Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca²⁺ concentration inside pancreatic acinar cells ([Ca²⁺]_i), due to excessive release of Ca²⁺ stored inside the cells followed by Ca²⁺ entry from the interstitial fluid. The sustained [Ca²⁺]_i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca²⁺ release-activated Ca²⁺ channels (CRAC) would prevent sustained elevation of [Ca²⁺]_i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca²⁺ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca²⁺. Ca²⁺ entry then occurred upon admission of Ca²⁺ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca²⁺ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC₅₀ = 3.4 μM), but had little or no effect on the physiological Ca²⁺ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca²⁺]_i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca²⁺]_i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.     

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.     

Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism

Regulation of ion-transport in the Na⁺/Ca²⁺ exchanger (NCX) occurs via its cytoplasmic Ca²⁺-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca²⁺-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca²⁺-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca²⁺. Striking differences in the electrostatic potential between the Ca²⁺-bound and -free forms strongly suggest that Ca²⁺-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C₂-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca²⁺-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca²⁺ is influenced by the closely located cassette exons. We further propose that Ca²⁺-binding to CBD2 induces a second electrostatic switch, required to alleviate Na⁺-dependent inactivation of Na⁺/Ca²⁺ exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.     

Src family tyrosine kinases inhibit single L-type: Ca2+ channel activity in human atrial myocytes

OBJECTIVE: Tyrosine kinases (TKs) are important regulators of the L-type Ca(2+) channel (LTCC) current in various cell types. However, there are no data addressing the role of TKs in the control of single LTCC activity in human atrial cardiac myocytes, where changes in LTCC gating properties have been described in a number of disease states. METHODS AND RESULTS: Single LTCC activity was recorded in isolated human atrial myocytes. The broad-spectrum TK inhibitor genistein and the Src family-selective TK inhibitor PP1 significantly enhanced single LTCC ensemble average current, availability, and open probability; the latter was due to significant increases of mean open time and mode 2 gating. Conversely, the tyrosine phosphatase inhibitor bisperoxo-phenanthroline-vanadate inhibited single LTCC activity, indicating that LTCC gating properties in human atrial myocytes are controlled by TKs and tyrosine phosphatases in a reciprocal fashion. The effects of genistein on single LTCC activity were not affected by stimulation (8Br-cAMP) or inhibition (Rp-8-CPT-cAMPS) of protein kinase A (PKA) or by inhibition of serine/threonine phosphatases types I and IIa (okadaic acid), indicating that TKs inhibit LTCC gating in human atrial myocytes independent of PKA and phosphatases types I and IIa. However, inhibition of protein kinase C (PKC) by staurosporine or bisindolylmaleimide reversed the stimulatory effects of genistein on single LTCC gating properties, indicating that PKC is required for the inhibitory effect of TKs on single LTCC activity. CONCLUSION: Src family TKs inhibit single LTCC activity in human atrial myocytes via PKC-dependent, but PKA and phosphatase types I and IIa-independent, molecular pathways.     

Structural basis for the high Ca2+ affinity of the ubiquitous SERCA2b Ca2+ pump

Sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) Ca²⁺ transporters pump cytosolic Ca²⁺ into the endoplasmic reticulum, maintaining a Ca²⁺ gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for Ca²⁺ ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca²⁺ affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure–function analysis of SERCA2b mutants and SERCA1a2b chimera revealed how the 2b-tail controls Ca²⁺ affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the Ca²⁺-bound E1 conformation and alters Ca²⁺-transport kinetics, which provides the rationale for the higher apparent Ca²⁺ affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the α- and β-subunits of Na⁺,K⁺-ATPase. The 2b-tail interaction site may represent a novel target to increase the Ca²⁺ affinity of malfunctioning SERCA2a in the failing heart to improve contractility.     

Ca2+-dependent release of Munc18-1 from presynaptic mGluRs in short-term facilitation

Short-term synaptic facilitation plays an important role in information processing in the central nervous system. Although the crucial requirement of presynaptic Ca²⁺ in the expression of this plasticity has been known for decades, the molecular mechanisms underlying the plasticity remain controversial. Here, we show that presynaptic metabotropic glutamate receptors (mGluRs) bind and release Munc18-1 (also known as rbSec1/nSec1), an essential protein for synaptic transmission, in a Ca²⁺-dependent manner, whose actions decrease and increase synaptic vesicle release, respectively. We found that mGluR4 bound Munc18-1 with an EC₅₀ for Ca²⁺ of 168 nM, close to the resting Ca²⁺ concentration, and that the interaction was disrupted by Ca²⁺-activated calmodulin (CaM) at higher concentrations of Ca²⁺. Consistently, the Munc18-1-interacting domain of mGluR4 suppressed both dense-core vesicle secretion from permeabilized PC12 cells and synaptic transmission in neuronal cells. Furthermore, this domain was sufficient to induce paired-pulse facilitation. Obviously, the role of mGluR4 in these processes was independent of its classical function of activation by glutamate. On the basis of these experimental data, we propose the following model: When neurons are not active, Munc18-1 is sequestered by mGluR4, and therefore the basal synaptic transmission is kept low. After the action potential, the increase in the Ca²⁺ level activates CaM, which in turn liberates Munc18-1 from mGluR4, causing short-term synaptic facilitation. Our findings unite and provide a new insight into receptor signaling and vesicular transport, which are pivotal activities involved in a variety of cellular processes.     

Experimental diabetes enhances Ca2+ mobilization and glutamate exocytosis in cerebral synaptosomes from mice

The present study was conducted to investigate the effects of the diabetic condition on the Ca²⁺ mobilization and glutamate release in cerebral nerve terminals (synaptosomes). Diabetes was induced in male mice by intraperitoneal injection of streptozotocin. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in synaptosomes were determined using fura-2 and enzyme-linked fluorometric assay, respectively. Diabetes significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. In addition, diabetes significantly enhanced K⁺- and 4-AP-evoked Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) inhibited K⁺- or 4-AP-induced increases in [Ca²⁺]_i and Ca²⁺-dependent glutamate release in synaptosomes from the control and diabetic mice to a similar extent, respectively. These results indicate that diabetes enhances a K⁺- or 4-AP-evoked Ca²⁺-dependent glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through both P- and N-type Ca²⁺ channels.     

Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation

Ca²⁺ efflux by Ca²⁺ cation antiporter (CaCA) proteins is important for maintenance of Ca²⁺ homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na⁺/Ca²⁺ exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca²⁺ efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca²⁺/H⁺ antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca²⁺-binding site. A hydrophobic “seal” closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca²⁺/H⁺ exchange pathway. In the Ca²⁺-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na⁺-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca²⁺/H⁺ exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.     

CaBP1, a neuronal Ca2+ sensor protein,inhibits inositol trisphosphate receptors by clamping intersubunit interactions

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca²⁺ channels, including inositol 1,4,5-trisphosphate receptors (InsP₃Rs). CaBP1 alone does not affect InsP₃R activity, but it inhibits InsP₃-evoked Ca²⁺ release by slowing the rate of InsP₃R opening. The inhibition is enhanced by Ca²⁺ binding to both the InsP₃R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1–604) of InsP₃R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca²⁺ binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the β-domain of the InsP₃-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP₃R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP₃R pore. InsP₃ activates InsP₃Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a “hot-spot” loop in the suppressor domain (residues 1–223) and the InsP₃-binding core β-domain. Targeted cross-linking of residues that contribute to this interface show that InsP₃ attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP₃R activity by restricting the intersubunit movements that initiate gating.     

Hydrogen peroxide-induced vascular relaxation in porcine coronary arteries is mediated by Ca2+-activated K+ channels

Summary Hydrogen peroxide (H₂O₂) elicited concentration-dependent relaxation of endothelium-denuded rings of porcine coronary arteries. The relaxation induced by the H₂O₂ was markedly attenuated by 10μM 1H-[1,2,4]oxadiazolo [4,3,a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, or by 100nM charybdotoxin, an inhibitor of large-conductance Ca²⁺-activated K⁺ (K_Ca) channels. A combination of the ODQ and charybdotoxin abolished the H₂O₂-induced relaxation. Pretreatment with 25 μM of an Rp stereoisomer of adenosine-3′,5′-cyclic monophosphothioate (Rp-cAMPS), 20μM glibenclamide, or 1mM 4-aminopyridine did not affect the vascular response to H₂O₂. The presence of catalase at 1000U/ml significantly attenuated the H₂O₂-induced relaxation. Exposure of cultured smooth muscle cells to H₂O₂ activated K_Ca channels in a concentration-dependent manner in cell-attached patches. Pretreatment with catalase significantly inhibited the activation of K_Ca channels. Rp-cAMPS did not inhibit the H₂O₂-induced activation of K_Ca channels. The activation of K_Ca channels by H₂O₂ was markedly decreased in the presence of ODQ. However, even in the presence of ODQ, H₂O₂ activated K_Ca channels in a concentration-dependent manner. In inside-out patches, H₂O₂ significantly activated K_Ca channels through a process independent of cyclic guanosine 3′,5′-monophosphate (cGMP). In conclusion, H₂O₂ elicits vascular relaxation due to activation of K_Ca channels, which is mediated partly by a direct action on the channel and partly by activation of soluble guanylate cyclase, resulting in the generation of cGMP.     

Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight

Neuronal Ca²⁺ signals can affect excitability and neural circuit formation. Ca²⁺ signals are modified by Ca²⁺ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca²⁺ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca²⁺ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP₃R) can partially compensate for their loss of flight. Ca²⁺ measurements show that Orai gain-of-function contributes to the quanta of Ca²⁺-release through mutant InsP₃Rs and elevates store-operated Ca²⁺ entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca²⁺ in neurons is required for Drosophila flight.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Targeted expression of the inositol 1,4,5-triphosphate receptor (IP3R) ligand-binding domain releases Ca2+ via endogenous IP3R channels

Virtually all functions of a cell are influenced by cytoplasmic [Ca(2+)] increases. Inositol 1,4,5-trisphosphate receptor (IP(3)R) channels, located in the endoplasmic reticulum (ER), release Ca(2+) in response to binding of the second messenger, IP(3).IP(3)Rs thus are part of the information chain interpreting external signals and transforming them into cytoplasmic Ca(2+) transients. IP(3)Rs function as tetramers, each unit comprising an N-terminal ligand-binding domain (LBD) and a C-terminal channel domain linked by a long regulatory region. It is not yet understood how the binding of IP(3) to the LBD regulates the gating properties of the channel. Here, we use the expression of IP(3) binding protein domains tethered to the surface of the endoplasmic reticulum (ER) to show that the all-helical domain of the IP(3)R LBD is capable of depleting the ER Ca(2+) pools by opening the endogenous IP(3)Rs, even without IP(3) binding. This effect requires the domain to be within 50 A of the ER membrane and is impaired by the presence of the N-terminal inhibitory segment on the LBD. These findings raise the possibility that the helical domain of the LBD functions as an effector module possibly interacting with the channel domain, thereby being part of the gating mechanisms by which the IP(3)-induced conformational change within the LBD regulates Ca(2+) release.     

Loss of luminal Ca2+ activation in the cardiac ryanodine receptor is associated with ventricular fibrillation and sudden death

Different forms of ventricular arrhythmias have been linked to mutations in the cardiac ryanodine receptor (RyR)2, but the molecular basis for this phenotypic heterogeneity is unknown. We have recently demonstrated that an enhanced sensitivity to luminal Ca(2+) and an increased propensity for spontaneous Ca(2+) release or store-overload-induced Ca(2+) release (SOICR) are common defects of RyR2 mutations associated with catecholaminergic polymorphic or bidirectional ventricular tachycardia. Here, we investigated the properties of a unique RyR2 mutation associated with catecholaminergic idiopathic ventricular fibrillation, A4860G. Single-channel analyses revealed that, unlike all other disease-linked RyR2 mutations characterized previously, the A4860G mutation diminished the response of RyR2 to activation by luminal Ca(2+), but had little effect on the sensitivity of the channel to activation by cytosolic Ca(2+). This specific impact of the A4860G mutation indicates that the luminal Ca(2+) activation of RyR2 is distinct from its cytosolic Ca(2+) activation. Stable, inducible HEK293 cells expressing the A4860G mutant showed caffeine-induced Ca(2+) release but exhibited no SOICR. Importantly, HL-1 cardiac cells transfected with the A4860G mutant displayed attenuated SOICR activity compared with cells transfected with RyR2 WT. These observations provide the first evidence that a loss of luminal Ca(2+) activation and SOICR activity can cause ventricular fibrillation and sudden death. These findings also indicate that although suppressing enhanced SOICR is a promising antiarrhythmic strategy, its oversuppression can also lead to arrhythmias.     

Regulation of sarcolemmal Ca2+ pump by endogenous protein phosphatases

Summary The function of several key sarcolemmal proteins is modulated through phosphorylation-dephosphorylation of serine/threonine residues. While the involvement of sarcolemma-associated protein kinases in the phosphorylation of these proteins has been established, the nature of the protein phosphatases controlling these proteins has not been investigated. Rat heart sarcolemma contains two protein phosphatase isozymes, protein phosphatase 1 and 2A, which are distinguished on the basis of their susceptibility of inhibitor 2. Both isozymes elute from a Bio Gel A-0.5 column in association with the highest molecular weight protein fraction. However, some protein phosphatase 1 activity elutes with a smaller molecular weight fraction of about 37000, suggesting that the native enzyme exists as a catalytic subunit in complex with an anchor protein. Inhibition of the protein phosphatases using standard inhibitors leads to a stimulation in both the rate and extent of³²P incorporation into isolated sarcolemma. Also affected by inhibition of protein phosphatase activity is the rate of ATP-dependent calcium uptake, which is stimulated following exposure to either inhibitor 2, a classical protein phosphatase 1 inhibitor, and microcystin, a protein phosphatase 1 and 2A inhibitor. The data suggest that the protein phosphatases regulate the dephosphorylation of sarcolemmal proteins Through this mechanism they serve as important modulators of the sarcolemmal Ca²⁺ pump.     

CSN5/Jab1 inhibits cardiac L-type Ca2+ channel activity through protein-protein interactions

L-type Ca(2+) channels have a wide tissue distribution and play essential roles in physiological responses. Recent studies have indicated that regulation of L-type Ca(2+) channels involves the assembly of macromolecular signaling complexes such as the beta(2)-adrenergic receptor signaling complex, the small G-protein kir/Gem and the BK channel. Here, we report the previously unidentified role of another protein in binding to the II-III linker of the alpha(1C) subunit of the L-type Ca(2+) channel. This protein is COP9 signalosome subunit 5 (CSN5)/Jun activation domain-binding protein 1 (Jab1). We have demonstrated that CSN5 interacts specifically with the II-III linker of the alpha(1C) subunit in a yeast two-hybrid system. The alpha(1C) subunit and CSN5 were coimmunoprecipitated in rat heart and both proteins were colocalized in sarcolemmal membranes and transverse tubules of cardiac myocytes. Silencing of CSN5 mRNA using siRNA decreased the endogenous protein level of CSN5 and activated L-type Ca(2+) channels expressed in COS7 cells. These data indicate that CSN5 is a protein that plays a newly defined functional role in association with the cardiac L-type Ca(2+) channel.     

Relationship between Ca2+-affinity and shielding of bulk water in the Ca2+-pump from molecular dynamics simulations

The sarcoplasmic reticulum Ca(2+)-ATPase transports two Ca(2+) per ATP hydrolyzed from the cytoplasm to the lumen against a large concentration gradient. During transport, the pump alters the affinity and accessibility for Ca(2+) by rearrangements of transmembrane helices. In this study, all-atom molecular dynamics simulations were performed for wild-type Ca(2+)-ATPase in the Ca(2+)-bound form and the Gln mutants of Glu771 and Glu908. Both of them contribute only one carboxyl oxygen to site I Ca(2+), but only Glu771Gln completely looses the Ca(2+)-binding ability. The simulations show that: (i) For Glu771Gln, but not Glu908Gln, coordination of Ca(2+) was critically disrupted. (ii) Coordination broke at site II first, although Glu771 and Glu908 only contribute to site I. (iii) A water molecule bound to site I Ca(2+) and hydrogen bonded to Glu771 in wild-type, drastically changed the coordination of Ca(2+) in the mutant. (iv) Water molecules flooded the binding sites from the lumenal side. (v) The side chain conformation of Ile775, located at the head of a hydrophobic cluster near the lumenal surface, appears critical for keeping out bulk water. Thus the simulations highlight the importance of the water molecule bound to site I Ca(2+) and point to a strong relationship between Ca(2+)-coordination and shielding of bulk water, providing insights into the mechanism of gating of ion pathways in cation pumps.     

Epinephrine-induced Ca2+ influx in vascular endothelial cells is mediated by CNGA2 channels

Epinephrine, through its action on β-adrenoceptors, may induce endothelium-dependent vascular dilation, and this action is partly mediated by a cytosolic Ca²⁺ ([Ca²⁺]_i) change in endothelial cells. In the present study, we explored the molecular identity of the channels that mediate epinephrine-induced endothelial Ca²⁺ influx and subsequent vascular relaxation. Patch clamp recorded an epinephrine- and cAMP-activated cation current in the primary cultured bovine aortic endothelial cells (BAECs) and H5V endothelial cells. L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated channels, diminished this cation current. Furthermore, this cation current was greatly reduced by a CNGA2-specific siRNA in H5V cells. With the use of fluorescent Ca²⁺ dye, it was found that epinephrine and isoprenaline, a β-adrenoceptor agonist, induced endothelial Ca²⁺ influx in the presence of bradykinin. This Ca²⁺ influx was inhibited by L-cis-diltiazem and LY-83583, and by a β₂-adrenoceptor antagonist ICI-118551. CNGA2-specific siRNA also diminished this Ca²⁺ influx in H5V cells. Furthermore, L-cis-diltiazem and LY-83583 inhibited the endothelial Ca²⁺ influx in isolated mouse aortic strips. L-cis-diltiazem also markedly reduced the endothelium-dependent vascular dilation to isoprenaline in isolated mouse aortic segments. In summary, CNG channels, CNGA2 in particular, mediate β-adrenoceptor agonist-induced endothelial Ca²⁺ influx and subsequent vascular dilation.