Impact Of Ca2 Research Articles

Mg2+/Ca2+ Induced Changes in Structure, Dynamics and Stability of Dream Protein

Downstream Regulatory Element Antagonist Modulator (DREAM) is a member of the neuronal calcium sensor family that controls activity of potassium voltage channels and regulates c-fos and prodynorphine gene transcription in a Ca2+ dependent manner. Here, we have investigated the impact of Mg2+ and Ca2+ binding on the structure, stability and dynamics of DREAM and DREAM C-terminal domain (DREAM-C). Mg2+ binding to the apoDREAM does not alter the secondary or tertiary structure as based on CD and Trp emission spectra whereas the association of Mg2+ to either EF-3 or EF-4 in apoDREAM-C results in an increase in protein secondary structure and alteration of the tertiary structure. Ca2+ binding to either apo- or Mg2+DREAM and apo- or Mg2+DREAM-C triggers larger conformational changes as evident from the blue-shift in emission spectra and the decrease of Stern-Volmer constant. Ca2+ triggered changes in DREAM conformational dynamics were characterized by time-resolved fluorescence. In apoDREAM, single tryptophan residue exhibits two lifetimes (τ1=3.38 ns, f1=73% and τ2=7.72 ns, f2=27%). Ca2+ binding to apo- and Mg2+DREAM leads to the shortening of the first lifetime and decrease of the fractional contribution (τ1 = 1.75 ns, f1 = 47% and τ2 = 7. ns, f2 = 53%). Furthermore, the impact of Ca2+/Mg2+ on DREAM stability was determined in equilibrium folding studies. The binding of Ca2+ increase the protein stability by ∼ 7 kcal mol−1 whereas the impact of Mg2+ on DREAM stability is significantly smaller, ∼ 1 kcal mol−1. The stability of the C terminal domain in both apo and Ca2+ bound form is significantly smaller compared to the full length protein, suggesting that the inter-domain interactions significantly contribute to the structural and functional properties of DREAM.

Highly Pathogenic H5N1 Avian Influenza Virus Induces Extracellular Ca 2+ Influx, Leading to Apoptosis in Avian Cells

In this study, we show that the highly pathogenic H5N1 avian influenza virus (AIV) (A/crow/Kyoto/53/04 and A/chicken/Egypt/CL6/07) induced apoptosis in duck embryonic fibroblasts (DEF). In contrast, apoptosis was reduced among cells infected with low-pathogenic AIVs (A/duck/HK/342/78 [H5N2], A/duck/HK/820/80 [H5N3], A/wigeon/Osaka/1/01 [H7N7], and A/turkey/Wisconsin/1/66 [H9N2]). Thus, we investigated the molecular mechanisms of apoptosis induced by H5N1-AIV infection. Caspase-dependent and -independent pathways contributed to the cytopathic effects. We further showed that, in the induction of apoptosis, the hemagglutinin of H5N1-AIV played a major role and its cleavage sequence was not critical. We also observed outer membrane permeabilization and loss of the transmembrane potential of the mitochondria of infected DEF, indicating that mitochondrial dysfunction was caused by the H5N1-AIV infection. We then analyzed Ca(2+) dynamics in the infected cells and demonstrated an increase in the concentration of Ca(2+) in the cytosol ([Ca(2+)](i)) and mitochondria ([Ca(2+)](m)) after H5N1-AIV infection. Regardless, gene expression important for regulating Ca(2+) efflux from the endoplasmic reticulum did not significantly change after H5N1-AIV infection. These results suggest that extracellular Ca(2+) may enter H5N1-AIV-infected cells. Indeed, EGTA, which chelates extracellular free Ca(2+), significantly reduced the [Ca(2+)](i), [Ca(2+)](m), and apoptosis induced by H5N1-AIV infection. In conclusion, we identified a novel mechanism for influenza A virus-mediated cell death, which involved the acceleration of extracellular Ca(2+) influx, leading to mitochondrial dysfunction and apoptosis. These findings may be useful for understanding the pathogenesis of H5N1-AIV in avian species as well as the impact of Ca(2+) homeostasis on influenza A virus infection.

Local influence of mitochondrial calcium transport in retinal amacrine cells

Ca2+-dependent synaptic transmission from retinal amacrine cells is thought to be initiated locally at dendritic processes. Hence, understanding the spatial and temporal impact of Ca2+ transport is fundamental to understanding how amacrine cells operate. Here, we provide the first examination of the local effects of mitochondrial Ca2+ transport in neuronal processes. By combining mitochondrial localization with measurements of cytosolic Ca2+, the local impacts of mitochondrial Ca2+ transport for two types of Ca2+ signals were investigated. Disruption of mitochondrial Ca2+ uptake with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) produces cytosolic Ca2+ elevations. The amplitudes of these elevations decline with distance from mitochondria suggesting that they are related to mitochondrial Ca2+ transport. The time course of the FCCP-dependent Ca2+ elevations depend on the availability of ER Ca2+ and we provide evidence that Ca2+ is released primarily via nearby ryanodine receptors. These results indicate that interactions between the ER and mitochondria influence cytosolic Ca2+ in amacrine cell processes and cell bodies. We also demonstrate that the durations of glutamate-dependent Ca2+ elevations are dependent on their proximity to mitochondria in amacrine cell processes. Consistent with this observation, disruption of mitochondrial Ca2+ transport alters the duration of glutamate-dependent Ca2+ elevations near mitochondria but not at sites more than 10 microm away. These results indicate that mitochondria influence local Ca2+-dependent signaling in amacrine cell processes.

Functional role of calcium signals for microglial function

In this review we summarize mechanisms of Ca(2+) signaling in microglial cells and the impact of Ca(2+) signaling and Ca(2+) levels on microglial function. So far, Ca(2+) signaling has been only characterized in cultured microglia and thus these data refer rather to activated microglia as observed in pathology when compared with the resting form found under physiological conditions. Purinergic receptors are the most prominently expressed ligand-gated Ca(2+)-permeable channels in microglia and control several microglial functions such as cytokine release in a Ca(2+)-dependent fashion. A large variety of metabotropic receptors are linked to Ca(2+) release from intracellular stores. Depletion of these intracellular stores triggers a capacitative Ca(2+) entry. While microglia are already in an activated state in culture, they can be further activated, for example, by exposure to bacterial endotoxin. This activation leads to a chronic increase of [Ca(2+)](i) and this Ca(2+) increase is a prerequisite for the release of nitric oxide and cytokines. Moreover, several factors (TNFalpha, IL-1beta, and IFN-gamma) regulate resting [Ca(2+)](i) levels.

P-978: Impact of Ca2+ flux inhibitors on acrosome reaction of hamster spermatozoa in vitro

Ca2+ plays a predominant role regulating many functional processes of spermatozoa capacitation, acrosome reaction and fertilization. Despite the importance of Ca2+ to the events, there are still many controversies in Ca2+ flux during sperm capacitation and acrosome reaction, such as Ca2+ channel, Ca2+-ATPase and Ca2+/Na+ exchanger. The purpose of the present study is to further define the function of Ca2+ channel and its possible role in sperm capacitation and acrosome reaction, which might help to determine calcium’s role. Prospective laboratory study. Testes and epididymis were obtained from adult healthy male hamster. Spermatozoa were washed with modified Tyrode’s medium supplemented with 0.3% BSA, adjusted to 4.5 x 107 motile sperm/ml, and then incubated in 200μl droplets for 3-4 hrs at 37oC in the presence of either the trifluoperazine (inhibitor for camoduline), TFP (25, 50, 100, 150 nM/ml, respectively), or verapamil (inhibitor for plasma membrane Ca2+-channel), VP (25, 50, 100, 150 nM/ml, respectively), or nifedipine (inhibitor for voltage-dependent Ca2+-channel, NF (50, 100, 200, 400nM/ml, respectively). The samples of the testes and sperm cells were performed with acrosome assessment by using Coomassie Brilliant Blue staining techniques. The data showed that the incubation of sperm with calcium antagonists significantly reduced the acrosome reaction in experimental groups (TFP: 88±2.3%, 65±2.0%, 60±2.2%, 54 ± 2.2%, respectively; VP: 45±1.3%, 23 ± 1.2%, 12±1.0%, 8±0.6%, respectively; NF: 11 ± 0.8%, 9±0.3%, 7.0±0.1%, 6.0±0.1%, respectively) as compared with control group (95 ± 3.0% acrosome reaction) after 3-4 hrs. However, the inhibitions of TFP and NF on the acrosome reaction were not significantly increased with the their concentration raise. These results suggested the antagonists may block the acrosome reaction by affecting the Ca2+ channels, especially in voltage-dependent Ca2+ channel.

The Role of Mitochondria for Ca2+ Refilling of the Endoplasmic Reticulum

Endoplasmic reticulum (ER) Ca2+ refilling is an active process to ensure an appropriate ER Ca2+ content under basal conditions and to maintain or restore ER Ca2+ concentration during/after cell stimulation. The mechanisms to achieve successful ER Ca2+ refilling are multiple and built on a concerted action of processes that provide a suitable reservoir for Ca2+ sequestration into the ER. Despite mitochondria having been found to play an essential role in the maintenance of capacitative Ca2+ entry by buffering subplasmalemmal Ca2+, their contribution to ER Ca2+ refilling was not subjected to detailed analysis so far. Thus, this study was designed to elucidate the involvement of mitochondria in Ca2+ store refilling during and after cell stimulation. ER Ca2+ refilling was found to be accomplished even during continuous inositol 1,4,5-trisphosphate (IP3)-triggered ER Ca2+ release by an agonist. Basically, ER Ca2+ refilling depended on the presence of extracellular Ca2+ as the source and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity. Interestingly, in the presence of an IP3-generating agonist, ER Ca2+ refilling was prevented by the inhibition of trans-mitochondrial Ca2+ flux by CGP 37157 (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one) that precludes the mitochondrial Na+/Ca2+ exchanger as well as by mitochondrial depolarization using a mixture of oligomycin and antimycin A. In contrast, after the removal of the agonist, ER refilling was found to be largely independent of trans-mitochondrial Ca2+ flux. Under these conditions, ER Ca2+ refilling took place even without an associated Ca2+ elevation in the deeper cytosol, thus, indicating that superficial ER domains mimic mitochondrial Ca2+ buffering and efficiently sequester subplasmalemmal Ca2+ and consequently facilitate capacitative Ca2+ entry. Hence, these data point to different contribution of mitochondria in the process of ER Ca2+ refilling based on the presence or absence of IP3, which represents the turning point for the dependence or autonomy of ER Ca2+ refilling from trans-mitochondrial Ca2+ flux.

Invited review: mechanisms of calcium handling in smooth muscles.

The concentration of cytoplasmic Ca(2+) regulates the contractile state of smooth muscle cells and tissues. Elevations in global cytoplasmic Ca(2+) resulting in contraction are accomplished by Ca(2+) entry and release from intracellular stores. Pathways for Ca(2+) entry include dihydropyridine-sensitive and -insensitive Ca(2+) channels and receptor and store-operated nonselective channels permeable to Ca(2+). Intracellular release from the sarcoplasmic reticulum (SR) is accomplished by ryanodine and inositol trisphosphate receptors. The impact of Ca(2+) entry and release on cytoplasmic concentration is modulated by Ca(2+) reuptake into the SR, uptake into mitochondria, and extrusion into the extracellular solution. Highly localized Ca(2+) transients (i.e., sparks and puffs) regulate ionic conductances in the plasma membrane, which can provide feedback to cell excitability and affect Ca(2+) entry. This short review describes the major transport mechanisms and compartments that are utilized for Ca(2+) handling in smooth muscles.

Interactions between neurotransmitters that regulate cAMP and intracellular Ca2+ levels in the CNS.

Two major second messenger signalling systems play prominent roles in the regulation of neuronal activity — that which modulates intracellular cAMP levels and that which results in an elevation in intracellular Ca2+ concentrations. Intracellular Ca2+ is now known to be mobilized as a consequence of receptor modulation of inositol phosphate metabolism.1 It is widely recognised that both of these pathways can lead to changes in the level of intracellular protein phosphorylation through the mediation of distinct protein kinases -sometimes converging on a single target protein, as is the situation in the case of tyrosine hydroxylase.2 However, less widely recognized is the fact that these signalling systems also interact more immediately at the level of the generation of their rimary signals. Recent studies — for instance those by Jakobs et al3 — indicate that, in some cells, the Ca2+-activated, protein kinase C, eliminates hormone inhibition of adenylate cyclase which is mediated by inhibitory GTP regulatory elements. It has also been recognised for some time that Ca2+/calmodulin regulates a specific cyclic AMP phosphodiesterase in neuronal tissue; this interaction provides a mechanism whereby Ca2+ signals can decrease cAMP levels.4,5 Thus, the potential for interaction between systems that utilize Ca2+ and cAMP has already been established. In order to explore other potential mechanisms by which these two systems interact at the level of signal generation, recent studies from these laboratories have focussed on two major issues: 1) additional functions that may be served by putative “cyclase-inhibitory” receptors in the CNS. The physiological significance of adenylate cyclase inhibition in neuronal tissue has often been questioned, since the magnitude of the effect is so modest -rarely exceeding 25%. In the present studies additional functions supported by “cyclase inhibitory” receptors are investigated; and, 2) the impact of Ca2+-signals on the regulation of adenylate cyclase by cyclase-linked receptors. Over the past few years, a focus in one of these laboratories ha been the fact — first reported by Brostrom et al.6 — that Ca2+/calmodulin can regulate neuronal adenylate cyclase — particularly in terms of its implications for neurotransmitter regulation of adenylate cyclase. Initial studies on the hippocampal system had indicated that adenosine-Al receptor-mediated inhibition of adenylate cyclase could occur only upon Ca2+/calmodulin stimulation of activity.7 Current studies have attempted to determine how widespread this phenomenon is within the nervous system, whether it applies to all inhibitory neurotransmitters8,9 and the implications of this regulation for stimulatory neurotransmission.KeywordsAdenylate CyclasePertussis ToxinInositol PhosphateAdenylate Cyclase ActivityAdenosine AnalogueThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.