2 years follow-up. Compared to wild type and mock-transfected HAC15 adrenocortical cells, those expressing the mutant KCNJ5 showed increased CYP11B2 expression (expression fold change: 2.9±0.3, p<0.05 vs mock transfected cells) and aldosterone secretion (260 pg/μg RNA, p<0.05 vs mock cells). The HEK293 cells expressing the mutated KCNJ5-insT149 channel exhibited a strong Na+ inward current, and a substantial rise in intracellular Ca2+. The L-type Ca2+ channel blocker verapamil [10 μM] inhibited by 50% the pathological Na+ inward current, while both the Na+/Ca2+ exchanger blocker KB-R7943 [10 μM] and the removal of extracellular Na+ abolished it. Conclusions: We identified a novel mutation of the Kir3.4 channelopathy located after the pore α-helix preceding the selectivity filter, which causes pathological Na+ permeability, membrane depolarization, raised cytosolic Ca2+, and constitutive hypersecretion of aldosterone resulting in ensuing pseudo resistant hypertension.

Basal cGMP regulates the resting pacemaker potential frequency of cultured mouse colonic interstitial cells of Cajal

Cyclic guanosine 3',5'-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca(2+) imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-N(G)-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K(+) channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca(2+) ([Ca(2+)]i) oscillations revealed that ODQ and L-NAME increased [Ca(2+)]i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca(2+)]i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca(2+) release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.

Premises for fowl sperm preservation based on applied bioenergetics1

The primary goal of this work was to test whether the sperm mobility assay could be used to derive mathematical relationships from which predictions could be made about sperm cell function. A precondition was random sampling from a pool of sperm. This precondition was met by centrifuging mobile sperm through 12% (wt/vol) Accudenz containing the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and then holding washed sperm at 20°C within buffered potassium chloride. These 2 conditions rendered washed sperm immobile at 20°C. Resumption of sperm mobility was independent of time (P > 0.8558) when sperm were reactivated at body temperature with 2 mM Ca(2+) in isotonic sodium chloride at pH 7.4. Reactivated sperm mobility was 93% of the prewash control. Subsequent experiments served to define a dose response, predict optimal conditions for in vitro sperm mobility, and show how sperm can recover from an imposed non-physiological condition. Thus, functions were derived from which predictions were made. Whereas the utility of BAPTA treatment was confirmed in a new context, such utility did not address the question of whole-cell Ca(2+) flux during sperm cell manipulation. This issue is pivotal for the application of bioenergetics to fowl sperm preservation. Therefore, the secondary goal of this research was to investigate sperm cell Ca(2+) flux using a simulation of conditions encountered by sperm during centrifugation through 12% (wt/vol) Accudenz. These conditions included a temperature of 30°C, a Ca(2+) sink, and no exogenous substrate. Sperm motion was measured with a Hobson SpermTracker. Data points conformed to parabolic functions when motile concentration and velocity were plotted as functions of time. In each case, maximums were observed, e.g., 26 min for motile concentration. The upswing was attributed to a redistribution of intracellular Ca(2+) whereas the downswing was attributed to sperm cell Ca(2+) depletion. A pronounced isothermal increase was observed for each variable when the Ca(2+) sink was overcome with exogenous Ca(2+). Experimental outcomes supported four testable premises applicable to fowl sperm preservation research: 1) the importance of sperm mobility phenotype, 2) the relationship between mitochondrial Ca(2+) cycling and sperm mobility, 3) the utility of the sperm mobility assay for predicting experimental outcomes, and 4) understanding mitochondrial Ca(2+) cycling in terms of whole-cell Ca(2+) flux.

K+ Channel-Interacting Protein 2 Deficient mice have a Rate Dependent Prolongation of Left Ventricular CA2+ Transients

In the heart, K+ channel-interacting protein 2 (KChIP2) stabilizes Kv4 channels in the membrane resulting in larger transient outward potassium currents (Ito). Recently it has been demonstrated that KChIP2 also enhances Cav1.2 and that the KChIP2-/- mouse has reduced inward L-type Ca2+ current. In this study, we test the impact of KChIP2-/- on cardiac contraction and on underlying Ca2+ transients.Through ultrasound examination, we found comparable left ventricular ejection fraction and fractional shortening in the heart of wild-type (WT) and KChIP2-/- mice, indicating that the function of the left ventricle was not impaired despite reduced L-type Ca2+ current. Confocal line scan Ca2+ imaging was performed using the Cal-520TM fluorophore. Whole-cell Ca2+ transients were recorded from disaggregated left ventricular cardiomyocytes at 37°C, field stimulated at cycle lengths between 80 and 1000 ms. A decrease in the relative peak of the Ca2+ transient was found in KChIP2-/- versus WT (0.58±0.06 vs. 0.71±0.07 F/F0, P<0.05) and the time to 50% decay of the transient was prolonged (133±5 vs. 121±3 ms, P<0.05). These differences were rate-dependent and diminished at cycle lengths shorter than 1000 ms. However, at the fastest pacing rate more cardiomyocytes were alternating in KChIP2-/- compared with WT (cycle length: 80 ms; WT: 12/18, KChIP2-/-: 23/23). The fractional Ca2+ release was not different between the two genotypes, suggesting that the differences in Ca2+ transients are not a result of differential SR load.In conclusion, although cardiac contraction is not impaired in the KChIP2-/- mouse, some abnormal Ca2+ handling is apparent in individual KChIP2-/- cardiomyocytes, including a higher propensity to develop Ca2+ transient alternans. If this is manifested significantly at the tissue level, it could contribute to the development of repolarization gradients across the myocardium.

Learning the Kinetics of Amyloid β Pore in Alzheimer's Disease Pathology

Increased synthesis of self-aggregating amyloid beta (Aβ) peptides caused by abnormal processing of amyloid precursor protein (APP) is a hallmark of Alzheimer's disease (AD) pathogenesis. Aβ mediates it's effect by disrupting the integrity of cell membrane and interacting with plasma membrane channels. The soluble form of Aβ aggregates into calcium (Ca2+) - permeable pores in the membrane. Aβ pores promote uncontrolled increase in the cytoplasmic Ca2+ concentration by allowing Ca2+ influx to the cell, in addition to enhancing the activity of Ca2+- permeable channels on the plasma membrane and intracellular compartments. The Ca2+ influx through Aβ pores upsets the otherwise fine-tuned micrometer-sized elementary Ca2+ release events and whole-cell Ca2+ response. The disrupted Ca2+ signaling in turn has the potential to alter cell function in many ways.Here, we have used computational modeling in conjunction with TIRF microscopy to study the function of Aβ pores in AD cells. TIRF microscopy was used to image Ca2+ flux through thousands of Aβ pores in parallel at the millisecond scale and single channel resolution. The fluorescence time-series from individual pores was idealized by extending the Maximum Likelihood-based method developed for separating signal from baseline in noisy quantal data (Bruno et al. 2013, Biophys. J. 105:68). The idealized data was used to developed data-driven models for the kinetics of Aβ pore at different stages of it's life. In addition to providing deep insights into the kinetics of Aβ pore, this study demonstrates that the massive imaging data obtained from thousands of channels in parallel using TIRF microscopy can be utilized for single molecule modeling in the same manner as electrical patch-clamp data. Employing the optical patch-clamp data for Markov chain modeling has the added advantage of the experiments being done under physiological conditions.

Atrial Excitation-Contraction Coupling and Ca Wave Propagation in Normal and Failing Hearts

We compared excitation-contraction coupling and Ca wave propagation in normal and heart failure (HF) rabbit atrial myocytes. Cytosolic Ca transients (Fluo-4 or Rhod-2), sarcoplasmic reticulum (SR) [Ca] (Fluo-5N) and mitochondrial localization (Mitotracker Red) were recorded by confocal microscopy. SEA and Ru360 were used to inhibit sarcolemmal Na/Ca exchange (NCX) or mitochondrial Ca uptake, respectively. In normal and HF rabbit atrial myocytes transverse tubules are missing, and the peripheral junctional SR (j-SR) is separated from the central non-junctional SR (nj-SR) by a 1-2 µm wide gap that is largely devoid of mitochondria and SR structures. During action potential (AP) stimulation SR Ca release initiated from the j-SR and propagated in centripetal direction via Ca-induced Ca release (CICR) from nj-SR. The centripetal Ca propagation velocity was highest across the gap between j-SR and nj-SR, slowed during propagation across the nj-SR and increased after Ru360 application. In HF, the centripetal propagation velocity was higher and mitochondrial density was decreased. Spontaneous Ca waves propagated with a leading wave front located to the cell periphery, i.e. arising from j-SR Ca release. Nearly half of all Ca waves in normal cells, but <10% in normal cells pretreated with SEA and none in ventricular cells triggered an AP, followed by a whole-cell Ca transient (termed here 'arrhythmogenic Ca waves'). The incidence of spontaneous Ca waves, the fraction of arrhythmogenic Ca waves and NCX activity were significantly increased in HF. In summary, the lack of mitochondria in the gap between j-SR and nj-SR enables rapid propagation of CICR from the j-SR to the nj-SR. Decreased mitochondrial Ca uptake in HF contributes to an increased centripetal Ca propagation velocity. Atrial myocytes are prone to develop NCX-dependent arrhythmogenic Ca waves, which is further accentuated in HF.

Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2Sequestration

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration.