A NOX2-independent mechanism of Hv1 channel activation promotes inflammatory cytokine release from BV-2 microglia via intracellular Ca2+ mobilisation.

Received July 2, 2009; accepted October 5, 2009. Heart failure (HF) is a major health problem in Western countries. Despite significant progress in pharmacological and device-based treatment, the disease burden imposed continues to increase, particularly as the population ages. HF incidence approaches 10 per 1000 after age 65 years.1 Congestive HF is the final consequence of diverse cardiovascular disorders, including atherosclerosis, cardiomyopathy, and hypertension. Described as a complex pathophysiological syndrome that involves interactions of the circulatory, neurohormonal, and renal systems, HF is first a disease of the myocardium, although it soon induces defects in other systems. Current treatments for HF, focused on blocking neurohormonal pathways, improve survival, but they do not halt the progression of HF. Late-stage HF has a poor prognosis, and therapeutic options are limited. Faced with these challenges, researchers are exploring novel therapeutic options. Chronic HF is associated with increased sympathetic outflow, which may be compensatory early on, but long-term neurohormonal activation induces significant damage to the heart; in addition, it results in multiple alterations in the β-adrenergic receptor (β-AR) signaling cascade, including receptor downregulation, upregulation of receptor kinases, and increased inhibitory G-protein function.2 The amplitude and velocity of Ca2+ cycling are regulated by a dynamic balance of phosphorylation and dephosphorylation through kinases and phosphatases. Activation of β-ARs stimulates cAMP production and results in protein kinase A (PKA) phosphorylation of key regulators of excitation-contraction coupling, such as L-type Ca2+ channels, phospholamban, troponin I, ryanodine receptors (RyR), myosin-binding protein C, and protein phosphatase inhibitor-1 (I-1; Figure), which leads to increased amplitude and velocity of Ca2+ cycling and increased contractility on a beat-to-beat basis.3 Protein phosphatases PP1 and PP2A counterbalance phosphorylation of these proteins. There is clear evidence that alterations in sarcoplasmic reticulum (SR) Ca2+ cycling are a component of the impaired …

The precise control of many T cell functions relies on cytosolic Ca(2+) dynamics that is shaped by the Ca(2+) release from the intracellular store and extracellular Ca(2+) influx. The Ca(2+) influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca(2+) concentration ([Ca(2+)](i)) necessary for T cell activation, whereas the role of intracellular Ca(2+) release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca(2+)](i) elevation observed upon activation of the store-operated Ca(2+) entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca(2+) release channels were activated in parallel with SOCE and contributed to global [Ca(2+)](i) elevation. Pretreating cells with (-)-xestospongin C (10 microM) or ryanodine (400 microM), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca(2+)](i) elevation evoked by the passive store depletion or TCR ligation. Although the Ca(2+) release from the IP3R can be activated by TCR stimulation, the Ca(2+) release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca(2+)](i) signaling in T cells, that is SOCE-dependent Ca(2+) release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca(2+)-dependent functions of T lymphocytes.

Elevations of sperm intracellular pH and Ca(2+) regulate sperm motility, chemotaxis, capacitation and the acrosome reaction, and play a vital role in the ability of the sperm cell to reach and fertilise the egg. In human spermatozoa, the flagellar voltage-gated proton channel Hv1 is the main H(+) extrusion pathway that controls sperm intracellular pH, and the pH-dependent flagellar Ca²(+) channel CatSper is the main pathway for Ca²(+) entry as measured by the whole-cell patch clamp technique. Hv1 and CatSper channels are co-localized within the principal piece of the sperm flagellum. Hv1 is dedicated to proton extrusion from flagellum and is activated by membrane depolarisation, an alkaline extracellular environment, the endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. The CatSper channel is strongly potentiated by intracellular alkalinisation. Since Hv1 and CatSper channels are located in the same subcellular domain, proton extrusion via Hv1 channels should induce intraflagellar alkalinisation and activate CatSper ion channels. Therefore the combined action of Hv1 and CatSper channels in human spermatozoa can induce elevation of both intracellular pH and Ca²(+) required for sperm activation in the female reproductive tract. Here, we discuss how Hv1 and CatSper channels regulate human sperm physiology and the differences in control of sperm intracellular pH and Ca²(+) between species.

Voltage-gated proton (Hv1) channels play important roles in the respiratory burst, in pH regulation, in spermatozoa, in apoptosis, and in cancer metastasis. Unlike other voltage-gated cation channels, the Hv1 channel lacks a centrally located pore formed by the assembly of subunits. Instead, the proton permeation pathway in the Hv1 channel is within the voltage-sensing domain of each subunit. The gating mechanism of this pathway is still unclear. Mutagenic and fluorescence studies suggest that the fourth transmembrane (TM) segment (S4) functions as a voltage sensor and that there is an outward movement of S4 during channel activation. Using thermodynamic mutant cycle analysis, we find that the conserved positively charged residues in S4 are stabilized by countercharges in the other TM segments both in the closed and open states. We constructed models of both the closed and open states of Hv1 channels that are consistent with the mutant cycle analysis. These structural models suggest that electrostatic interactions between TM segments in the closed state pull hydrophobic residues together to form a hydrophobic plug in the center of the voltage-sensing domain. Outward S4 movement during channel activation induces conformational changes that remove this hydrophobic plug and instead insert protonatable residues in the center of the channel that, together with water molecules, can form a hydrogen bond chain across the channel for proton permeation. This suggests that salt bridge networks and the hydrophobic plug function as the gate in Hv1 channels and that outward movement of S4 leads to the opening of this gate.

Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2 +-ATPase activity and perturb Ca2 + homeostasis in human coronary artery endothelial cells

Targeting the Activating Mutations of NOTCH1 in T-Cell Lymphoblastic Leukemia with a New SERCA Inhibitor CAD204520

Membrane voltage and intracellular ion concentrations change under various cell states. Changes of both membrane potential and ion concentration can be sensed by the same elements in cell membranes. The calcium-activated potassium channels are a typical example. In hippocampal neurons, action potential waveforms are adapted to repetitive electrical inputs based on calcium-dependent repolarization. This may occur quickly or slowly. In the fast process, the duration of a single action potential becomes shorter. In the slow process, spike frequency and patterns are changed. The fast process is mainly achieved by activation of a class of Kv channel, a BK-type calcium-activated potassium channel. Its activities are regulated by both increase of intracellular calcium and depolarization. In fact, the BK channel has a voltage sensor domain and a cytoplasmic calcium-sensing domain. BK channels are often co-localized with voltage-gated calcium channels and can sense local and rapid calcium increase following opening of a single voltage-gated calcium channel during an action potential. The slower process of modification of neuronal firing depends on another class of potassium channel, the small conductance calcium-activated potassium channel (the SK channel), which binds calmodulin on the intracellular side. In contrast with the BK channel, the SK channel lacks intrinsic voltage dependence. Synergy between membrane voltage change and ions is not restricted to neurons and myocytes. In phagocytes such as the neutrophil or macrophage, reactive oxygen species are produced by the actions of NADPH oxidase. This activity induces remarkable depolarization since superoxide anions are transferred to the external space leaving protons inside. Thus both depolarization and intracellular acidification occur by the oxidase's activity. The voltage-gated proton (Hv) channel serves as an ideal player for cancelling both outcomes of the oxidase activity. The Hv channel is activated by both depolarization and intracellular acidification. Voltage dependence of steady-state open probability measured as the conductance–voltage (G–V) curve is shifted dependent on both intracellular and extracellular pH values. Recently, the molecular correlate of the Hv channel was identified. Knockout mice for the Hv channel exhibit reduced production of superoxide anions in neutrophils, consistent with the view that the Hv channel plays a role in promoting NADPH oxidase activity. More roles of the Hv channel in other cell types are also emerging. A study of knockout mice for the Hv channel led to the identification of unexpected roles in the signals of antibody production in B-lymphocytes. Another surprise was its presence and potential role in human sperm. Many ion channel species are expressed in sperm, but how ion channels contribute to sperm physiology, such as motility, has remained unknown. Patch clamping of sperm has now brought about a revolution in this field. This series of reviews covers three ion channel topics that explore the sites of synergy between voltage and ions. Jianmin Cui reviews recent findings of the structure–function relationship of the BK-type calcium-activated potassium channel (Cui, 2010). The interaction between S4, a key transmembrane segment of the voltage sensor domain, and the cytoplasmic metal ion-binding sites is one focus and the recently resolved X-ray structure of the cytoplasmic domain for calcium sensing is also discussed. Demaurex & Chemaly (2010) review the historical background of the Hv channel in leukocytes and recent molecular studies. Finally, Lishko & Kirichok (2010) review the recent discovery of the Hv channel in human sperm and its functional coupling with the CatSper channel, another class of voltage sensor containing channel that operates as an alkalization-activated calcium channel.

Intracellular pH (pHi) and Ca2+ regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca2+. The sources of the Ca2+ increase are from the endoplasmic reticulum (ER) Ca2+ pools as well as from Ca2+ influx. The store-mobilization component of the Ca2+ increase induced by the pHi rise was not sensitive to antagonists for either IP3-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), leading to depletion of the ER Ca2+ store. We further showed that the physiological consequence of depletion of the ER Ca2+ store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca2+ influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca2+ leak from ER pools followed by Ca2+ influx via SOCs.

The submicromolar levels of free Ca(2+) ions in animal cells are believed to be maintained in the long term by two different plasma membrane transport mechanisms. These are Na-Ca exchange, driven by the sodium gradient, and a Na-independent Ca pump, driven by ATP. There is good evidence from red blood cells, and indirect evidence from other non-neuronal preparations, that the Ca-ATPase exchanges internal Ca(2+) for external H(+). Although Ca extrusion from nerve cells is inhibited by high external pH, there as yet is no evidence for the counter-transport of H(+). We have used both pH- and calcium-sensitive microelectrodes on the cell surface, and the Ca indicator fura-2 intracellularily, to investigate how snail neurons regulate cytoplasmic free Ca(2+). We now report that in snail neurons the recovery of intracellular Ca(2+) after an increase coincides with both the expected increase in surface Ca(2+) and a decrease in surface H+. Recovery of intracellular Ca and the changes in surface pH and Ca are all blocked by intracellular vanadate. We conclude that snail neurons regulate intracellular Ca mainly by a Ca-H ATPase, and suggest that this Ca-H exchange may account for many of the reported extracellular pH changes seen with neuronal excitation.

Regulation of cardiac calcium signaling by newly identified calcium pump modulators

In atrial myocytes excitation-contraction coupling is strikingly different from ventricle because atrial myocytes lack a transverse tubule membrane system: Ca2+ release starts in the cell periphery and propagates towards the cell centre by Ca2+ -induced Ca2+ release from the sarcoplasmic reticulum (SR) Ca2+ store. The cytosolic Ca2+ sensitivity of the ryanodine receptor (RyRs) Ca2+ release channel is low and it is unclear how Ca2+ release can be activated in the interior of atrial cells. Simultaneous confocal imaging of cytosolic and intra-SR calcium revealed a transient elevation of store Ca2+ that we termed 'Ca2+ sensitization signal'. We propose a novel paradigm of atrial ECC that is based on tandem activation of the RyRs by cytosolic and luminal Ca2+ through a 'fire-diffuse-uptake-fire' (or FDUF) mechanism: Ca2+ uptake by SR Ca2+ pumps at the propagation front elevates Ca2+ inside the SR locally, leading to luminal RyR sensitization and lowering of the cytosolic Ca2+ activation threshold. In atrial myocytes Ca2+ release during excitation-contraction coupling (ECC) is strikingly different from ventricular myocytes. In many species atrial myocytes lack a transverse tubule system, dividing the sarcoplasmic reticulum (SR) Ca2+ store into the peripheral subsarcolemmnal junctional (j-SR) and the much more abundant central non-junctional (nj-SR) SR. Action potential (AP)-induced Ca2+ entry activates Ca2+ -induced Ca2+ release (CICR) from j-SR ryanodine receptor (RyR) Ca2+ release channels. Peripheral elevation of [Ca2+ ]i initiates CICR from nj-SR and sustains propagation of CICR to the cell centre. Simultaneous confocal measurements of cytosolic ([Ca2+ ]i ; with the fluorescent Ca2+ indicator rhod-2) and intra-SR ([Ca2+ ]SR ; fluo-5N) Ca2+ in rabbit atrial myocytes revealed that Ca2+ release from j-SR resulted in a cytosolic Ca2+ transient of higher amplitude compared to release from nj-SR; however, the degree of depletion of j-SR [Ca2+ ]SR was smaller than nj-SR [Ca2+ ]SR . Similarly, Ca2+ signals from individual release sites of the j-SR showed a larger cytosolic amplitude (Ca2+ sparks) but smaller depletion (Ca2+ blinks) than release from nj-SR. During AP-induced Ca2+ release the rise of [Ca2+ ]i detected at individual release sites of the nj-SR preceded the depletion of [Ca2+ ]SR , and during this latency period a transient elevation of [Ca2+ ]SR occurred. We propose that Ca2+ release from nj-SR is activated by cytosolic and luminal Ca2+ (tandem RyR activation) via a novel 'fire-diffuse-uptake-fire' (FDUF) mechanism. This novel paradigm of atrial ECC predicts that Ca2+ uptake by sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) at the propagation front elevates local [Ca2+ ]SR , leading to luminal RyR sensitization and lowering of the activation threshold for cytosolic CICR.

Intracellular pH (pHi) and Ca 2+ regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca 2+ . The sources of the Ca 2+ increase are from the endoplasmic reticulum (ER) Ca 2+ pools as well as from Ca 2+ influx. The store-mobilization component of the Ca 2+ increase induced by the pHi rise was not sensitive to antagonists for either IP 3receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA), leading to depletion of the ER Ca 2+ store. We further showed that the physiological consequence of depletion of the ER Ca 2+ store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca 2+ influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca 2+ leak from ER pools followed by Ca 2+ influx via SOCs.

Platelet-derived growth factor (PDGF) signaling is implicated in a wide range of diseases, and PDGF drives the pathological responses in many vascular disorders and fibrotic diseases such as atherosclerosis, restenosis, pulmonary arterial hypertension, and pulmonary fibrosis. In vascular smooth muscle cells (VSMC), PDGF is a potent mitogen which promotes proliferation and migration and contributes to pathological vascular remodeling. Recent studies have demonstrated a role for PDGF in stromal interaction molecule 1 (STIM1)/Orai1-mediated Ca2+ influx in VSMC.1 Regulation of cytosolic Ca2+ concentration ([Ca2+]cyt) is critical for many cellular processes. A rise in [Ca2+]cyt is a major trigger for VSMC proliferation and contraction. One of the major routes of Ca2+ entry into cells is through store-operated Ca2+ (SOC) channels. On depletion of Ca2+ from the stores (sarcoplasmic or endoplasmic reticulum, SR/ER), a Ca2+ deficiency signal is transmitted to the SOC channels in the plasma membrane via STIM1. This causes the SOC to open, allowing Ca2+ to flow into the cytosol, a process referred to as store-operated Ca2+ entry (SOCE). The cytosolic Ca2+ is then sequestered into the stores by the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA), thus replenishing the stores. Studies have demonstrated a role for Ca2+ release-activated Ca2+ (CRAC) channels in SOCE, with STIM1 and Orai1 as the major components.2 Article, see p 66 The majority of STIM1 is expressed in the SR/ER membrane, where it senses decreased Ca2+ concentration in the SR/ER when inositol 1,4,5-triphosphate (IP3)-mediated activation of IP3 receptor induces Ca2+ release. STIM1 then undergoes a conformational change allowing STIM1 to multimerize and translocate …

Familial Alzheimer disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutant PS expression has several cellular consequences, including exaggerated intracellular Ca(2+) ([Ca(2+)](i)) signaling due to enhanced agonist sensitivity and increased magnitude of [Ca(2+)](i) signals. The mechanisms underlying these phenomena remain controversial. It has been proposed that PSs are constitutively active, passive endoplasmic reticulum (ER) Ca(2+) leak channels and that FAD PS mutations disrupt this function resulting in ER store overfilling that increases the driving force for release upon ER Ca(2+) release channel opening. To investigate this hypothesis, we employed multiple Ca(2+) imaging protocols and indicators to directly measure ER Ca(2+) dynamics in several cell systems. However, we did not observe consistent evidence that PSs act as ER Ca(2+) leak channels. Nevertheless, we confirmed observations made using indirect measurements employed in previous reports that proposed this hypothesis. Specifically, cells lacking PS or expressing a FAD-linked PS mutation displayed increased area under the ionomycin-induced [Ca(2+)](i) versus time curve (AI) compared with cells expressing WT PS. However, an ER-targeted Ca(2+) indicator revealed that this did not reflect overloaded ER stores. Monensin pretreatment selectively attenuated the AI in cells lacking PS or expressing a FAD PS allele. These findings contradict the hypothesis that PSs form ER Ca(2+) leak channels and highlight the need to use ER-targeted Ca(2+) indicators when studying ER Ca(2+) dynamics.

Subunit Interactions during Cooperative Opening of Voltage-Gated Proton Channels