Whole-cell Ca2 Research Articles

Long-term hypoxia modulates depolarization activation of BKCa currents in fetal sheep middle cerebral arterial myocytes.

Previous evidence indicates that gestational hypoxia disrupts cerebrovascular development, increasing the risk of intracranial hemorrhage and stroke in the newborn. Due to the role of cytosolic Ca2+ in regulating vascular smooth muscle (VSM) tone and fetal cerebrovascular blood flow, understanding Ca2+ signals can offer insight into the pathophysiological disruptions taking place in hypoxia-related perinatal cerebrovascular disease. This study aimed to determine the extent to which gestational hypoxia disrupts local Ca2+ sparks and whole-cell Ca2+ signals and coupling with BKCa channel activity. Confocal imaging of cytosolic Ca2+ and recording BKCa currents of fetal sheep middle cerebral arterial (MCA) myocytes was performed. MCAs were isolated from term fetal sheep (∼140 days of gestation) from ewes held at low- (700 m) and high-altitude (3,801 m) hypoxia (LTH) for 100+ days of gestation. Arteries were depolarized with 30 mM KCl (30K), in the presence or absence of 10 μM ryanodine (Ry), to block RyR mediated Ca2+ release. Membrane depolarization increased Ry-sensitive Ca2+ spark frequency in normoxic and LTH groups along with BKCa activity. LTH reduced Ca2+ spark and whole-cell Ca2+ activity and induced a large leftward shift in the voltage-dependence of BKCa current activation. The influence of LTH on the spatial and temporal aspects of Ca2+ sparks and whole-cell Ca2+ responses varied. Overall, LTH attenuates Ca2+ signaling while increasing the coupling of Ca2+ sparks to BKCa activity; a process that potentially helps maintain oxygen delivery to the developing brain.

Modulation of intracellular calcium activity in interstitial cells of Cajal by inhibitory neural pathways within the internal anal sphincter.

The internal anal sphincter (IAS) functions to maintain continence. Previous studies utilizing mice with cell-specific expression of GCaMP6f revealed two distinct subtypes of intramuscular interstitial cells of Cajal (ICC-IM) with differing Ca2+ activities in the IAS. The present study further examined Ca2+ activity in ICC-IM and its modulation by inhibitory neurotransmission. The spatiotemporal properties of Ca2+ transients in Type II ICC-IM mimicked those of smooth muscle cells (SMCs), indicating their joint participation in the "SIP" syncytium. Electrical field stimulation (EFS; atropine present) abolished localized and whole cell Ca2+ transients in Type I and II ICC-IM. The purinergic antagonist MRS2500 did not abolish EFS responses in either cell type, whereas the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine (l-NNA) abolished responses in Type I but not Type II ICC-IM. Combined antagonists abolished EFS responses in Type II ICC-IM. In both ICC-IM subtypes, the ability of EFS to inhibit Ca2+ release was abolished by l-NNA but not MRS2500, suggesting that the nitrergic pathway directly inhibits ICC-IM by blocking Ca2+ release from intracellular stores. Since inositol (1,4,5)-trisphosphate receptor-associated cGMP kinase substrate I (IRAG1) is expressed in ICC-IM, it is possible that it participates in the inhibition of Ca2+ release by nitric oxide. Platelet-derived growth factor receptor α (PDGFRα)+ cells but not ICC-IM expressed P2Y1 receptors (P2Y1R) and small-conductance Ca2+-activated K+ channels (SK3), suggesting that the purinergic pathway indirectly blocks whole cell Ca2+ transients in Type II ICC-IM via PDGFRα+ cells. This study provides the first direct evidence for functional coupling between inhibitory motor neurons and ICC-IM subtypes in the IAS, with contractile inhibition ultimately dependent upon electrical coupling between SMCs, ICC, and PDGFRα+ cells via the SIP syncytium.NEW & NOTEWORTHY Two intramuscular interstitial cells of Cajal (ICC-IM) subtypes exist within the internal anal sphincter (IAS). This study provides the first evidence for direct coupling between nitrergic motor neurons and both ICC-IM subtypes as well as indirect coupling between purinergic inputs and Type II ICC-IM. The spatiotemporal properties of whole cell Ca2+ transients in Type II ICC-IM mimic those of smooth muscle cells (SMCs), suggesting that ICC-IM modulate the activity of SMCs via their joint participation in a SIP syncytium (SMCs, ICC, and PDGFRα+ cells).

Chronic Hypoxia during Gestation Disrupts Spontaneous Ca2+ Oscillations in Middle Cerebral Arterial Myocytes of Fetal Sheep

Long Term Hypoxic (LTH) stress is associated with an elevated risk of intracranial hemorrhage and stroke in newborns. Existing evidence illustrates that fetal sheep subjected to LTH conditions exhibit compromised cerebrovascular reactivity and arterial development, leading to perturbations in cerebral blood flow. Given the critical role of whole-cell Ca2+ waveforms in the contraction of middle cerebral arterial (MCA) myocytes and the regulation of cerebral blood flow, a comprehensive understanding of Ca2+ signaling patterns can provide insight into hypoxia-related cerebrovascular disease in neonates. This study was designed to test the hypothesis that intrauterine LTH disrupts whole-cell Ca2+ oscillations in fetal MCA myocytes. The hypothesis was addressed using MCAs isolated from term fetal sheep (approximately 140 days of gestation) delivered by cesarean section. These fetuses were carried by ewes residing at low-altitude (700 m) or high-altitude conditions (3801 m) for over 100 days of gestation. Ca2+ oscillatory activity was recorded using confocal fluorescence imaging techniques in an en face myocyte preparation. Spontaneous Ca2+ oscillations were evaluated in the presence and absence of 30 mM K+ to depolarize the myocytes, with or without 10 μM ryanodine, which blocks ryanodine receptor mediated Ca2+ release. The results of the study reveal that LTH decreases oscillatory activity and restricts the augmentation of activity following membrane depolarization. Furthermore, LTH induces dysregulation in spatial and temporal Ca2+ signals, manifesting as reduced event amplitude, shortened event duration, and decreased area under the curve (AUC). These findings highlight the impact of gestational hypoxia on multiple aspects of Ca2+ signaling in fetal lamb MCA, which may contribute to cerebrovascular dysfunction in the newborn. R01HL155295, R01HL149608, Advanced Imaging and Microscopy Core Loma Linda University School of Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Vasopressin differentially modulates the excitability of rat olfactory bulb neuron subtypes.

Vasopressin (VP) plays a crucial role in social memory even at the level of the olfactory bulb (OB), where OB VP cells are activated during social interactions. However, it remains unclear how VP modulates olfactory processing to enable enhanced discrimination of very similar odors, e.g., rat body odors. Thus far, it has been shown that VP reduces firing rates in mitral cells (MCs) during odor presentation in vivo and decreases the amplitudes of olfactory nerve-evoked excitatory postsynaptic potentials (ON-evoked EPSPs) in external tufted cells in vitro. We performed whole-cell patch-clamp recordings and population Ca2+ imaging on acute rat OB slices. We recorded ON-evoked EPSPs as well as spontaneous inhibitory postsynaptic currents (IPSCs) from two types of projection neurons: middle tufted cells (mTCs) and MCs. VP bath application reduced the amplitudes of ON-evoked EPSPs and the frequencies of spontaneous IPSCs in mTCs but did not change those in MCs. Therefore, we analyzed ON-evoked EPSPs in inhibitory interneurons, i.e., periglomerular cells (PGCs) and granule cells (GCs), to search for the origin of increased inhibition in mTCs. However, VP did not increase the amplitudes of evoked EPSPs in either type of interneurons. We next performed two-photon population Ca2+ imaging in the glomerular layer and the superficial GC layer of responses to stronger ON stimulation than during patch-clamp experiments that should evoke action potentials in the measured cells. We observed that VP application increased ON-evoked Ca2+ influx in juxtaglomerular cells and GC somata. Thus, our findings indicate inhibition by VP on projection neurons via strong ON input-mediated inhibitory interneuron activity. This neural modulation could improve representation of odors, hence, better discriminability of similar odors, e.g., conspecific body odors.

Effects of reversible SERCA inhibition on catecholamine exocytosis and intracellular [Ca2+] signaling in chromaffin cells from normotensive Wistar Kyoto rats and spontaneously hypertensive rats

Intracellular Ca2+ ([Ca2+]i) signaling and catecholamine (CA) exocytosis from adrenal chromaffin cells (CCs) differ between mammalian species. These differences partly result from the different contributions of Ca2+-induced Ca2+-release (CICR) from internal stores, which boosts intracellular Ca2+ signals. Transient inhibition of the sarcoendoplasmic reticulum (SERCA) Ca2+ pump with cyclopiazonic acid (CPA) reduces CICR. Recently, Martínez-Ramírez et al. found that CPA had contrasting effects on catecholamine secretion and intracellular Ca2+ signals in mouse and bovine CCs, where it enhanced and inhibited exocytosis, respectively. After CPA withdrawal, exocytosis diminished in mouse CCs and increased in bovine CCs. These differences can be explained if mouse CCs have weak CICR and strong Ca2+ uptake, and the reverse is true for bovine CCs. Surprisingly, CPA slightly reduced the amplitude of Ca2+ signals in both mouse and bovine CCs. Here we examined the effects of CPA on stimulated CA exocytosis and Ca2+ signaling in rat CCs and investigated if it alters differently the responses of CCs from normotensive (WKY) or hypertensive (SHR) rats, which differ in the gain of CICR. Our results demonstrate that CPA application strongly inhibits voltage-gated exocytosis and Ca2+ transients in rat CCs, regardless of strain (SHR or WKY). Thus, despite the greater phylogenetic distance from the most recent common ancestors, suppression of endoplasmic reticulum (ER) Ca2+ uptake through CPA inhibits the CA secretion in rat CCs more similarly to bovine than mouse CCs, unveiling divergent evolutionary relationships in the mechanism of CA exocytosis of CCs between rodents. Agents that inhibit the SERCA pump, such as CPA, suppress catecholamine secretion equally well in WKY and SHR CCs and are not potential therapeutic agents for hypertension. Rat CCs display Ca2+ signals of varying widths. Some even show early and late Ca2+ components. Narrowing the Ca2+ transients by CPA and ryanodine suggests that the late component is mainly due to CICR. Simultaneous recordings of Ca2+ signaling and amperometry in CCs revealed the existence of a robust and predictable correlation between the kinetics of the whole-cell intracellular Ca2+ signal and the rate of exocytosis at the single-cell level.

TRPML channel activation and recovery of calcium signals in pulmonary arterial myocytes of fetal and adult sheep disrupted by long term hypoxia

Ca 2+ signals are important for regulating vasodilatory function in the pulmonary vasculature. Previous studies demonstrate that there are developmental changes in rapid Ca 2+ sparks and slower whole-cell Ca 2+ oscillations. Long-term hypoxia (LTH) causes pulmonary hypertension and aberrations in these Ca 2+ signals are thought to be important to the etiology of the disease. The LTH-induced dysregulation in Ca 2+ sparks is linked to loss in communication between L-type Ca 2+ channels on the plasma membrane and ryanodine receptors on the sarcoplasmic reticulum. Recent studies illustrate that Ca 2+ filled lysosomes can be closely associated with the sarcoplasmic reticulum. Previous work shows that activation of TRPML channels on lysosomes releases Ca 2+ into the cytosol and can trigger ryanodine receptor mediated Ca 2+ sparks. The aim of this study was to test if TRPML channel activation recovers aberrant Ca2+ sparks and whole-cell Ca 2+ oscillations after LTH exposure. Ca 2+ sparks and whole-cell Ca 2+ oscillations were measured on live fetal or adult sheep pulmonary arterial myocytes loaded with fluo-4 in an en face arterial preparation using confocal microscopy. The sheep were housed at either normoxic (700m) or hypoxic (3801m) conditions. Myocyte Ca 2+ was measured in the absence and then presence of 10 μM MLSA-1, a selective TRPML channel agonist. Treatment with MLSA-1 increased overall spark and oscillatory activity. In the fetal hypoxic group, MLSA-1 significantly increased the number of cells with oscillatory activity. MLSA-1 also significantly increased Ca 2+ spark activity in fetal and adult hypoxic, as well as adult normoxic myocytes. Interestingly, MLSA-1 did not influence amplitude of Ca 2+ sparks or the area of whole-cell Ca 2+ oscillatory events. These results suggest that activation of lysosomal TRPML channels may be a therapeutic target for pulmonary hypertension, through recovery of Ca 2+ signals that regulate pulmonary vascular reactivity. Still, further exploration is needed to fully understand the impact of LTH on lysosomal function, the mechanisms by which aberrant Ca 2+ signaling activity is recovered, and the effect on pulmonary vascular resistance and pressure. NIH R01HL155295, R01HL149608 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Two distinct calcium signaling behaviors are present in interstitial cells of Cajal of the gastroesophageal junction

Background/Objectives: The lower esophageal sphincter (LES) is located within the gastroesophageal junction (GEJ) and generates tone to prevent gastric reflux into the esophagus. Previously, we demonstrated that the generation and maintenance of tone in the LES clasp muscle involves intramuscular interstitial cells of Cajal (ICC-IM) (Drumm et al., 2022; PMID: 35229888). The aim of the current study was to further characterize Ca2+ signaling in ICC-IM within the GEJ. We hypothesize that Ca2+ release from the endoplasmic reticulum (ER) activates Ca2+-activated Cl- channels (ANO1) in ICC leading to depolarization that is conducted to smooth muscle cells (SMCs) via gap junctions; this in turn leads to Ca2+ influx via L-type Ca2+ channels (CavL) and the development of tone. Methods: Confocal spinning disc microscopy was used to image Ca2+ transients in the LES and distal esophagus of mice expressing GCaMP6f in ICC (Kit-GCaMP6f). Muscles of the GEJ imaged with the submucosal surface upwards at 20x. Ani9 (3mM), nifedipine (1mM) and CPA (10mM) were used to inhibit ANO1, CavL and SERCA, respectively. Pinacidil (10mM) was used to activate KATP channels. Results: ICC-IM within the GEJ displayed two types of Ca2+ signaling behaviors. Ca2+ transients in ICC-IM of the LES clasp muscle were stochastic, arising from multiple firing sites within the same cell. These Ca2+ transients were not synchronized within the same cell or between adjacent cells. In contrast, two Ca2+ signaling patterns were observed in ICC-IM of the distal esophagus where smooth muscle transitions to skeletal muscle. One ICC-IM population (Type I) shared the Ca2+ signaling behaviors of ICC-IM in the LES clasp. Another ICC population (Type II) exhibited rhythmic whole-cell Ca2+ transients that were synchronized between adjacent Type II ICC. Whole cell Ca2+ transients in Type II ICC were abolished by Ani9, nifedipine or pinacidil, revealing underlying Ca2+ transients similar to those observed in Type I ICC. In contrast, Ca2+ transients in Type I ICC were resistant to Ani9, nifedipine and pinacidil. Ca2+ transients were abolished in both types of ICC by removal of extracellular Ca2+, however the activity in Type I ICC persisted longer than that of Type II ICC. Discussion/Conclusions: This study demonstrates two distinct Ca2+ signaling behaviors in ICC-IM of the GEJ with differing pharmacology. This is similar to what has been previously described within the internal anal sphincter (IAS) (Hannigan et al., 2020; PMID: 32587396). In the IAS, it has been suggested that Type II ICC-IM generate slow waves however their role in the GEJ remains unclear as slow waves have not been described within this region of the mouse. Thus, the functional role of ICC-IM in the GEJ, particularly Type II ICC, warrants further investigation. NIH DK129528 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Probenecid affects muscle Ca2+ homeostasis and contraction independently from pannexin channel block.

Tight control of skeletal muscle contractile activation is secured by the excitation-contraction (EC) coupling protein complex, a molecular machinery allowing the plasma membrane voltage to control the activity of the ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. This machinery has been shown to be intimately linked to the plasma membrane protein pannexin-1 (Panx1). We investigated whether the prescription drug probenecid, a widely used Panx1 blocker, affects Ca2+ signaling, EC coupling, and muscle force. The effect of probenecid was tested on membrane current, resting Ca2+, and SR Ca2+ release in isolated mouse muscle fibers, using a combination of whole-cell voltage-clamp and Ca2+ imaging, and on electrically triggered contraction of isolated muscles. Probenecid (1 mM) induces SR Ca2+ leak at rest and reduces peak voltage-activated SR Ca2+ release and contractile force by 40%. Carbenoxolone, another Panx1 blocker, also reduces Ca2+ release, but neither a Panx1 channel inhibitory peptide nor a purinergic antagonist affected Ca2+ release, suggesting that probenecid and carbenoxolone do not act through inhibition of Panx1-mediated ATP release and consequently altered purinergic signaling. Probenecid may act by altering Panx1 interaction with the EC coupling machinery, yet the implication of another molecular target cannot be excluded. Since probenecid has been used both in the clinic and as a masking agent for doping in sports, these results should encourage evaluation of possible effects on muscle function in treated individuals. In addition, they also raise the question of whether probenecid-induced altered Ca2+ homeostasis may be shared by other tissues.

BIN1 is essential for β-adrenergic regulation of cardiac function.

The number and activity of voltage gated L-type CaV1.2 Ca2+ channels in the sarcolemma of cardiomyocytes tunes the magnitude of Ca2+-induced Ca2+ release and subsequent contraction. Previous studies from our group revealed that β-adrenergic receptor stimulation in mouse ventricular myocytes triggers mobilization of CaV1.2 channels from early and recycling endosomal reservoirs to augment sarcolemmal expression and clustering and enhancing excitation-contraction coupling. In the heart, the membrane curvature-mediating protein bridging integrator 1 (BIN1) mediates t-tubule micro-folding, and targeted delivery of CaV1.2 channels to t-tubules via anchored microtubules. BIN1 deficits are associated with heart failure. In neurons, BIN1 is known to play an essential role in tubulation of endosomes to permit cargo exit and recycling. We used myocytes isolated from cardiac-specific BIN1 knockout mice, to test the hypothesis that expressional deficits in BIN1 impairs isoproterenol-stimulated endosomal recycling of CaV1.2, reducing the ability to respond to acute stress. Super-resolution Airyscan imaging of immunostained ventricular myocytes revealed that CaV1.2 pools on early and recycling endosomes are mobilized to the sarcolemma to bolster channel expression in response to β-adrenergic stimulation with isoproterenol in WT but not BIN1-/- cells. Moreover, single molecule localization microscopy revealed that isoproterenol-stimulated CaV1.2 upregulation and super-clustering at the t-tubule sarcolemma occurs in wild-type myocytes but is reduced in BIN1+/- and absent in BIN1-/- myocytes. To assess the functional implications on β-adrenergic regulation of CaV1.2 and excitation-contraction coupling, we performed voltage-clamp electrophysiological recordings of whole-cell Ca2+ currents and measured Ca2+transients from paced myocytes finding impaired responses to isoproterenol in BIN1 knockout cells versus wild-type controls. Based on these data, we propose that tight regulation of BIN1 expression is required to support β-adrenergic receptor stimulated CaV1.2 channel trafficking and recycling to increase Ca2+ influx and inotropy in times of acute stress.

Genetic deletion of STAC2 adaptor protein alters electrical activity of mouse chromaffin cells.

Voltage-gated Cav1.2 and Cav1.3 L-type Ca2+ channels (LTCCs) regulate action potential (AP) firing and catecholamine release in mouse chromaffin cells (MCCs). Src homology 3 and cysteine rich domain adaptor proteins (Stac1-Stac3) have recently been identified as novel regulators of neuronal LTCCs expression and biophysical properties. Upon overexpression in cultured hippocampal neurons Stac2 abolished LTCC Ca2+ dependent inactivation via an allosteric inhibition of calmodulin binding. Additionally, in Drosophila, deletion of the homologous DStac gene resulted in deficient LTCC Ca2+ transients and reduced neuropeptide release. Here we investigated the effect of Stac2 genetic ablation on neuronal LTCC function, electrical activity, and vesicle exocytosis in MCCs. Constitutive deletion of Stac2 does not affect the MCCs resting membrane potential or spontaneous firing frequency. However, the AP depolarization threshold was significantly reduced in Stac2-/- MCCs compared to WT (WT = −35.6 ± 1.2 mV, Stac2-/- = −40.6 ± 0.6 mV; ∗∗ P < 0.01). Additionally, step current injection elicited an electrical activity with higher initial AP firing frequency in Stac2-/- compared to WT that led to earlier depolarization block (F0 ≅2 pA current injection: WT = 0.28 ± 0.28 Hz, Stac2-/- = 0.89 ± 0.40 Hz; ∗ P < 0.05). Surprisingly, initial characterization shows that Stac2 deletion does not alter whole-cell Ca2+ current amplitude or inactivation kinetics but significantly shifts the voltage-dependence of activation to more hyperpolarized potential (V0.5: WT = −5.95 ± 1.65 mV, Stac2-/- = −15.25 ± 2.05 mV; ∗ P < 0.05). We are currently investigating possible effects of Stac2 deletion on other ionic conductances governing MCC excitability and the role on catecholamine vesicle exocytosis. Support: TWF F.18863, LFU 2021-CHEM-8 to SMG; FWF P31434, P36053, DOC30-B30 to PT; P33776 to MC.

Chronic angiotensin II signaling drives sustained PIP2 deficits and altered sarcolemmal Cav1.2 expression and function in cardiomyocytes.

Contractile dysfunction, hypertrophy, and cell death during heart failure are linked to altered Ca2+ handling and elevated levels of angiotensin II (AngII). This hormone signals through Gq-coupled AT1 receptors, initiating hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Previous work revealed acute AngII signaling and PIP2 depletion destabilizes CaV1.2 clusters in cardiomyocytes, triggering their internalization, and subsequent reduction of whole-cell Ca2+ current (ICa) and contractility. However, the effects of chronically elevated AngII on the phospholipid landscape and associated effects on ICa remain unknown. We subjected mice to sustained delivery of AngII to test the impact on phosphoinositides, ICa, and excitation-contraction coupling. We hypothesized that chronic AngII signaling leads to sustained PIP2 and CaV1.2 deficits, contributing to the diminished functional output associated with disease development. Phospholipid mass spectrometry revealed extensive alteration of phosphoinositide species, including a ∼44% reduction in PIP2 and ∼37% reduction in cardioprotective PIP3 in AngII-infused hearts compared to controls. To study the effects of this phospholipid imbalance on CaV1.2, we examined ICa, finding similar current density in both groups. However, super-resolution imaging revealed a ∼54% reduction in t-tubular CaV1.2 expression and a ∼23% reduction in cluster area. Conserved ICa density despite reduced t-tubular CaV1.2 suggests that either the channels have relocated to the sarcolemmal crest, or their Po has increased. We examined crest CaV1.2 and observed no change with AngII, thus the Po of the remaining channels must increase to maintain ICa. Western blots revealed enhanced phosphorylation of CaV1.2, phosphorylation of RyR2, and an increased expression of the catalytic subunit of PKA, suggesting a compensatory sympathetic activation underlying conserved ICa. These findings support a model wherein chronic elevation of AngII disrupts phosphoinositide and CaV1.2 levels, elucidating the role of chronic AngII in disease progression.

14-3-3 regulates cardiac CaV1.2 clustering, expression, and trafficking.

The voltage-gated L-type Ca2+ channel (CaV1.2) is essential for cardiac excitation-contraction coupling. CaV1.2 channel phosphorylation downstream of several physiological signaling cascades, regulates its activity, recycling, and trafficking to tune excitation-contraction coupling and contractility. A ubiquitously expressed protein called 14-3-3 has been previously proposed to affect CaV1.2 trafficking however the molecular details of this regulation are lacking. 14-3-3 preferentially binds phospho-serine/threonine residues on target proteins to affect many cellular processes and is known to regulate the trafficking and activity of other cardiac ion channels including NaV1.5 and hERG. Altered expression and function of 14-3-3 has additionally been implicated in cardiac pathologies including hypertrophy. Accordingly, we tested the hypothesis that 14-3-3 regulates cardiac CaV1.2 trafficking and activity in a phosphorylation-dependent manner, resulting in enhanced channel clustering that facilitates cooperative interactions and amplifies Ca2+ influx. Proximity ligation assays in cardiomyocytes revealed enhanced proximity between CaV1.2 and 14-3-3 upon stimulation with the β-adrenergic receptor agonist isoproterenol. Single molecule localization microscopy confirmed that a population of 14-3-3 colocalized with CaV1.2 and the level of colocalization increased with isoproterenol. Notably, only the 14-3-3-associated CaV1.2 population displayed increased cluster size with isoproterenol. In transfected tsA-201 cells, confocal imaging revealed reduced CaV1.2 surface expression when 14-3-3 was inhibited, and increased expression when 14-3-3 was overexpressed. Similarly, isoproterenol-stimulated augmentation of sarcolemmal CaV1.2 expression in cardiomyocytes was abrogated by pharmacological inhibition of 14-3-3. Functionally, voltage clamp experiments revealed whole-cell Ca2+ currents are reduced with 14-3-3 inhibition and increased when 14-3-3 was overexpressed. These data support a model where 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner to promote its enhanced trafficking/recycling, clustering, and activity during β-adrenergic stimulation.

Computational Model of Noradrenaline Modulation of Astrocyte Responses to Synaptic Activity

The mathematical modeling of synaptically connected neuronal networks is an established instrument for gaining insights into dynamics of neuronal ensembles and information processing in the nervous system. Recently, calcium signaling in astrocytes—glial cells controlling local tissue metabolism and synapse homeostasis—and its corresponding downstream effect on synaptic plasticity and neuromodulation appeared in the limelight of modeling studies. Here, we used mechanism-based mathematical modeling to disentangle signaling pathways and feedback loops in the astrocytic calcium response to noradrenaline, an important neuromodulator marking periods of heightened alertness and arousal. The proposed model is based on an experiment-based 2D representation of astrocyte morphology, discrete random glutamate synapses with placement probability defined by the morphology pattern, and spatially heterogeneous noradrenaline sources, reflecting axonal varicosities of the adrenergic axons. Both glutamate and noradrenaline drive Ca2+ dynamics in the astrocyte in an additive or synergistic manner. Our simulations replicate the global activation of astrocytes by noradrenaline and predict the generation of high-frequency Ca2+ waves in a dose-dependent manner and the preferred Ca2+ wave origination near noradrenaline release sites if they colocalise with high-density clusters of glutamate synapses. We tested positive feedback loops between noradrenaline release and glutamate spillover directly or mediated by gliotransmitter release from the activated astrocyte. The simulations suggest that the coupled stochastic drive by glutamate and noradrenaline release converges on the graded modulation of the IP3 level, which is translated into whole-cell Ca2+ waves of different frequencies. Thus, the proposed approach is supported by experimental data and can be used to address situations inaccessible directly by experiment, and is a starting point for a more detailed model that includes other signaling mechanisms providing negative feedback.

Novel CACNA1C R511Q mutation, located in domain Ⅰ-Ⅱ linker, causes non-syndromic type-8 long QT syndrome.

BackgroundGain-of-function mutations in CACNA1C encoding Cav1.2 cause syndromic or non-syndromic type-8 long QT syndrome (LQTS) (sLQT8 or nsLQT8). The cytoplasmic domain (D)Ⅰ-Ⅱ linker in Cav1.2 plays a pivotal role in calcium channel inactivation, and mutations in this site have been associated with sLQT8 (such as Timothy syndrome) but not nsLQT8.ObjectiveSince we identified a novel CACNA1C mutation, located in the DⅠ-Ⅱ linker, associated with nsLQTS, we sought to reveal its biophysical defects.MethodsTarget panel sequencing was employed in 24 genotype-negative nsLQTS probands (after Sanger sequencing) and three family members. Wild-type (WT) or R511Q Cav1.2 was transiently expressed in tsA201 cells, then whole-cell Ca2+ or Ba2+ currents (ICa or IBa) were recorded using whole-cell patch-clamp techniques.ResultsWe identified two CACNA1C mutations, a previously reported R858H mutation and a novel R511Q mutation located in the DⅠ-Ⅱ linker. Four members of one nsLQTS family harbored the CACNA1C R511Q mutation. The current density and steady-state activation were comparable to those of WT-ICa. However, persistent currents in R511Q-ICa were significantly larger than those of WT-ICa (WT at +20 mV: 3.3±0.3%, R511Q: 10.8±0.8%, P<0.01). The steady-state inactivation of R511Q-ICa was weak in comparison to that of WT-ICa at higher prepulse potentials, resulting in increased window currents in R511Q-ICa. Slow component of inactivation of R511Q-ICa was significantly delayed compared to that of WT-ICa (WT-tau at +20 mV: 81.3±3.3 ms, R511Q-tau: 125.1±5.0 ms, P<0.01). Inactivation of R511Q-IBa was still slower than that of WT-IBa, indicating that voltage-dependent inactivation (VDI) of R511Q-ICa was predominantly delayed.ConclusionsDelayed VDI, increased persistent currents, and increased window currents of R511Q-ICa cause nsLQT8. Our data provide novel insights into the structure-function relationships of Cav1.2 and the pathophysiological roles of the DⅠ-Ⅱ linker in phenotypic manifestations.

Long-Term Hypoxia Negatively Influences Ca2+ Signaling in Basilar Arterial Myocytes of Fetal and Adult Sheep.

Cerebral arterial vasoreactivity is vital to the regulation of cerebral blood flow. Depolarization of arterial myocytes elicits whole-cell Ca2+ oscillations as well as subcellular Ca2+ sparks due to activation of ryanodine receptors on the sarcoplasmic reticulum. Previous evidence illustrates that contraction of cerebral arteries from sheep and underlying Ca2+ signaling pathways are modified by age and that long-term hypoxia (LTH) causes aberrations in Ca2+ signaling pathways and downstream effectors impacting vasoregulation. We hypothesize that age and LTH affect the influence of membrane depolarization on whole-cell intracellular Ca2+ oscillations and sub-cellular Ca2+ spark activity in cerebral arteries. To test this hypothesis, we examined Ca2+ oscillatory and spark activities using confocal fluorescence imaging techniques of Fluo-4 loaded basilar arterial myocytes of low- and high-altitude term fetal (∼145 days of gestation) and adult sheep, where high-altitude pregnant and non-pregnant sheep were placed at 3,801 m for >100 days. Ca2+ oscillations and sparks were recorded using an in situ preparation evaluated in the absence or presence of 30 mM K+ (30K) to depolarize myocytes. Myocytes from adult animals tended to have a lower basal rate of whole-cell Ca2+ oscillatory activity and 30K increased the activity within cells. LTH decreased the ability of myocytes to respond to depolarization independent of age. These observations illustrate that both altitude and age play a role in affecting whole-cell and localized Ca2+ signaling, which are important to arterial vasoreactivity and cerebral blood flow.

Noise Exposure Potentiates Exocytosis From Cochlear Inner Hair Cells.

Noise-induced hearing loss has gained relevance as one of the most common forms of hearing impairment. The anatomical correlates of hearing loss, principally cell damage and/or death, are relatively well-understood histologically. However, much less is known about the physiological aspects of damaged, surviving cells. Here we addressed the functional consequences of noise exposure on the capacity of inner hair cells (IHCs) to release synaptic vesicles at synapses with spiral ganglion neurons (SGNs). Mice of either sex at postnatal day (P) 15–16 were exposed to 1–12 kHz noise at 120 dB sound pressure level (SPL), for 1 h. Exocytosis was measured by tracking changes in membrane capacitance (ΔCm) from IHCs of the apical cochlea. Upon IHC depolarization to different membrane potentials, ΔCm showed the typical bell-shaped curve that mirrors the voltage dependence of Ca2+ influx, in both exposed and unexposed cells. Surprisingly, from IHCs at 1-day after exposure (d.a.e.), we found potentiation of exocytosis at the peak of the bell-shaped curve. The increase in exocytosis was not accompanied by changes in whole-cell Ca2+ influx, suggesting a modification in coupling between Ca2+ channels and synaptic vesicles. Consistent with this notion, noise exposure also changed the Ca2+-dependence of exocytosis from linear to supralinear. Noise exposure did not cause loss of IHCs, but did result in a small reduction in the number of IHC-SGN synapses at 1-d.a.e. which recovered by 14-d.a.e. In contrast, a strong reduction in auditory brainstem response wave-I amplitude (representing synchronous firing of SGNs) and distortion product otoacoustic emissions (reflecting outer hair cell function) indicated a profound hearing loss at 1- and 14-d.a.e. To determine the role of glutamate release in the noise-induced potentiation of exocytosis, we evaluated vesicular glutamate transporter-3 (Vglut3) knock-out (KO) mice. Unlike WT, IHCs from Vglut3KO mice showed a noise-induced reduction in ΔCm and Ca2+ influx with no change in the Ca2+-dependence of exocytosis. Together, these results indicate that traumatic noise exposure triggers changes of IHC synaptic function including a Vglut3-dependent potentiation of exocytosis.

PDE1 Inhibition Modulates Cav1.2 Channel to Stimulate Cardiomyocyte Contraction.

[Figure: see text].

Optically activated, customizable, excitable cells.

Genetically encoded fluorescent biosensors are powerful tools for studying complex signaling in the nervous system, and now both Ca2+ and voltage sensors are available to study the signaling behavior of entire neural circuits. There is a pressing need for improved sensors, but improving them is challenging because testing them involves a low throughput, labor-intensive processes. Our goal was to create synthetic, excitable cells that can be activated with brief pulses of blue light and serve as a medium throughput platform for screening the next generation of sensors. In this live cell system, blue light activates an adenylyl cyclase enzyme (bPAC) that increases intracellular cAMP (Stierl M et al. 2011). In turn, the cAMP opens a cAMP-gated ion channel. This produces slow, whole-cell Ca2+ transients and voltage changes. To increase the speed of these transients, we add the inwardly rectifying potassium channel Kir2.1, the bacterial voltage-gated sodium channel NAVROSD, and Connexin-43. The result is a highly reproducible, medium-throughput, live cell system that can be used to screen voltage and Ca2+ sensors.

Spontaneous Depolarization-Induced Action Potentials of ON-Starburst Amacrine Cells during Cholinergic and Glutamatergic Retinal Waves.

Correlated spontaneous activity in the developing retina (termed “retinal waves”) plays an instructive role in refining neural circuits of the visual system. Depolarizing (ON) and hyperpolarizing (OFF) starburst amacrine cells (SACs) initiate and propagate cholinergic retinal waves. Where cholinergic retinal waves stop, SACs are thought to be driven by glutamatergic retinal waves initiated by ON-bipolar cells. However, the properties and function of cholinergic and glutamatergic waves in ON- and OFF-SACs still remain poorly understood. In the present work, we performed whole-cell patch-clamp recordings and Ca2+ imaging from genetically labeled ON- and OFF-SACs in mouse flat-mount retinas. We found that both SAC subtypes exhibited spontaneous rhythmic depolarization during cholinergic and glutamatergic waves. Interestingly, ON-SACs had wave-induced action potentials (APs) in an age-dependent manner, but OFF-SACs did not. Simultaneous Ca2+ imaging and patch-clamp recordings demonstrated that, during a cholinergic wave, APs of an ON-SAC appeared to promote the dendritic release of acetylcholine onto neighboring ON- and OFF-SACs, which enhances their Ca2+ transients. These results advance the understanding of the cellular mechanisms underlying correlated spontaneous activity in the developing retina.

Relative role of T-tubules disruption and decreased SERCA2 on contractile dynamics of isolated rat ventricular myocytes

AimsVentricular myocytes (VM) depolarization activates L-type Ca2+ channels (LCC) allowing Ca2+ influx (ICa) to synchronize sarcoplasmic reticulum (SR) Ca2+ release, via Ca2+-release channels (RyR2). The resulting whole-cell Ca2+ transient triggers contraction, while cytosolic Ca2+ removal by SR Ca2+ pump (SERCA2) and sarcolemmal Na+/Ca2+ exchanger (NCX) allows relaxation.In diseased hearts, extensive VM remodeling causes heterogeneous, blunted and slow Ca2+ transients. Among remodeling changes are: A) T-tubules disorganization. B) Diminished SERCA2 and low SR Ca2+. However, those often overlap, hindering their relative contribution to contractile dysfunction (CD). Furthermore, few studies have assessed their specific impact on the spatiotemporal Ca2+ transient properties and contractile dynamics simultaneously. Therefore, we sought to perform a quantitative comparison of how heterogeneous and slow Ca2+ transients, with different underlying determinants, affect contractile performance. MethodsWe used two experimental models: A) formamide-induced acute “detubulation”, where VM retain functional RyR2 and SERCA2, but lack T-tubules-associated LCC and NCX. B) Intact VM from hypothyroid rats, presenting decreased SERCA2 and SR Ca2+, but maintained T-tubules. By confocal imaging of Fluo-4-loaded VM, under field-stimulation, simultaneously acquired Ca2+ transients and shortening, allowing direct correlations. Key findingsWe found near-linear correlations among key parameters of altered Ca2+ transients, caused independently by T-tubules disruption or decreased SR Ca2+, and shortening and relaxation, SignificanceUnrelated structural and molecular alterations converge in similarly abnormal Ca2+ transients and CD, highlighting the importance of independently reproduce disease-specific alterations, to quantitatively assess their impact on Ca2+ signaling and contractility, which would be valuable to determine potential disease-specific therapeutic targets.