μM Free Ca2 Research Articles

Amyloid-β Protein Oligomer at Low Nanomolar Concentrations Activates Microglia and Induces Microglial Neurotoxicity

Amyloid-β Protein Oligomer at Low Nanomolar Concentrations Activates Microglia and Induces Microglial Neurotoxicity

Calcium-Dependent Operation of the Human BK Channel Gating Ring Apparatus

The large cytoplasmic C-terminal domain (CTD) of the human BK channel forms a gating ring structure composed by two tandem RCK domains serving as a signal transducer for intracellular Ca2+ and other ligands. However, the mechanism of the gating ring's operation remains unknown. We have used steady-state and time-resolved spectroscopy in combination with dynamic light scattering to the Ca-induced conformational changes of the purified CTD of human BK channel. The CTD domain of the human BK channel assembles as a tetrameric gating ring structure (MW∼310 kDa) with a hydrodynamic radius (RH) ∼10.5 nm. In the presence of 35 μM free Ca2+, RH reversibly decreases to ∼7.5 nm. The modulation of the gating ring hydrodynamic shape suggests that it undergoes Ca2+-induced conformational transitions, which we further, characterized using time-correlated single-photon counting spectroscopy. Increasing free [Ca2+] up to 35 μM shortened the average Trp fluorescence lifetime (τavg) of the wild-type gating ring from ∼2.6ns to ∼1.9ns, while the neutralization of the high-affinity Ca-binding site within RCK2 (Ca bowl, D894-898N) attenuated the effect of Ca2+. Steady-state fluorescence analysis revealed that these ligand-dependent structural rearrangements of the gating ring possess strong divalent cation selectivity. The gating ring exhibited i) high-affinity Ca2+-binding (Khalf_1 ∼0.3μM and Khalf_2 ∼4μM); ii) significantly lowered affinity for Mg2+ (Khalf ∼200μM) and iii) no Ba2+ sensitivity up to 13mM, consistent with the lack of Ba2+-dependent BK channel activation. Interestingly, Ca2+ bowl neutralization eliminated Mg2+ sensing up to 12mM. In summary, under physiologically-relevant conditions, these ligand-induced conformational transitions are strongly ion-specific and associated with changes in the hydrodynamic properties of the BK gating ring and likely represent the ligand-induced molecular events underlying channel activation.

Imaging ensemble activity in arthropod olfactory receptor neurons in situ

We show that lobster olfactory receptor neurons (ORNs), much like their vertebrate counterparts, generate a transient elevation of intracellular calcium (Ca i) in response to odorant activation that can be used to monitor ensemble ORN activity. This is done in antennal slice preparation in situ maintaining the polarity of the cells and the normal micro-environment of the olfactory cilia. The Ca i signal is ligand-specific and increases in a dose-dependent manner in response to odorant stimulation. Saturating stimulation elicits a robust increase of up to 1 μM free Ca i within 1–2 s of stimulation. The odor-induced Ca i response closely follows the discharge pattern of extracellular spikes elicited by odorant application, with the maximal rise in Ca i matching the peak of the spike generation. The Ca i signal can be used to track neuronal activity in a functional subpopulation of rhythmically active ORNs and discriminate it from that of neighboring tonically active ORNs. Being able to record from many ORNs simultaneously over an extended period of time not only allows more accurate estimates of neuronal population activity but also dramatically improves the ability to identify potential new functional subpopulations of ORNs, especially those with more subtle differences in responsiveness, ligand specificity, and/or transduction mechanisms.

Characterization of the Oligomeric Structure of the Ca2+-activated Cl− Channel Ano1/TMEM16A

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca(2+)-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl(-) secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca(2+), suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.

TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca2+ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca2+ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca2+ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca2+ that will enable it to act as a Ca2+ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca2+] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca2+ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca2+ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

Allosteric Block of KCa2 Channels by Apamin

Activation of small conductance calcium-activated potassium (K(Ca)2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K(Ca)2.2 and K(Ca)2.3 with the same high affinity (K(D) approximately 5 pM for both subtypes) but requires significantly higher concentrations to block functional current (IC(50) values of approximately 100 pM and approximately 5 nM, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K(Ca)2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.

Expression of KCa2.x and KCa3.1 channels in vascular smooth muscle cells from mouse portal vein

Calcium‐activated K+ channels are key regulators of smooth muscle membrane potential, and therefore are essential to smooth muscle function. Small and intermediate conductance Ca2+‐activated K+ channels (KCa2.x and KCa3.1) have been reported in vascular myocytes only during proliferation. Portal vein is an atypical blood vessel as it shows action potentials (AP). As KCa2.x channels have been shown to be involved in the control of AP in different cell types, we investigated the presence of KCa2.x and KCa3.1 channels in vascular smooth muscle cells from mouse portal vein (mPV). PCR experiments showed the presence of mRNA for all KCa2.x and KCa3.1 channels in smooth muscle cells from mPV. In Patch clamp experiments, freshly isolated myocytes were dialyzed with a pipette solution containing 1 μM free Ca2+ to stimulate KCa channels. To minimize the contribution of BKCa channels, paxilline (10 μM) was added in the pipette as well as in the superfusion solution. Exposure of myocytes to TRAM‐34 (10 μM), an inhibitor of KCa3.1 channels, induced a decrease of ≈ 65% in membrane currents. However, Apamin (300 nM), a KCa2.x channels blocker, had little if any effect on membrane currents. These data suggest that KCa2.x and KCa3.1 channels are expressed in mPV and may play an important role in the control of membrane potential. This study is financially supported by FRSQ and the Fondation de l'Institut de Cardiologie de Montréal.

α5β1 Integrin Engagement Increases Large Conductance, Ca2+-activated K+ Channel Current and Ca2+ Sensitivity through c-src-mediated Channel Phosphorylation

Large conductance, calcium-activated K(+) (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous alpha5beta1 integrin ligand, enhances BK channel current through both Ca(2+)- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel alpha-subunits (mSlo) are acutely potentiated following alpha5beta1 integrin activation. The effect occurs in a Ca(2+)-dependent manner, 1-3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca(2+) concentrations >or=1 microm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca(2+) sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by alpha5beta1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel alpha-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca(2+) sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions.

Modification of Cardiac Ryanodine Receptors by Reactive Carbonyl Species Alter Conductance and Gating

Previously, we and others found that ventricular myocytes isolated from streptozotocin (STZ)-induced diabetic rats exhibited enhanced spontaneous Ca2+ releases. To date, molecular mechanisms underlying this phenomenon remains incompletely understood. This study was designed to determine whether carbonyl adducts previously found on RyR2 during diabetes contribute to its dysregulation. Male Sprague-Dawley rats were injected with STZ. Six weeks later, diabetic rats were divided into two groups: one group was treated with insulin for two weeks while the other group received no treatment. Non-diabetic controls were run alongside. After eight weeks, RyR2 was isolated and proteoliposomes prepared. Following incorporation into the lipid bilayer, diabetic RyR2, which contained elevated levels of carbonyl adducts, activated to a greater extent with 1.0 μM Ca2+ than control RyR2 (Po was 5X greater in diabetic RyR2). Two weeks of insulin treatment blunted the enhanced Ca2+ responsiveness. When added to the cis chamber the potent reactive carbonyl species (RCS), 80 μM methylglyoxal increased the open probability (Po) of RyR2 3-fold (0.05 to 0.16) within 10 min and this increase was independent of holding potential. Increasing [MGO] further to 160 μM, reduced the conductance of RyR2 by 25% without changing Po. Incubating RyR2 with MGO (5-500 μM with 200 μM free Ca2+ in buffer) dose-dependently reduced its ability to binding [3H]ryanodine. Singly mutating R1611, R2190 and K2888 to W or Y, to mimic adducts previously found on them during diabetes, resulted in gain-of-function of RyR2 (Po increased >2-fold at 3.3 μM Ca2+ ). Mutating c-terminal R4462, and R4683 to W or Y resulted in loss-of function of RyR2. We conclude that modification of RyR2 by RCS during diabetes is responsible in part for its dysregulation. (This work was funded by NIH and Nebraska Redox Biology Center)

Simultaneous Optical-Electrical Measurement of the Delay between formation of the Fusion Pore and Proton Equilibration in Exocytosis of Single Vesicles

Optical detetction of neutralization of pH in granules or vesicles is often used to define exocytotic events. However, combined measurents of ensemble capacitance and pH-dependent vesicular fluorescence changes have suggested that the movement of protons only becomes possible after fusion pore expansion with a mean delay of > 300 ms (1). To enhance the temporal resolution of such measurements, we have combined capacitance recordings of single vesicle fusion in RBL cells transfected with synapthpHluorin as a reporter of vesicular pH. To monitor cell capacitance steps due to exocytosis of single granules in whole cell patch-clamp mode, we used the piecewise linear technique. Internal solution contained 10 μM free Ca2+ and 300 μM GTPγS.View Large Image | View Hi-Res Image | Download PowerPoint Slide Before establishing whole cell recordings, punctate fluorescence signals could be detected with excitation at 460 nm, while during perfusion with internal solution and excitation at 480 nm, punctate fluorescence signals gradually appeared at corresponding sites. Fluorescence increases clearly lagged capacitance steps by several 100 ms- seconds, supporting the idea that pH equilibration through the fusion pore is delayed.(1) Barg et al.: Neuron, 33, 287-299, 2002.

Phospholipid-Binding Protein EhC2A Mediates Calcium-Dependent Translocation of Transcription Factor URE3-BP to the Plasma Membrane of Entamoeba histolytica

The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) is a transcription factor that binds DNA in a Ca(2+)-inhibitable manner. The protein is located in both the nucleus and the cytoplasm but has also been found to be enriched in the plasma membrane of amebic trophozoites. We investigated the reason for the unusual localization of URE3-BP at the amebic plasma membrane. Here we identify and characterize a 22-kDa Ca(2+)-dependent binding partner of URE3-BP, EhC2A, a novel member of the C2-domain superfamily. Immunoprecipitations of URE3-BP and EhC2A showed that the proteins interact and that such interaction was enhanced in the presence of Ca(2+). Recombinant and native EhC2A bound phospholipid liposomes in a Ca(2+)-dependent manner, with half-maximal binding occurring at 3.4 muM free Ca(2+). A direct interaction between EhC2A and URE3-BP was demonstrated by the ability of recombinant EhC2A to recruit recombinant URE3-BP to phospholipid liposomes in a Ca(2+)-dependent manner. URE3-BP and EhC2A were observed to translocate to the amebic plasma membrane upon an increase in the intracellular Ca(2+) concentration of trophozoites, as revealed by subcellular fractionation and immunofluorescent staining. Short hairpin RNA-mediated knockdown of EhC2A protein expression significantly modulated the mRNA levels of URE3-BP-regulated transcripts. Based on these results, we propose a model for EhC2A-mediated regulation of the transcriptional activities of URE3-BP via Ca(2+)-dependent anchoring of the transcription factor to the amebic plasma membrane.

Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K+ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an "active" conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca2+ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca2+ sensor, conferring Ca2+ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.

PTP1B Dephosphorylates N-Ethylmaleimide-sensitive Factor and Elicits SNARE Complex Disassembly during Human Sperm Exocytosis

The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.

The conductive properties of exponentially dividing LNCaP prostate cancer cells

The intermediate conductance Ca2+‐activated K+ channel (hIK1) is thought to be involved in the cell division of cancer cells. However, direct measurements of the expression and properties of hIK1 during the cell cycle have not been made. Therefore, as an initial step in defining the role of hIK1 in the cell cycle we have measured the conductive properties of exponentially growing LNCaP cells using the whole cell patch clamp technique. Flow cytometry analysis indicated that 24 h after plating 84% of the cells were in the G1 phase, and 7% and 8% of the cells were in the S and G2/M phases, respectively (n = 4). In these cells three distinct conductances were identified in different cells. The majority of the cells (18 out of 29 cells) exhibited an outwardly rectified K+ current. In the presence of 500 μM free Ca2+, this current was inhibited by 10 mM tetraethylammonium (TEA) (n = 8, P < 0.0001) but not the hIK1 inhibitor clotrimazole (10 μM, n = 3, P = 0.11). Furthermore, 1‐EBIO (100 μM), an activator of hIK1, did not stimulate an increase in current in these cells (n = 3, P = 0.27). In a second group of cells (5 of 29) an outwardly rectified current was also seen, but this was relatively insensitive to TEA. In the remaining 5 cells the dominant current was inwardly rectified. 1‐EBIO increased this current from ‐2.7 ± 1.3 to ‐10.8 ± 6.7 pA pF−1 at ERev‐60 mV (n = 3, P = 0.2), but the increase was not significant suggesting the lack of hIK1 activity. Therefore, if hIK1 is involved in proliferation of LNCaP cells, there must be a dramatic change in the expression level or biophysical properties of the channel as the cells proceed through the cell cycle. This work was funded by Grants from the HS & JC Anderson Trust and Lotteries Health.

Calcium Signaling in Brain Mitochondria

Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate alpha-ketoglutarate (alphaKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that alphaKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in alphaKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of alphaKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo.

Ca2+-calmodulin Increases RyR2 Open Probability Yet Reduces Ryanoid Association With RyR2

We have previously demonstrated that low, physiological concentrations of Ca2+-calmodulin (Ca2+CaM; 50–100 nM) activate RyR2 channels incorporated into bilayers (Sigalas & Sitsapesan (2007) Biophys.J., 88a). In contrast, we find that Ca2+CaM dose-dependently inhibits [3H]ryanodine binding to cardiac HSR. Since it appears that the [3H]ryanodine binding assay is not accurately reporting the effects of Ca2+CaM on RyR2 Po we have investigated, using the reversible ryanoid, ryanodol, whether Ca2+CaM can directly influence the binding of ryanoid compounds to RyR2 independently of Po. Purified single sheep RyR2 channels were incorporated into artificial membranes under voltage-clamp conditions. In symmetrical solutions containing 250 mM K+, 100 μM free Ca2+, pH 7.2, and 20 mM caffeine to clamp Po to high levels, 1 μM CaM had no significant effect on Po. This agrees with our previous work on native RyR2 channels demonstrating that high levels of CaM can reverse the activating effects of low [Ca2+CaM] but do not significantly lower Po below control values. We find that Ca2+CaM significantly reduces the rate of association of ryanodol to RyR2 but does not affect the rate of dissociation. Ca2+CaM also significantly alters channel behaviour within the ryanodol-modified conductance state. Our results lead to two important conclusions: 1. A Ca2+CaM-induced decrease in the rate of association of ryanoids to RyR2 is consistent with the reduction in [3H]ryanodine binding caused by Ca2+CaM, providing an explanation why the functional effects of Ca2+CaM at the single-channel level are not faithfully mirrored in the [3H]ryanodine binding studies. 2. The important, wider implications of this study are that [3H]ryanodine binding assays, in the absence of other supporting data, may not provide reliable information about the Po of RyR channel populations. Supported by the BHF.

A New Conformation in Sarcoplasmic Reticulum Calcium Pump and Plasma Membrane Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic Probe

The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca(2+) pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2'-[(125)I]iodo-4'-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([(125)I]TID-PC/16) under different conditions. We determined the following. 1) Incorporation of [(125)I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca(2+). This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca(2+) and calmodulin decreases by approximately the same amount. However, incubation with Ca(2+) alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [(125)I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E(1) conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca(2+) + C28 for SERCA and Ca(2+) alone for PMCA), and one in which the enzyme is fully active (Ca(2+) for SERCA and Ca(2+)-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.

Ca-dependent Nonsecretory Vesicle Fusion in a Secretory Cell

We have compared Ca-dependent exocytosis in excised giant membrane patches and in whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Stable patches of 2–4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A. Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100–200 μM free Ca. Capacitance and amperometric recording show that large secretory granules (SGs) containing serotonin are mostly lost from patches. Small vesicles that are retained (non-SGs) do not release serotonin or other substances detected by amperometry, although their fusion is reduced by tetanus toxin light chain. Non-SG fusion is unaffected by N-ethylmaleimide, phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P2) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P2, PI(3)P, and PI(4)P antibodies. In patch recordings, but not whole-cell recordings, fusion can be strongly reduced by ATP removal and by the nonspecific PI-kinase inhibitors wortmannin and adenosine. In whole-cell recording, non-SG fusion is strongly reduced by osmotically induced cell swelling, and subsequent recovery after shrinkage is then inhibited by wortmannin. Thus, membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole-cell fusion responses. Regarding Ca sensors for non-SG fusion, fusion remains robust in synaptotagmin (Syt) VII−/− mouse embryonic fibroblasts (MEFs), as well as in PLCδ1, PLC δ1/δ4, and PLCγ1−/− MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (KD ∼71 μM) than expected for these C2 domain–containing proteins. In summary, we find that non-SG membrane fusion behaves and is regulated substantially differently from SG fusion, and we have identified an ATP-dependent process that restores non-SG fusion capability after it is perturbed by membrane stretch or cell dilation.

Fluorescence assay for mitochondrial permeability transition in cardiomyocytes cultured in a microtiter plate

Mitochondrial permeability transition pore (MPTP) is a voltage-dependent, large-conductance channel of the inner mitochondrial membrane with an important role in a range of pathophysiological conditions. To facilitate studies of pharmacological pore modulation, we describe an assay in a model using neonatal cardiomyocytes in a 96-well microtiter plate format. In the presence of mitochondrial membrane potential ΔΨm, accumulation of rhodamine-123 in mitochondria (40,000 cells/well, 2.6 μM rhodamine-123) caused fluorescence signal quenching. Following substitution of dye-free buffer, dequenching occurred on the distribution of rhodamine-123 into the extracellular volume. The addition of a small buffer volume containing digitonin (final concentration 10 μg/ml) and Ca 2+ (final concentrations up to 100 μM free Ca 2+) caused dequenching (Δ F) due to ΔΨm dissipation by MPTP, as evidenced by inhibition in the presence of cyclosporin A (0.2–2 μM) and facilitation by pH 6.2. Δ F due to ΔΨm-dissipating agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or alamethicin (10 μM) was insensitive to either pH or cyclosporin A. Inhibition of Ca 2+-induced (but not of FCCP- or alamethicin-induced) Δ F by glycogen synthase kinase 3β (GSK3β) antagonist SB216763 and adenosine, acting at the level of intracellular signaling and plasma membrane receptors, respectively, is shown to illustrate potential applications of this assay. Limitation of the assay to cells with energized mitochondria is stressed.

Anionic lipids activate group IVA cytosolic phospholipase A2 via distinct and separate mechanisms

Previously, ceramide-1-phosphate (C1P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] were demonstrated to be potent and specific activators of group IVA cytosolic phospholipase A2 (cPLA2alpha). In this study, we hypothesized that these anionic lipids functionally activated the enzyme by distinctly different mechanisms. Indeed, surface plasmon resonance and surface dilution kinetics demonstrated that C1P was a more potent effector than PI(4,5)P2 in decreasing the dissociation constant of the cPLA2alpha-phosphatidylcholine (PC) interaction and increasing the residence time of the enzyme on the vesicles/micelles. PI(4,5)P2, in contrast to C1P, decreased the Michaelis-Menten constant, increasing the catalytic efficiency of the enzyme. Furthermore, PI(4,5)P2 activated cPLA2alpha with a stoichiometry of 1:1 versus C1P at 2.4:1. Lastly, PI(4,5)P2, but not C1P, increased the penetration ability of cPLA2alpha into PC-rich membranes. Therefore, this study demonstrates two distinct mechanisms for the activation of cPLA2alpha by anionic lipids. First, C1P activates cPLA2alpha by increasing the residence time of the enzyme on membranes. Second, PI(4,5)P2 activates the enzyme by increasing catalytic efficiency through increased membrane penetration.