Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states

We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in mammalian cells. Orai1 was detected predominantly as dimers under resting conditions and as tetramers when coexpressed with C-STIM1 to activate Ca(2+) influx. Orai1 was also found to be tetrameric when coexpressed with STIM1 and evaluated following fixation. We show that fixation rapidly causes release of Ca(2+), redistribution of STIM1 to the plasma membrane, and STIM1/Orai1 puncta formation, and may cause the channel to be in the activated state. Consistent with this possibility, Orai1 was found predominantly as a dimer when coexpressed with STIM1 in living cells under resting conditions. We further show that Orai3, like Orai1, is dimeric under resting conditions and is predominantly tetrameric when activated by C-STIM1. Interestingly, a dimeric Orai3 stoichiometry was found both before and during application of 2-aminoethyldiphenyl borate (2-APB) to activate a nonselective cation conductance in its STIM1-independent mode. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca(2+)-selective pore during store-operated activation and that Orai3 forms a dimeric nonselective cation pore upon activation by 2-APB.     

Phospholipase D in human platelets: Presence of isoenzymes and participation of autocrine stimulation during thrombin activation

Phospholipase D (PLD), which hydrolyzes phosphatidylcholine to phosphatidic acid (PA) and choline, is present in human platelets. Thrombin and other agonists have been shown to activate PLD but the precise mechanisms of activation and PLDs role in platelet activation remains unclear. We measured thrombin-stimulated PLD activity in platelets as formation of phosphatidylethanol. Since no specific PLD inhibitors exist, we investigated possible roles for PLD in platelets by correlating PLD activity with platelet responses such as thrombin-mediated secretion and F-actin formation (part of platelet shape change). Extracellular Ca²⁺ potentiated thrombin-stimulated PLD, but did not stimulate PLD in the absence of thrombin. Thrombin-induced PLD activity was enhanced by secreted ADP and binding of fibrinogen to its receptors. In contrast to others, we also found a basal PLD activity. Comparison of time courses and dose responses of platelets with PLD showed many points of correlation between PLD activation and lysosomal secretion and F-actin formation. The finding of different PLD activities suggested that different PLD isoenzymes exist in platelets as reported for other cells. Here we present evidence for the presence of both PLD1 and PLD2 in platelets by use of specific antibodies with immunoblotting and immunohistochemistry. Both isoforms were randomly localized in resting platelets, but became rapidly translocated to the proximity of the plasma membrane upon thrombin stimulation, thus indicating a role for PLD in platelet activation.     

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca²⁺ mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca²⁺ channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca²⁺]_i in these cells, suggesting that they do not possess TG-sensitive Ca²⁺ pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca²⁺ oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca²⁺]_i of type III adenoma cells that were also characterized by a low resting [Ca²⁺]_i and by the absence of spontaneous Ca²⁺ fluctuations. K⁺-induced depolarization provoked a reduced Ca²⁺ influx, whereas TG had no effect on the [Ca²⁺]_i of type III adenoma cells. The variety of [Ca²⁺]_i response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.     

H(2)O(2)-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation

Reactive oxygen species (ROS) and intracellular Ca²⁺ overload play key roles in myocardial ischemia-reperfusion (IR) injury but the relationships among ROS, Ca²⁺ overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H₂O₂ impairs LV function by causing Ca²⁺ overload by increasing late sodium current (I_Na), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca²⁺ concentrations (d[Ca²⁺]_i and s[Ca²⁺]_i) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H₂O₂ (100 μM, 30 min) increased d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently before declining gradually to 17%. Ouabain (80 μM) exerted similar effects to ATX-II. Late I_Na inhibitors, lidocaine (10 μM) or R56865 (2 μM), reduced effects of ATX-II on [Ca²⁺]_i and LV function, but did not alter effects of H₂O₂. The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H₂O₂-induced LV dysfunction, but did not alter [Ca²⁺]_i. Paradoxically, further increases in [Ca²⁺]_i by ATX-II or ouabain, given 10 min after H₂O₂, improved function. The failure of late I_Na inhibitors to prevent H₂O₂-induced LV dysfunction, and the ability of MPG to prevent H₂O₂-induced LV dysfunction independent of changes in [Ca²⁺]_i indicate that impaired contractility is not due to Ca²⁺ overload. The ability of further increases in [Ca²⁺]_i to reverse H₂O₂-induced LV dysfunction suggests that Ca²⁺ desensitization is the predominant mechanism of ROS-induced contractile dysfunction.     

胰淀素抑制高糖刺激INS-1细胞内钙离子浓度升高的作用机制

 目的 分析阐明胰淀素短时间作用于INS-1细胞、抑制高糖刺激的细胞内钙离子浓度（［Ca²⁺］_i）升高的机制。方法 采用钙离子荧光指示剂Fluo-4/AM负载细胞后,激光共聚焦显微镜下连续动态观察INS-1细胞经胰淀素孵育前后在葡萄糖、KCl、咖啡因和卡巴胆碱存在条件下［Ca²⁺］_i荧光强度变化。结果 （1）单纯16.7 mmol/L葡萄糖刺激使细胞内钙离子荧光强度变化的曲线下面积（AUC）为990±16;0.5 μmol/L胰淀素使16.7 mmol/L葡萄糖刺激的胞内荧光强度有所下降（AUC为831±10）, 1.0、5.0、10.0 μmol/L胰淀素均可使细胞内荧光强度显著降低（AUC分别为555±9、535±6、531±5）,与单纯葡萄糖刺激组比较差异有统计学意义,且呈现剂量依赖趋势。（2）10.0 μmol/L胰淀素可使30 mmol/L KCl刺激的荧光强度明显减低,与单纯KCl刺激组相比（AUC：168±5比311±11）差异有统计学意义。（3） 10.0 μmol/L胰淀素预处理后咖啡因和卡巴胆碱刺激的胞内荧光强度与单纯咖啡因和卡巴胆碱刺激组相比差异无统计学意义。结论 高浓度胰淀素的短时间作用对INS-1细胞经咖啡因和卡巴胆碱刺激的内质网鱼尼丁受体(ryanodine receptor,RyR)、三磷酸肌醇(IP₃)钙库释放没有影响,但可使高糖及KCl刺激的胞内荧光强度降低。推测胰淀素短时间作用使 ［Ca²⁺］_i减少的现象,主要是通过影响膜上钙通道实现的,与胞内钙库的释放无直接相关性。     

Passive transport pathways for Ca and Co in human red blood cells. Co as a tracer for Ca influx

The passive transport of calcium and cobalt and their interference were studied in human red cells using ⁴⁵Ca and ⁵⁷Co as tracers. In ATP-depleted cells, with the ATP concentration reduced to about 1 μM, the progress curve for ⁴⁵Ca uptake at 1 mM rapidly levels off with time, consistent with a residual Ca-pump activity building up at increasing [Ca^T]_c to reach at [Ca^T]_c about 5 μmol (l cells)^{− 1} a maximal pump rate that nearly countermands the passive Ca influx, resulting in a linear net uptake at a low level. In ATP-depleted cells treated with vanadate, supposed to cause Ca-pump arrest, a residual pump activity is still present at high [Ca^T]_c. Moreover, vanadate markedly increases the passive Ca²⁺ influx. The residual Ca-pump activity in ATP-depleted cells is fuelled by breakdown of the large 2,3-DPG pool, rate-limited by the sustainable ATP-turnover at about 40–50 μmol (l cells)^{− 1} h^{− 1}. The apparent Ca²⁺ affinity of the Ca-pump appears to be markedly reduced compared to fed cells. The 2,3-DPG breakdown can be prevented by inhibition of the 2,3-DPG phosphatase by tetrathionate, and under these conditions the ⁴⁵Ca uptake is markedly increased and linear with time, with the unidirectional Ca influx at 1 mM Ca²⁺ estimated at 50–60 μmol (l cells)^{− 1} h^{− 1}. The Ca influx increases with the extracellular Ca²⁺ concentration with a saturating component, with K_½(Ca) about 0.3 mM, plus a non-saturating component. From ⁴⁵Ca-loaded, ATP-depleted cells the residual Ca-pump can also be detected as a vanadate- and tetrathionate-sensitive efflux. The ⁴⁵Ca efflux is markedly accelerated by external Ca²⁺, both in control cells and in the presence of vanadate or tetrathionate, suggesting efflux by carrier-mediated Ca/Ca exchange.The ⁵⁷Co uptake is similar in fed cells and in ATP-depleted cells (exposed to iodoacetamide), consistent with the notion that Co²⁺ is not transported by the Ca-pump. The transporter is thus neither SH-group nor ATP or phosphorylation dependent. The ⁵⁷Co uptake shows several similarities with the ⁴⁵Ca uptake in ATP-depleted cells supplemented with tetrathionate. The uptake is linear with time, and increases with the cobalt concentration with a saturating component, with J_max about 16 μmol (l cells)^{− 1} h^{− 1} and K_½(Co) about 0.1 mM, plus a non-saturating component. The ⁵⁷Co and ⁴⁵Ca uptake shows mutual inhibition, and at least the stochastic Ca²⁺ influx is inhibited by Co²⁺. The ⁵⁷Co and ⁴⁵Ca uptake are both insensitive to the 1,4-dihydropyridine Ca-channel blocker nifedipine, even at 100 μM. The ⁵⁷Co uptake is increased at high negative membrane potentials, indicating that the uptake is at least partially electrogenic. The ⁵⁷Co influx amounts to about half the ⁴⁵Ca influx in ATP-depleted cells. It is speculated that the basal Ca²⁺ and Co²⁺ uptake could be mediated by a common transporter, probably with a channel-like and a carrier-mediated component, and that ⁵⁷Co could be useful as a tracer for at least the channel-like Ca²⁺ entry pathway in red cells, since it is not itself transported by the Ca-pump and, moreover, is effectively buffered in the cytosol by binding to hemoglobin, without interfering with Ca²⁺ buffering. The molecular identity of the putative common transporter(s) remains to be defined.     

Action potential prolongation in cardiac myocytes of old rats is an adaptation to sustain youthful intracellular Ca2+ regulation

Advanced age in rats is accompanied by reduced expression of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA-2). The amplitudes of intracellular Ca2+ (Ca2+(i)) transients and contractions in ventricular myocytes isolated from old (23-24-months) rats (OR), however, are similar to those of young (4-6-months) rat myocytes (YR). OR myocytes also manifest slowed inactivation of L-type Ca2+ current (I(CaL)) and marked prolongation of action potential (AP) duration. To determine whether and how age-associated AP prolongation preserves the Ca2+(i) transient amplitude in OR myocytes, we employed an AP-clamp technique with simultaneous measurements of I(CaL) (with Na+ current, K+ currents and Ca2+ influx via sarcolemmal Na+-Ca2+ exchanger blocked) and Ca2+(i) transients in OR rat ventricular myocytes dialyzed with the fluorescent Ca2+ probe, indo-1. Myocytes were stimulated with AP-shaped voltage clamp waveforms approximating the configuration of prolonged, i.e. the native, AP of OR cells (AP-L), or with short AP waveforms (AP-S), typical of YR myocytes. Changes in SR Ca2+ load were assessed by rapid, complete SR Ca2+ depletions with caffeine. As expected, during stimulation with AP-S vs AP-L, peak I(CaL) increased, by 21+/-4%, while the I(CaL) integral decreased, by 19+/-3% (P<0.01 for each). Compared to AP-L, stimulation of OR myocytes with AP-S reduced the amplitudes of the Ca2+(i) transient by 31+/-6%, its maximal rate of rise (+dCa2+(i)/dt(max); a sensitive index of SR Ca2+ release flux) by 37+/-4%, and decreased the SR Ca2+ load by 29+/-4% (P<0.01 for each). Intriguingly, AP-S also reduced the maximal rate of the Ca2+(i) transient relaxation and prolonged its time to 50% decline, by 35+/-5% and 33+/-7%, respectively (P<0.01 for each). During stimulation with AP-S, the gain of Ca2+-induced Ca2+ release (CICR), indexed by +dCa2+(i)/dt(max)/I(CaL), was reduced by 46+/-4% vs AP-L (P<0.01). We conclude that the effects of an application of a shorter AP to OR myocytes to reduce +dCa2+(i)/dt(max) and the Ca2+ transient amplitude are attributable to a reduction in SR Ca2+ load, presumably due to a reduced I(CaL) integral and likely also to an increased Ca2+ extrusion via sarcolemmal Na+-Ca2+ exchanger. The decrease in the Ca2+(i) transient relaxation rate in OR cells stimulated with shorter APs may reflect a reduction of Ca2+/calmodulin-kinase II-regulated modulation of Ca2+ uptake via SERCA-2, consequent to a reduced local Ca2+ release in the vicinity of SERCA-2, also attributable to reduced SR Ca2+ load. Thus, the reduction of CICR gain during stimulation with AP-S is the net result of both a diminished SR Ca2+ release and an increased peak I(CaL). These results suggest that ventricular myocytes of old rats utilize AP prolongation to preserve an optimal SR Ca2+ loading, CICR gain and relaxation of Ca2+(i) transients.     

Remodeled cardiac calcium channels

Cardiac calcium channels play a pivotal role in the proper functioning of cardiac cells. In response to various pathologic stimuli, they become remodeled, changing how they function, as they adapt to their new environment. Specific features of remodeled channels depend upon the particular disease state. This review will summarize what is known about remodeled cardiac calcium channels in three disease states: hypertrophy, heart failure and atrial fibrillation. In addition, it will review the recent advances made in our understanding of the function of the various molecular building blocks that contribute to the proper functioning of the cardiac calcium channel.     

Ca2+-dependent modulation of single human cardiac L-type calcium channels by the calcineurin inhibitor cyclosporine

OBJECTIVE: Activity of single L-type calcium channels (LTCC) is enhanced in human failing myocardium (Circulation 98 (1998) 969.), most likely due to impaired dephosphorylation. Protein phosphatase 2B (calcineurin) has recently been shown to be involved in heart failure pathophysiology. We now focus on the regulation of single LTCC by calcineurin that were prevented by Ca(2+)-free experimental conditions in our previous study. METHODS: Single LTCC currents were recorded in myocytes from human atrium and ventricle. Charge carriers were 70 mM Ba(2+), or a mixture of 30 mM Ca(2+) and 60 mM Ba(2+) to facilitate Ca(2+) permeation through recorded channels. The calcineurin inhibitor cyclosporine (10 microM) was used to reveal a putative role for calcineurin in regulation of LTCC. RESULTS: A mixture of Ca(2+) and Ba(2+) as charge carriers allowed for Ca(2+) permeation through recombinant human embryonic kidney cells and native (atrial and ventricular) human cardiac LTCC. With only Ba(2+) as the charge carrier, activities of both ventricular and atrial LTCC were strongly decreased by cyclosporine. In contrast, channel activity remained constant when Ca(2+) permeation was provided. In the presence of thapsigargin and (S)-BayK 8644, cyclosporine here even increased channel activity. CONCLUSIONS: We propose a dual cyclosporine effect on human cardiac LTCC. A non-specific inhibitory effect prevails with Ba(2+) permeation but can be compensated or overcome by a specific Ca(2+)-dependent stimulation with Ca(2+) permeation. More complete restoration of physiological Ca(2+) movements (e.g., Ca(2+) release from sarcoplasmic reticulum) will help to define even more precisely the involvement of calcineurin in regulation of human cardiac LTCC.     

The human CASQ2 mutation K206N is associated with hyperglycosylation and altered cellular calcium handling

Mutations in the human cardiac calsequestrin gene (CASQ2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT-2). This inherited disorder is characterized by life-threatening arrhythmias induced by physical and emotional stress in young patients. Here we identified a novel heterozygous missense mutation (K206N) in the CASQ2 gene in a symptomatic family in which one member died of cardiac arrest. The functional properties of CSQ^K206N were investigated in comparison to the wild-type form of CASQ2 (CSQ^WT) by expression in eukaryotic cell lines and neonatal mouse myocytes. The mutation created an additional N-glycosylation site resulting in a higher molecular weight form of the recombinant protein on immunoblots. The mutation reduced the Ca²⁺ binding capacity of the protein and exhibited an altered aggregation state. Consistently, CSQ^K206N-expressing myocytes exhibited an impaired response to caffeine administration, suggesting a lower Ca²⁺ load of the sarcoplasmic reticulum (SR). The interaction of the mutated CSQ with triadin and the protein levels of the ryanodine receptor were unchanged but the maximal specific [³H]ryanodine binding was increased in CSQ^K206N-expressing myocytes, suggesting a higher opening state of the SR Ca²⁺ release channel. Myocytes with expression of CSQ^K206N showed a higher rate of spontaneous SR Ca²⁺ releases under basal conditions and after β-adrenergic stimulation. We conclude that CSQ^K206N caused a reduced Ca²⁺ binding leading to an abnormal regulation of intracellular Ca²⁺ in myocytes. This may then contribute to the increased propensity to trigger spontaneous Ca²⁺ transients in CSQ^K206N-expressing myocytes.     

Aging differentially modifies agonist-evoked mouse detrusor contraction and calcium signals

Although aging-induced changes in urinary bladder neurotransmission have been studied in some detail, information regarding alterations in detrusor muscle is scanty and addresses only partial aspects of the myogenic response of detrusor. Rodent bladder aging shows several features similar to those reported in humans. The aim of this study was to characterize in aged mouse the alterations of detrusor muscle contraction and the putative underlying changes in Ca²⁺ signals. We studied in vitro the myogenic contraction induced by agonists in detrusor strips from adult (3 months old) or aged (23–25 months old) mice. In addition, we determined the agonist-induced [Ca²⁺]_i signals by epifluorescence microscopy in fura-2 loaded isolated detrusor cells. Aging impaired the contractile response of bladder strips to cholinergic stimulation with bethanechol and to chemical depolarization with KCl-containing solutions. On the contrary, the response to purinergic stimulation (ATP) was enhanced. Aging also diminished the transient Ca²⁺ signal evoked by bethanechol and the Ca²⁺ influx induced by KCl in bladder strips. Treatments aimed to release calcium from intracellular stores (caffeine and a low level of ionomycin in Ca²⁺-free medium) showed that aging reduces the size of agonist-releasable stores. Similar to contraction, the mobilization of Ca²⁺ by ATP was increased in aged cells. Therefore, the differential effects of aging on detrusor contraction are associated to alterations of [Ca²⁺]_i signals: the cholinergic inhibition is due to inhibition of voltage-operated Ca²⁺ influx and reduction of the size of intracellular Ca²⁺ stores, while the age-induced ATP response is accompanied by an enhanced Ca²⁺ mobilization.     

Changes of intra-mitochondrial Ca in adult ventricular cardiomyocytes examined using a novel fluorescent Ca indicator targeted to mitochondria

In this study a Ca²⁺ sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca²⁺-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca²⁺ sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca²⁺ transient. Cytoplasmic (Fura-2) and mitochondrial Ca²⁺ (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca²⁺ loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca²⁺ sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K_d of  220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca²⁺ transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca²⁺ release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by  65% and increased the time to 50% decay, whilst cytosolic Ca²⁺ transients were unchanged. The mitochondrial Ca²⁺ uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca²⁺. The mitochondrial-targeted Ca²⁺ probe indicates sustained and transient phases of mitochondrial Ca²⁺ signal, which are dependent on cytoplasmic Ca²⁺ levels and require a functional mCU.     

Functional heterogeneity of the four voltage sensors of a human L-type calcium channel

Excitation-evoked Ca²⁺ influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca²⁺ entry are thought to be functions of voltage-gated calcium (Ca_V) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I–IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of Ca_V1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in Ca_V1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.Voltage-gated Ca²⁺ (Ca_V) channels respond to membrane depolarization by catalyzing Ca²⁺ influx. Ca_V-mediated elevation of intracellular [Ca²⁺] regulates such critical physiological functions as neurotransmitter and hormone release, axonal outgrowth, muscle contraction, and gene expression (1). Their relevance to human physiology is evident from the broad phenotypic consequences of Ca_V channelopathies (2). The voltage dependence of Ca_V-driven Ca²⁺ entry relies on the modular organization of the channel-forming α₁ subunit (Fig. 1), which consists of four repeated motifs (I–IV), each comprising six membrane-spanning helical segments (S1–S6) (Fig. 1A). Segments S1–S4 form a voltage-sensing domain (VSD), whereas segments S5 and S6 contribute to the Ca²⁺-conductive pore (1). The VSDs surround the central pore (Fig. 1B). VSDs are structurally and functionally conserved modules (3–5) capable of transducing a change in the cell membrane electrical potential into a change of ion-specific permeability or enzyme activity. VSDs sense depolarization by virtue of a signature motif of positively charged Arg or Lys at every third position of helix S4 (Fig. 1D), which rearranges in response to depolarization (4, 6–10). In contrast to voltage-gated K⁺ (K_V) channels but similar to pseudotetrameric voltage-gated Na⁺ (Na_V) channels, the amino acid sequences encoding each VSD have evolved independently (Fig. 1D). In addition to their distinct primary structure, the four Ca_V VSDs may also gain distinct functional properties from the asymmetrical association of auxiliary subunits, such as β, α₂δ, and calmodulin (1, 11–16) (Fig. 1C). The structural divergence among VSD-driven channels was foreseen by the classical Hodgkin–Huxley model (17), in which four independent “gating particles” control the opening of homotetrameric K_V channels and only three seem sufficient to open Na_V channels. An early study by Kostyuk et al. (18) suggested that only two gating particles are coupled to Ca_V channel opening. We recognize today that gating particles correspond to VSDs, and in Na_V channels, VSDs I–III control Na⁺ influx, whereas VSD IV is associated with fast inactivation (19–21).Open in a separate windowFig. 1.Ca_V membrane topology, putative structure, and S4 helix homology. (A) Ca_V channel-forming α₁ subunits consist of four concatenated repeats, each encompassing one voltage sensor domain (VSD) and a quarter of the central pore domain (PD) (1). Stars indicate the positions of fluorophore labeling. (B) The atomic structure of an Na_V channel (Protein Data Bank ID code 4EKW; top view) (56) shown as a structural representation for the Ca_V α₁ subunit. (C) The α₁ subunit asymmetrically associates with auxiliary β, α₂δ, and calmodulin (CaM) subunits (11–16). (D) Sequence alignment of VSD helix S4 from each of four Ca_V1.2 repeats and the archetypal homotetrameric Shaker K⁺ channel. Conserved, positively charged Arg or Lys is in blue. Residues substituted by Cys for fluorescent labeling are marked: F231 (VSD I), L614 (VSD II), V994 (VSD II), and S1324 (VSD IV).In this study, we used fluorometry to probe the properties of four individual VSDs in a human L-type calcium channel Ca_V1.2, which is a widely expressed regulator of physiological processes, such as cardiac and smooth muscle contractility (22). Although the collective transition of the Ca_V VSDs and the pore has been investigated in studies measuring total charge displacement (gating currents) (23, 24), the activation properties and functional roles of each VSD are unknown. Evidence for the role of each VSD in L-type Ca_V channel operation has been presented from charge neutralization studies, but a clear picture has yet to emerge. Work on a chimeric L-type channel suggests that VSDs I and III drive channel opening (25), whereas other studies on Ca_V1.2 favored the involvement of VSD II over VSD I, with the roles of VSDs III and IV remaining unclear (26, 27).The individual optical reports of four Ca_V1.2 VSDs revealed that each operates with distinct biophysical parameters. We found that VSDs II and III exhibit voltage- and time-dependent characteristics compatible with channel opening and that they can be considered rate-limiting for activation. We compared the voltage and time dependence of the fluorescent signals and ionic currents with the predictions of thermodynamic models relevant to Ca_V domain organization. We found that Ca_V1.2 activation is compatible with a model of allosteric VSD–pore coupling, where VSDs II and III are the primary drivers of channel opening with a smaller contribution by VSD I. We discuss the mechanism of Ca_V1.2 voltage sensitivity, which exhibits similarities to but also clear differences from the related pseudotetrameric Na_V1.4 channels.     

Activated human platelet products induce proarrhythmic effects in ventricular myocytes

Sudden cardiac death remains one of the most prevalent modes of death and is mainly caused by ventricular fibrillation (VF) in the setting of acute ischemia resulting from coronary thrombi. Animal experiments have shown that platelet activation may increase susceptibility of ischemic myocardium to VF, but the mechanism is unknown. In the present study, we evaluated the effects of activated blood platelet products (ABPPs) on electrophysiological properties and intracellular Ca²⁺ (Ca²⁺_i) homeostasis. Platelets were collected from healthy volunteers. After activation, their secreted ABPPs were added to superfusion solutions. Rabbit ventricular myocytes were freshly isolated, and membrane potentials and Ca²⁺_i were recorded using patch-clamp methodology and indo-1 fluorescence measurements, respectively. ABPPs prolonged action potential duration and induced early and delayed afterdepolarizations. ABPPs increased L-type Ca²⁺ current (I_Ca,L) density, but left densities of sodium current, inward rectifier K⁺ current, transient outward K⁺ current, and rapid component of the delayed rectifier K⁺ current unchanged. ABPPs did not affect kinetics or (in)activation properties of membrane currents. ABPPs increased systolic Ca²⁺_i, Ca²⁺_i transient amplitude, and sarcoplasmic reticulum Ca²⁺ content. ABPPs did not affect the Na⁺− Ca²⁺ exchange current (I_NCX) in Ca²⁺-buffered conditions. Products secreted from activated human platelets induce changes in I_Ca,L and Ca²⁺_i, which result in action potential prolongation and the occurrence of early and delayed afterdepolarizations in rabbit myocytes. These changes may trigger and support reentrant arrhythmias in ischemia models of coronary thrombosis.     

Ca-sensitive tyrosine kinase Pyk2/CAK β-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy

G-protein-coupled receptor agonists including endothelin-1 (ET-1) and phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Others and we previously reported that Rac1 signaling pathway plays an important role in this agonist-induced cardiomyocyte hypertrophy. In this study reported here, we found that a Ca(2+)-sensitive non-receptor tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2)/cell adhesion kinase beta (CAKbeta), is involved in ET-1- and PE-induced cardiomyocyte hypertrophy medicated through Rac1 activation. ET-1, PE or the Ca(2+) inophore, ionomycin, stimulated a rapid increase in tyrosine phosphorylation of Pyk2. The tyrosine phosphorylation of Pyk2 was suppressed by the Ca(2+) chelator, BAPTA. ET-1- or PE-induced increases in [(3)H]-leucine incorporation and expression of atrial natriuretic factor and the enhancement of sarcomere organization. Infection of cardiomyocytes with an adenovirus expressing a mutant Pyk2 which lacked its kinase domain or its ability to bind to c-Src, eliminated ET-1- and PE-induced hypertrophic responses. Inhibition of Pyk2 activation also suppressed Rac1 activation and reactive oxygen species (ROS) production. These findings suggest that the signal transduction pathway leading to hypertrophy involves Ca(2+)-induced Pyk2 activation followed by Rac1-dependent ROS production.     

Enhanced L-type calcium currents in cardiomyocytes from transgenic rats overexpressing SERCA2a

BACKGROUND:

Previous research reported that transgenic rats overexpressing the sarco(endo)plasmic reticulum Ca²⁺-ATPase SERCA2a exhibit improved contractile function of the myocardium. Furthermore, impaired Ca²⁺ uptake and reduced relaxation rates in rats with diabetic cardiomyopathy were partially rescued by transgenic expression of SERCA2a in the heart.

OBJECTIVE:

To explore whether enhanced Ca²⁺ cycling in the cardiomyocytes of SERCA2a transgenic rats is associated with changes in L-type Ca²⁺ (I_Ca-L) currents.

METHODS:

The patch-clamp technique was used to measure whole-cell currents in cardiomyocytes from transgenic rats overexpressing SERCA2a and from wild-type (nontransgenic) animals.

RESULTS:

The amplitudes of I_Ca-L currents at depolarizing pulses ranging from −45 mV to 0 mV (350 ms duration, 1 Hz) were significantly higher in cardiomyocytes of SERCA2a transgenic rats than in nontransgenic rats (1985±48 pA [n=32] versus 1612±55 pA [n=28], respectively). The inactivation kinetics of I_Ca-L showed subtle differences with increased tau fast and tau slow decay constants in cardiomyocytes of SERCA2a transgenic animals. Beta-adrenergic stimulation with 50 nM isoproterenol reduced tau fast and tau slow decay constants in cardiomyocytes of transgenic rats to values that were not significantly different from those in normal cardiomyocytes. Furthermore, isoproterenol enhanced I_Ca-L currents 3.2-fold and 2.3-fold in cardiomyocytes with and without the SERCA2a transgene, respectively, and this effect was abolished by buffering intracellular Ca²⁺ with BAPTA.

CONCLUSIONS:

These findings indicate that enhanced Ca²⁺ cycling in the hearts of SERCA2a transgenic rats, both under normal conditions and during beta-adrenergic stimulation, involves changes in I_Ca-L currents. Modified I_Ca-L kinetics may contribute, to some extent, to the improved contractile function of the myocardium of transgenic rats.     

Mg2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK channels

The voltage-sensor domain (VSD) of voltage-dependent ion channels and enzymes is critical for cellular responses to membrane potential. The VSD can also be regulated by interaction with intracellular proteins and ligands, but how this occurs is poorly understood. Here, we show that the VSD of the BK-type K(+) channel is regulated by a state-dependent interaction with its own tethered cytosolic domain that depends on both intracellular Mg(2+) and the open state of the channel pore. Mg(2+) bound to the cytosolic RCK1 domain enhances VSD activation by electrostatic interaction with Arg-213 in transmembrane segment S4. Our results demonstrate that a cytosolic domain can come close enough to the VSD to regulate its activity electrostatically, thereby elucidating a mechanism of Mg(2+)-dependent activation in BK channels and suggesting a general pathway by which intracellular factors can modulate the function of voltage-dependent proteins.     

Voltage-gating and cytosolic Ca2+ activation mechanisms of Arabidopsis two-pore channel AtTPC1

Arabidopsis thaliana two-pore channel AtTPC1 is a voltage-gated, Ca²⁺-modulated, nonselective cation channel that is localized in the vacuolar membrane and responsible for generating slow vacuolar (SV) current. Under depolarizing membrane potential, cytosolic Ca²⁺ activates AtTPC1 by binding at the EF-hand domain, whereas luminal Ca²⁺ inhibits the channel by stabilizing the voltage-sensing domain II (VSDII) in the resting state. Here, we present 2.8 to 3.3 Å cryoelectron microscopy (cryo-EM) structures of AtTPC1 in two conformations, one in closed conformation with unbound EF-hand domain and resting VSDII and the other in a partially open conformation with Ca²⁺-bound EF-hand domain and activated VSDII. Structural comparison between the two different conformations allows us to elucidate the structural mechanisms of voltage gating, cytosolic Ca²⁺ activation, and their coupling in AtTPC1. This study also provides structural insight into the general voltage-gating mechanism among voltage-gated ion channels.

Voltage-gated ion channels (VGICs), such as voltage-gated potassium channel (Kv), sodium channel (Nav), and calcium channel (Cav), are activated by depolarization of membrane potential and play essential roles in electrical signal transduction (1–3). VGICs sense the membrane potential by voltage-sensing domains (VSDs), which consist of four transmembrane helices S1 to S4. In most VGICs, VSDs are stabilized in the resting state by hyperpolarizing (negative) membrane potential, and the channel gate stays closed. At depolarizing (relatively positive) membrane potential, VSDs are activated, and their depolarization-induced conformational changes are coupled to the S5–S6 pore domain, resulting in the opening of the channel gate.While dozens of VGIC structures have been determined over the past two decades, only very few VSDs in these structures were captured in the resting state. That is because VGICs for structural studies in vitro are solubilized in detergent micelle or lipid nanodisc, making it difficult to recapitulate the resting state under hyperpolarizing (negative) membrane potential. The structure of plant two-pore channel (TPC) from Arabidopsis thaliana (AtTPC1) was among the first to capture a resting-state VSD (4, 5). In addition, mutagenesis combined with cross-linking, ion bridge, or toxin binding have been used to trap the structures of VGICs in resting state in several recent studies, including structures of the bacterial sodium channel NavAb (6), the eukaryotic sodium channel chimera Nav1.7-NavPaS (7), and the hyperpolarization-activated cyclic nucleotide-gated ion channel HCN (8). To fully understand the similarities and differences of the voltage-gating mechanism among different VGICs, it will be essential to visualize the structures of various VGICs in both activated and resting state. To this end, we are using AtTPC1 as a model to elucidate the structural mechanism of voltage gating.TPCs belong to the VGIC superfamily and are ubiquitously expressed in animals and plants (9, 10). While animal TPCs (TPC1 and TPC2) are endolysosomal sodium channels, the plant TPC (TPC1) is a nonselective cation channel responsible for generating the slow vacuole (SV) current (11, 12). TPCs function as homodimer with each subunit comprising two homologous Shaker-like 6-transmembrane segment domains (6-TM I and 6-TM II) (9, 10, 13), thereby equivalent to a classical VGIC with four VSDs and one pore domain.Plant TPC is involved in many important physiological processes, such as germination and stomatal opening (12), jasmonate biosynthesis (14, 15), modulation of Ca²⁺ waves induced by salinity stress (16), and plant–pathogen interaction (17). AtTPC1, the most well-studied plant TPC from A. thaliana, is activated by the membrane depolarization and cytosolic Ca²⁺ but inhibited by vacuolar Ca²⁺ (18, 19). Previously, we determined the crystal structure of AtTPC1 in closed state (Protein Data Bank [PDB]: 5E1J, AtTPC1_5E1J) (4). We demonstrated that between the two VSDs within each AtTPC1 subunit, only the second one (VSDII) senses the membrane potential and adopts a resting state in the structure whereas the first one (VSDI) lacks several key features essential for voltage sensing and therefore does not contribute to the voltage-dependent gating. Ca²⁺ activation occurs at the EF-hand domain containing two EF-hand motifs. However, Ca²⁺ binding at EF hand 1 appears to play a structural role and does not contribute to Ca²⁺ activation; Ca²⁺ binding at EF hand 2 is central for Ca²⁺ activation and it adopts an unbound state in the structure (4, 19). We also identified the luminal divalent inhibition site in AtTPC1 where Ca²⁺ or Ba²⁺ binding can stabilize the voltage-sensing VSDII in a resting state. Based on our structural and electrophysiological analysis, we proposed that the conformational changes triggered by the binding of Ca²⁺ to cytosolic EF-hand domain are coupled with the pair pore-lining inner helices from the 6-TM I (IS6), whereas the conformational changes of VSDII activated by membrane potential are coupled with the pair of inner helices from the 6-TM II (IIS6) (4). In order to understand the structural basis underlying multistimuli gating of AtTPC1, here we determined AtTPC1 structures in both closed and partially open conformation under different Ca²⁺ conditions, revealing the structural mechanism of voltage gating and Ca²⁺ modulation of AtTPC1.