Steady-state Ca2 Research Articles

Evaluation of four KCNMA1 channelopathy variants on BK channel current under CaV1.2 activation

ABSTRACT Variants in KCNMA1, encoding the voltage- and calcium-activated K+ (BK) channel, are associated with human neurological disease. The effects of gain-of-function (GOF) and loss-of-function (LOF) variants have been predominantly studied on BK channel currents evoked under steady-state voltage and Ca2+ conditions. However, in their physiological context, BK channels exist in partnership with voltage-gated Ca2+ channels and respond to dynamic changes in intracellular Ca2+ (Ca2+ i). In this study, an L-type voltage-gated Ca2+ channel present in the brain, CaV1.2, was co-expressed with wild type and mutant BK channels containing GOF (D434G, N999S) and LOF (H444Q, D965V) patient-associated variants in HEK-293T cells. Whole-cell BK currents were recorded under CaV1.2 activation using buffering conditions that restrict Ca2+ i to nano- or micro-domains. Both conditions permitted wild type BK current activation in response to CaV1.2 Ca2+ influx, but differences in behavior between wild type and mutant BK channels were reduced compared to prior studies in clamped Ca2+ i. Only the N999S mutation produced an increase in BK current in both micro- and nano-domains using square voltage commands and was also detectable in BK current evoked by a neuronal action potential within a microdomain. These data corroborate the GOF effect of N999S on BK channel activity under dynamic voltage and Ca2+ stimuli, consistent with its pathogenicity in neurological disease. However, the patient-associated mutations D434G, H444Q, and D965V did not exhibit significant effects on BK current under CaV1.2-mediated Ca2+ influx, in contrast with prior steady-state protocols. These results demonstrate a differential potential for KCNMA1 variant pathogenicity compared under diverse voltage and Ca2+ conditions.

Calcium flow at ER-TGN contact sites facilitates secretory cargo export.

Ca2+ influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca2+-ATPase SPCA1 and the luminal Ca2+ binding protein Cab45. Cab45 oligomerizes upon local Ca2+ influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca2+ flux into the lumen of the TGN is achieved remains mysterious, as the cytosol has a nanomolar steady-state Ca2+ concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), allowing protein-mediated exchange of molecular species such as lipids. Here, we show that the TGN export of secretory proteins requires the integrity of ER-TGN MCS and inositol 3 phosphate receptor (IP3R)-dependent Ca2+ fluxes in the MCS, suggesting Ca2+ transfer between these organelles. Using an MCS-targeted Ca2+ FRET sensor module, we measure the Ca2+ flow in these sites in real time. These data show that ER-TGN MCS facilitates the Ca2+ transfer required for Ca2+-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology.

Ratiometric measurement of MAM Ca2+ dynamics using a modified CalfluxVTN

Mitochondria-associated ER membrane (MAM) is a structure where these calcium-regulating organelles form close physical contact sites for efficient Ca2+ crosstalk. Despite the central importance of MAM Ca2+ dynamics in diverse biological processes, directly and specifically measuring Ca2+ concentrations inside MAM is technically challenging. Here, we develop MAM-Calflux, a MAM-specific BRET-based Ca2+ indicator. The successful application of the bimolecular fluorescence complementation (BiFC) concept highlights Ca2+-responsive BRET signals in MAM. The BiFC strategy imparts dual functionality as a Ca2+ indicator and quantitative structural marker specific for MAM. As a ratiometric Ca2+ indicator, MAM-Calflux estimates steady-state MAM Ca2+ levels. Finally, it enables the visualization of uneven intracellular distribution of MAM Ca2+ and the elucidation of abnormally accumulated MAM Ca2+ from the neurons of Parkinson’s disease mouse model in both steady-state and stimulated conditions. Therefore, we propose that MAM-Calflux can be a versatile tool for ratiometrically measuring dynamic inter-organellar Ca2+ communication.

Constraints of vigilance-dependent noradrenergic signaling to mouse cerebellar Bergmann glia.

Behavioral state plays an important role in determining astroglia Ca2+ signaling. In particular, locomotion-mediated elevated vigilance has been found to trigger norepinephrine-dependent whole cell Ca2+ elevations in astroglia throughout the brain. For cerebellar Bergmann glia it has recently been found that locomotion-induced transient Ca2+ elevations depend on their α1A -adrenergic receptors. With increasing availability and implementation of locomotion as behavioral parameter it becomes important to understand the constraints of noradrenergic signaling to astroglia. Here we evaluated the effect of speed, duration and interval of locomotion on Ca2+ signals in Bergmann glia as well as cerebellar noradrenergic axon terminals. We found almost no dependence on locomotion speed, but following the initial Ca2+ transient prolonged locomotion events revealed a steady-state Ca2+ elevation. Comparison of time course and recovery of transient Bergmann glia and noradrenergic terminal Ca2+ dynamics suggested that noradrenergic terminal Ca2+ activity determines Bergmann glia Ca2+ activation and does not require noradrenergic receptor desensitization to account for attenuation during prolonged locomotion. Further, analyzing the correlation among Ca2+ dynamics within regions within the field of observation we found that coordinated activity among noradrenergic terminals accounts for fluctuations of steady-state Bergmann glia Ca2+ activity. Together, our findings will help to better understand astroglia Ca2+ dynamics during less controlled awake behavior and may guide the identification of behavioral contexts preferably dependent on astroglia Ca2+ signaling.

CaMKII-Dependent Contractile Dysfunction and Pro-Arrhythmic Activity in a Mouse Model of Obstructive Sleep Apnea.

Left ventricular contractile dysfunction and arrhythmias frequently occur in patients with sleep-disordered breathing (SDB). The CaMKII-dependent dysregulation of cellular Ca homeostasis has recently been described in SDB patients, but these studies only partly explain the mechanism and are limited by the patients' heterogeneity. Here, we analyzed contractile function and Ca homeostasis in a mouse model of obstructive sleep apnea (OSA) that is not limited by confounding comorbidities. OSA was induced by artificial tongue enlargement with polytetrafluorethylene (PTFE) injection into the tongue of wildtype mice and mice with a genetic ablation of the oxidative activation sites of CaMKII (MMVV knock-in). After eight weeks, cardiac function was assessed with echocardiography. Reactive oxygen species (ROS) and Ca transients were measured using confocal and epifluorescence microscopy, respectively. Wildtype PTFE mice exhibited an impaired ejection fraction, while MMVV PTFE mice were fully protected. As expected, isolated cardiomyocytes from PTFE mice showed increased ROS production. We further observed decreased levels of steady-state Ca transients, decreased levels of caffeine-induced Ca transients, and increased pro-arrhythmic activity (defined as deviations from the diastolic Ca baseline) only in wildtype but not in MMVV PTFE mice. In summary, in the absence of any comorbidities, OSA was associated with contractile dysfunction and pro-arrhythmic activity and the inhibition of the oxidative activation of CaMKII conveyed cardioprotection, which may have therapeutic implications.

Functional comparison of phosphomimetic S15D and T160D mutants of myosin regulatory light chain exchanged in cardiac muscle preparations of HCM and WT mice.

In this study, we investigated the rescue potential of two phosphomimetic mutants of the myosin regulatory light chain (RLC, MYL2 gene), S15D, and T160D RLCs. S15D-RLC mimics phosphorylation of the established serine-15 site of the human cardiac RLC. T160D-RLC mimics the phosphorylation of threonine-160, identified by computational analysis as a high-score phosphorylation site of myosin RLC. Cardiac myosin and left ventricular papillary muscle (LVPM) fibers were isolated from a previously generated model of hypertrophic cardiomyopathy (HCM), Tg-R58Q, and Tg-wild-type (WT) mice. Muscle specimens were first depleted of endogenous RLC and then reconstituted with recombinant human cardiac S15D and T160D phosphomimetic RLCs. Preparations reconstituted with recombinant human cardiac WT-RLC and R58Q-RLC served as controls. Mouse myosins were then tested for the actin-activated myosin ATPase activity and LVPM fibers for the steady-state force development and Ca2+-sensitivity of force. The data showed that S15D-RLC significantly increased myosin ATPase activity compared with T160D-RLC or WT-RLC reconstituted preparations. The two S15D and T160D phosphomimetic RLCs were able to rescue Vmax of Tg-R58Q myosin reconstituted with recombinant R58Q-RLC, but the effect of S15D-RLC was more pronounced than T160D-RLC. Low tension observed for R58Q-RLC reconstituted LVPM from Tg-R58Q mice was equally rescued by both phosphomimetic RLCs. In the HCM Tg-R58Q myocardium, the S15D-RLC caused a shift from the super-relaxed (SRX) state to the disordered relaxed (DRX) state, and the number of heads readily available to interact with actin and produce force was increased. At the same time, T160D-RLC stabilized the SRX state at a level similar to R58Q-RLC reconstituted fibers. We report here on the functional superiority of the established S15 phospho-site of the human cardiac RLC vs. C-terminus T160-RLC, with S15D-RLC showing therapeutic potential in mitigating a non-canonical HCM behavior underlined by hypocontractile behavior of Tg-R58Q myocardium.

ALG-2 and peflin regulate COPII targeting and secretion in response to calcium signaling

ER-to-Golgi transport is the first step in the constitutive secretory pathway, which, unlike regulated secretion, is believed to proceed nonstop independent of Ca2+ flux. However, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca2+ signaling by adopting one of several distinct activity states. Functionally, these states can adjust the rate of ER export of COPII-sorted cargos up or down by ∼50%. We found that at steady-state Ca2+, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. Upon Ca2+ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration—phenomena that could contribute to cellular growth and intercellular communication following secretory increases or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Furthermore, distinct Ca2+ signaling patterns in NRK cells produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased levels of the COPII outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPII outer shell and decreased peflin at ERES. These data provide insights into how PEF protein dynamics affect secretion of important physiological cargoes such as collagen I and significantly impact ER stress.

Finite element analysis predicts Ca2+ microdomains within tubular-sarcoplasmic reticular junctions of amphibian skeletal muscle

A finite element analysis modelled diffusional generation of steady-state Ca2+ microdomains within skeletal muscle transverse (T)-tubular-sarcoplasmic reticular (SR) junctions, sites of ryanodine receptor (RyR)-mediated SR Ca2+ release. It used established quantifications of sarcomere and T-SR anatomy (radial diameter d=220 , mathrm{n}mathrm{m}; axial distance w=12 , mathrm{n}mathrm{m}). Its boundary SR Ca2+ influx densities,{J}_{mathrm{influx}}, reflected step impositions of influxes, it {Phi }_{mathrm{influx}}={J}_{mathrm{influx}}left(frac{pi {d}^{2}}{4}right), deduced from previously measured Ca2+ signals following muscle fibre depolarization. Predicted steady-state T-SR junctional edge [Ca2+], [Ca2+]edge, matched reported corresponding experimental cytosolic [Ca2+] elevations given diffusional boundary effluxit it {Phi }_{mathrm{efflux}}=frac{D [ {{{mathrm{Ca}}^{2+}}}]_{mathrm{edge}}}{lambda } (pi dw), established cytosolic Ca2+ diffusion coefficients (D = 4 times {10}^{7} mathrm{nm}^{2}/mathrm{s}) and exit length lambda = 9.2 , mathrm{n}mathrm{m}. Dependences of predicted [Ca2+]edge upon {J}_{mathrm{influx}} then matched those of experimental [Ca2+] upon Ca2+ release through their entire test voltage range. The resulting model consistently predicted elevated steady-state T-SR junctional ~ µM-[Ca2+] elevations radially declining from maxima at the T-SR junction centre along the entire axial T-SR distance. These [Ca2+] heterogeneities persisted through 104- and fivefold, variations in D and w around, and fivefold reductions in d below, control values, and through reported resting muscle cytosolic [Ca2+] values, whilst preserving the flux conservation (it it {Phi }_{mathrm{influx}}={Phi }_{mathrm{efflux}}) condition, {left[mathrm{C}{mathrm{a}}^{2+}right]}_{mathrm{edge}}=frac{lambda {dJ}_{mathrm{influx}}}{4Dw}. Skeletal muscle thus potentially forms physiologically significant ~ µM-[Ca2+] T-SR microdomains that could regulate cytosolic and membrane signalling molecules including calmodulin and RyR, These findings directly fulfil recent experimental predictions invoking such Ca2+ microdomains in observed regulatory effects upon Na+ channel function, in a mechanism potentially occurring in similar restricted intracellular spaces in other cell types.

TRPM7 is an essential regulator for volume-sensitive outwardly rectifying anion channel

Animal cells can regulate their volume after swelling by the regulatory volume decrease (RVD) mechanism. In epithelial cells, RVD is attained through KCl release mediated via volume-sensitive outwardly rectifying Cl− channels (VSOR) and Ca2+-activated K+ channels. Swelling-induced activation of TRPM7 cation channels leads to Ca2+ influx, thereby stimulating the K+ channels. Here, we examined whether TRPM7 plays any role in VSOR activation. When TRPM7 was knocked down in human HeLa cells or knocked out in chicken DT40 cells, not only TRPM7 activity and RVD efficacy but also VSOR activity were suppressed. Heterologous expression of TRPM7 in TRPM7-deficient DT40 cells rescued both VSOR activity and RVD, accompanied by an increase in the expression of LRRC8A, a core molecule of VSOR. TRPM7 exerts the facilitating action on VSOR activity first by enhancing molecular expression of LRRC8A mRNA through the mediation of steady-state Ca2+ influx and second by stabilizing the plasmalemmal expression of LRRC8A protein through the interaction between LRRC8A and the C-terminal domain of TRPM7. Therefore, TRPM7 functions as an essential regulator of VSOR activity and LRRC8A expression.

A comprehensive overview of the complex world of the endo- and sarcoplasmic reticulum Ca2+-leak channels

Inside cells, the endoplasmic reticulum (ER) forms the largest Ca2+ store. Ca2+ is actively pumped by the SERCA pumps in the ER, where intraluminal Ca2+-binding proteins enable the accumulation of large amount of Ca2+. IP3 receptors and the ryanodine receptors mediate the release of Ca2+ in a controlled way, thereby evoking complex spatio-temporal signals in the cell. The steady state Ca2+ concentration in the ER of about 500 μM results from the balance between SERCA-mediated Ca2+ uptake and the passive leakage of Ca2+. The passive Ca2+ leak from the ER is often ignored, but can play an important physiological role, depending on the cellular context. Moreover, excessive Ca2+ leakage significantly lowers the amount of Ca2+ stored in the ER compared to normal conditions, thereby limiting the possibility to evoke Ca2+ signals and/or causing ER stress, leading to pathological consequences. The so-called Ca2+-leak channels responsible for Ca2+ leakage from the ER are however still not well understood, despite over 20 different proteins have been proposed to contribute to it. This review has the aim to critically evaluate the available evidence about the various channels potentially involved and to draw conclusions about their relative importance.

K+-specific importers Trk1 and Trk2 play different roles in Ca2+ homeostasis and signalling in Saccharomyces cerevisiae cells.

The maintenance of K+ and Ca2+ homeostasis is crucial for many cellular functions. Potassium is accumulated in cells at high concentrations, while the cytosolic level of calcium, to ensure its signalling function, is kept at low levels and transiently increases in response to stresses. We examined Ca2+ homeostasis and Ca2+ signalling in Saccharomyces cerevisiae strains lacking plasma-membrane K+ influx (Trk1 and Trk2) or efflux (Tok1, Nha1 and Ena1-5) systems. The lack of K+ exporters slightly increased the cytosolic Ca2+, but did not alter the Ca2+ tolerance or Ca2+-stress response. In contrast, the K+-importers Trk1 and Trk2 play important and distinct roles in the maintenance of Ca2+ homeostasis. The presence of Trk1 was vital mainly for the growth of cells in the presence of high extracellular Ca2+, whilst the lack of Trk2 doubled steady-state intracellular Ca2+ levels. The absence of both K+ importers highly increased the Ca2+ response to osmotic or CaCl2 stresses and altered the balance between Ca2+ flux from external media and intracellular compartments. In addition, we found Trk2 to be important for the tolerance to high KCl and hygromycin B in cells growing on minimal media. All the data describe new interconnections between potassium and calcium homeostasis in S. cerevisiae.

Total Matrix Ca2+ Modulates Ca2+ Efflux via the Ca2+/H+ Exchanger in Cardiac Mitochondria.

Mitochondrial Ca2+ handling is accomplished by balancing Ca2+ uptake, primarily via the Ru360-sensitive mitochondrial calcium uniporter (MCU), Ca2+ buffering in the matrix and Ca2+ efflux mainly via Ca2+ ion exchangers, such as the Na+/Ca2+ exchanger (NCLX) and the Ca2+/H+ exchanger (CHE). The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca2+ regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca2+ efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca2+ load via repetitive application of a finite CaCl2 bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca2+ efflux at different matrix Ca2+ loads was revealed by inhibiting Ca2+ uptake or reuptake with Ru360 after increasing number of CaCl2 boluses. In Na+-free experimental buffer and with Ca2+ uptake inhibited, the rate of Ca2+ efflux and steady-state free matrix Ca2+ [mCa2+]ss increased as the number of administered CaCl2 boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca2+ buffering while maintaining a constant [mCa2+]ss, decreased the rate of Ca2+ efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca2+ buffering rate and decreased [mCa2+]ss. A change in the pH of the external medium to a more acidic value from 7.15 to 6.8∼6.9 caused a twofold increase in the Ca2+ efflux rate, while an alkaline change in pH from 7.15 to 7.4∼7.5 did not change the Ca2+ efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca2+ efflux. The results indicate that Ca2+ efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca2+. A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca2+ buffering system and the matrix free Ca2+ concentration.

Novel TRPV6 mutations in the spectrum of transient neonatal hyperparathyroidism

Maternal–fetal calcium (Ca2+) transport in the placenta plays a critical role in maintaining fetal bone mineralization. Mutations in the gene encoding the transient receptor potential cation channel, subfamily V, member 6 (TRPV6) have been identified as causative mutations of transient neonatal hyperparathyroidism due to insufficient maternal–fetal Ca2+ transport in the placenta. In this study, we found two novel mutations in subjects that have transient neonatal hyperparathyroidism. TRPV6 carrying the mutation p.Arg390His that localizes to the outer edge of the first transmembrane domain (S1) showed impaired trafficking to the plasma membrane, whereas TRPV6 having the mutation p.Gly291Ser in the sixth ankyrin repeat (AR) domain had channel properties that were comparable those of WT channels, although the increases in steady-state intracellular Ca2+ concentration could have led to Ca2+ overload and subsequent death of cells expressing this mutant channel. These results indicate that the AR6 domain contributes to TRPV6-mediated maintenance of intracellular Ca2+ concentrations, and that this region could play a novel role in regulating the activity of TRPV6 Ca2+-selective channels.

Dysregulation of Endogenous and Paracrine Calcium Signaling Pathways by Rotaviruses and Caliciviruses

Despite causing severe, potentially life‐threatening diarrhea, most enteric viruses only infect a small percent of intestinal epithelial cells. Thus, the induction of paracrine signaling pathways to dysregulate both infected and neighboring uninfected cell remains a major premise of enteric virus pathogenesis. However, virus‐induced paracrine signaling has never been directly observed and therefore the mechanisms have yet to be defined. Rotavirus (RV) remains a major cause of severe diarrhea in children worldwide. A hallmark of RV infection is the activation of aberrant calcium (Ca2+) signaling, which is necessary for replication and activation of secretory pathways in GI epithelium. We sought to characterize RV‐induced calcium signaling dynamics and identify paracrine signaling pathways responsible for infected‐to‐uninfected cell signaling. We conducted live‐cell Ca2+ imaging throughout the entire infection using cell lines and human intestinal enteroids (HIEs) engineered to stably express the genetically‐encoded Ca2+ indicator GCaMP. We found that rotavirus significantly increases both steady‐state and transient Ca2+ signaling mediated by RV nonstructural protein 4 (NSP4) and mutation of the NSP4 ion channel domain altered the RV‐induced Ca2+ signaling pattern observed, particularly low amplitude Ca2+ puffs observed early in infected cells. Further, isolated rotavirus‐infected cells generated multiple intercellular calcium waves (ICWs), which was the most prominent paracrine signal observed in both MA104 cells and HIEs. RV infection induces several signaling molecules, such as enterotoxin NSP4, prostaglandins and nitric oxide, and in many cell types extracellular purinergic signaling by ATP/ADP are responsible for ICW propagation. We found that RV‐induced ICWs were abolished by blocking extracellular ATP/ADP signaling with apyrase or purinergic receptor inhibitors or by CRISPR/Cas9‐mediated deletion of the P2Y1 receptor, but not by blocking extracellular enterotoxin form of NSP4, nitric oxide, or prostaglandin signaling. Rotavirus‐induced paracrine signaling was critical for multiple aspects of rotavirus pathogenesis, including fluid secretion and serotonin release, as well as regulating host responses, such as upregulation of IL‐1α and mucin secretion. Finally, we investigated whether activation of aberrant Ca2+ signaling and paracrine purinergic signaling were features of enteric caliciviruses using Tulane virus (TV), a rhesus monkey calicivirus. Like RV, we found that TV encodes for a viral ion channel in the endoplasmic reticulum and induces aberrant Ca2+ signaling during infection. TV infected cells also trigger ICWs that are blocked by purinergic signaling inhibitors. Thus, enteric viruses employ mechanisms to not only disrupt signaling in the virus‐infected cell but also exploit paracrine purinergic signaling to generate ICWs that represent a potent mechanism to amplify the pathophysiological signals underlying viral diarrhea.

Multiple Dynamical Mechanisms of Phase-2 Early Afterdepolarizations in a Human Ventricular Myocyte Model: Involvement of Spontaneous SR Ca 2+ Release.

Early afterdepolarization (EAD) is known to cause lethal ventricular arrhythmias in long QT syndrome (LQTS). In this study, dynamical mechanisms of EAD formation in human ventricular myocytes (HVMs) were investigated using the mathematical model developed by ten Tusscher and Panfilov (Am J Physiol Heart Circ Physiol 291, 2006). We explored how the rapid (IKr) and slow (IKs) components of delayed-rectifier K+ channel currents, L-type Ca2+ channel current (ICaL), Na+/Ca2+ exchanger current (INCX), and intracellular Ca2+ handling via the sarcoplasmic reticulum (SR) contribute to initiation, termination and modulation of phase-2 EADs during pacing in relation to bifurcation phenomena in non-paced model cells. Parameter-dependent dynamical behaviors of the non-paced model cell were determined by calculating stabilities of equilibrium points (EPs) and limit cycles, and bifurcation points to construct bifurcation diagrams. Action potentials (APs) and EADs during pacing were reproduced by numerical simulations for constructing phase diagrams of the paced model cell dynamics. Results are summarized as follows: (1) A modified version of the ten Tusscher-Panfilov model with accelerated ICaL inactivation could reproduce bradycardia-related EADs in LQTS type 2 and β-adrenergic stimulation-induced EADs in LQTS type 1. (2) Two types of EADs with different initiation mechanisms, ICaL reactivation–dependent and spontaneous SR Ca2+ release–mediated EADs, were detected. (3) Termination of EADs (AP repolarization) during pacing depended on the slow activation of IKs. (4) Spontaneous SR Ca2+ releases occurred at higher Ca2+ uptake rates, attributable to the instability of steady-state intracellular Ca2+ concentrations. Dynamical mechanisms of EAD formation and termination in the paced model cell are closely related to stability changes (bifurcations) in dynamical behaviors of the non-paced model cell, but they are model-dependent. Nevertheless, the modified ten Tusscher-Panfilov model would be useful for systematically investigating possible dynamical mechanisms of EAD-related arrhythmias in LQTS.

Warmer, faster, stronger: Ca2+ cycling in avian myocardium.

Birds occupy a unique position in the evolution of cardiac design. Their hearts are capable of cardiac performance on par with, or exceeding that of mammals, and yet the structure of their cardiomyocytes resembles those of reptiles. It has been suggested that birds use intracellular Ca2+ stored within the sarcoplasmic reticulum (SR) to power contractile function, but neither SR Ca2+ content nor the cross-talk between channels underlying Ca2+-induced Ca2+ release (CICR) have been studied in adult birds. Here we used voltage clamp to investigate the Ca2+ storage and refilling capacities of the SR and the degree of trans-sarcolemmal and intracellular Ca2+ channel interplay in freshly isolated atrial and ventricular myocytes from the heart of the Japanese quail (Coturnix japonica). A trans-sarcolemmal Ca2+ current (ICa) was detectable in both quail atrial and ventricular myocytes, and was mediated only by L-type Ca2+ channels. The peak density of ICa was larger in ventricular cells than in atrial cells, and exceeded that reported for mammalian myocardium recorded under similar conditions. Steady-state SR Ca2+ content of quail myocardium was also larger than that reported for mammals, and reached 750.6±128.2 μmoll-1 in atrial cells and 423.3±47.2 μmoll-1 in ventricular cells at 24°C. We observed SR Ca2+-dependent inactivation of ICa in ventricular myocytes, indicating cross-talk between sarcolemmal Ca2+ channels and ryanodine receptors in the SR. However, this phenomenon was not observed in atrial myocytes. Taken together, these findings help to explain the high-efficiency avian myocyte excitation-contraction coupling with regard to their reptilian-like cellular ultrastructure.

The LRRC8/VRAC anion channel facilitates myogenic differentiation of murine myoblasts by promoting membrane hyperpolarization

Skeletal muscle myoblast differentiation involves elaborate signaling networks, including the activity of various ion channels and transporters. Several K+ and Ca2+ channels have been shown to affect myogenesis, but little is known about roles of Cl- channels in the associated processes. Here, we report that the leucine-rich repeat containing family 8 (LRRC8)/volume-regulated anion channel (VRAC) promotes mouse myoblast differentiation. All LRRC8 subunits of heteromeric VRAC were expressed during myotube formation of murine C2C12 myoblasts. Pharmacological VRAC inhibitors, siRNA-mediated knockdown of the essential VRAC subunit LRRC8A, or VRAC activity-suppressing overexpression of LRRC8A effectively reduced the expression of the myogenic transcription factor myogenin and suppressed myoblast fusion while not affecting myoblast proliferation. We found that inhibiting VRAC impairs plasma membrane hyperpolarization early during differentiation. At later times (more than 6 h after inducing differentiation), VRAC inhibition no longer suppressed myoblast differentiation, suggesting that VRAC acts upstream of K+ channel activation. Consequently, VRAC inhibition prevented the increase of intracellular steady-state Ca2+ levels that normally occurs during myogenesis. Our results may explain the mechanism for the thinning of skeletal muscle bundles observed in LRRC8A-deficient mice and highlight the importance of the LRRC8/VRAC anion channel in cell differentiation.

Deletion of the Ca2+ Channel Subunit α2δ3 Differentially Affects Cav2.1 and Cav2.2 Currents in Cultured Spiral Ganglion Neurons Before and After the Onset of Hearing.

Voltage-gated Ca2+ channels are composed of a pore-forming α1 subunit and auxiliary β and α2δ subunits, which modulate Ca2+ current properties and channel trafficking. So far, the partial redundancy and specificity of α1 for α2δ subunits in the CNS have remained largely elusive. Mature spiral ganglion (SG) neurons express α2δ subunit isoforms 1, 2, and 3 and multiple Ca2+ channel subtypes. Differentiation and in vivo functions of their endbulb of Held synapses, which rely on presynaptic P/Q channels (Lin et al., 2011), require the α2δ3 subunit (Pirone et al., 2014). This led us to hypothesize that P/Q channels may preferentially co-assemble with α2δ3. Using a dissociated primary culture, we analyzed the effects of α2δ3 deletion on somatic Ca2+ currents (ICa) of SG neurons isolated at postnatal day 20 (P20), when the cochlea is regarded to be mature. P/Q currents were the dominating steady-state Ca2+ currents (54% of total) followed by T-type, L-type, N-type, and R-type currents. Deletion of α2δ3 reduced P/Q- and R-type currents by 60 and 38%, respectively, whereas L-type, N-type, and T-type currents were not altered. A subset of ICa types was also analyzed in SG neurons isolated at P5, i.e., before the onset of hearing (P12). Both L-type and N-type current amplitudes of wildtype SG neurons were larger at P5 compared with P20. Deletion of α2δ3 reduced L-type and N-type currents by 23 and 44%, respectively. In contrast, small P/Q currents, which were just being up-regulated at P5, were unaffected by the lack of α2δ3. In summary, α2δ3 regulates amplitudes of L- and N-type currents of immature cultured SG neurons, whereas it regulates P/Q- and R-type currents at P20. Our data indicate a developmental switch from dominating somatic N- to P/Q-type currents in cultured SG neurons. A switch from N- to P/Q-type channels, which has been observed at several central synapses, may also occur at developing endbulbs of Held. In this case, reduction of both neonatal N- (P5) and more mature P/Q-type currents (around/after hearing onset) may contribute to the impaired morphology and function of endbulb synapses in α2δ3-deficient mice.

New Insights Into the Role of Cav2 Protein Family in Calcium Flux Deregulation in Fmr1-KO Neurons.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability (ID) and a leading cause of autism, results from the loss of expression of the Fmr1 gene which encodes the RNA-binding protein Fragile X Mental Retardation Protein (FMRP). Among the thousands mRNA targets of FMRP, numerous encode regulators of ion homeostasis. It has also been described that FMRP directly interacts with Ca2+ channels modulating their activity. Collectively these findings suggest that FMRP plays critical roles in Ca2+ homeostasis during nervous system development. We carried out a functional analysis of Ca2+ regulation using a calcium imaging approach in Fmr1-KO cultured neurons and we show that these cells display impaired steady state Ca2+ concentration and an altered entry of Ca2+ after KCl-triggered depolarization. Consistent with these data, we show that the protein product of the Cacna1a gene, the pore-forming subunit of the Cav2.1 channel, is less expressed at the plasma membrane of Fmr1-KO neurons compared to wild-type (WT). Thus, our findings point out the critical role that Cav2.1 plays in the altered Ca2+ flux in Fmr1-KO neurons, impacting Ca2+ homeostasis of these cells. Remarkably, we highlight a new phenotype of cultured Fmr1-KO neurons that can be considered a novel cellular biomarker and is amenable to small molecule screening and identification of new drugs to treat FXS.

Structural and functional impact of troponin C-mediated Ca2+ sensitization on myofilament lattice spacing and cross-bridge mechanics in mouse cardiac muscle

Acto-myosin cross-bridge kinetics are important for beat-to-beat regulation of cardiac contractility; however, physiological and pathophysiological mechanisms for regulation of contractile kinetics are incompletely understood. Here we explored whether thin filament-mediated Ca2+ sensitization influences cross-bridge kinetics in permeabilized, osmotically compressed cardiac muscle preparations. We used a murine model of hypertrophic cardiomyopathy (HCM) harboring a cardiac troponin C (cTnC) Ca2+-sensitizing mutation, Ala8Val in the regulatory N-domain. We also treated wild-type murine muscle with bepridil, a cTnC-targeting Ca2+ sensitizer. Our findings suggest that both methods of increasing myofilament Ca2+ sensitivity increase cross-bridge cycling rate measured by the rate of tension redevelopment (kTR); force per cross-bridge was also enhanced as measured by sinusoidal stiffness and I1,1/I1,0 ratio from X-ray diffraction. Computational modeling suggests that Ca2+ sensitization through this cTnC mutation or bepridil accelerates kTR primarily by promoting faster cross-bridge detachment. To elucidate if myofilament structural rearrangements are associated with changes in kTR, we used small angle X-ray diffraction to simultaneously measure myofilament lattice spacing and isometric force during steady-state Ca2+ activations. Within in vivo lattice dimensions, lattice spacing and steady-state isometric force increased significantly at submaximal activation. We conclude that the cTnC N-domain controls force by modulating both the number and rate of cycling cross-bridges, and that the both methods of Ca2+ sensitization may act through stabilization of cTnC's D-helix. Furthermore, we propose that the transient expansion of the myofilament lattice during Ca2+ activation may be an additional factor that could increase the rate of cross-bridge cycling in cardiac muscle. These findings may have implications for the pathophysiology of HCM.