Osmotic Stress Reduces Ca2+ Signals through Deformation of Caveolae.

Caveolae are membrane invaginations that can sequester various signaling proteins. Caveolae have been shown to provide mechanical strength to cells by flattening to accommodate increased volume when cells are subjected to hypo-osmotic stress. We have previously found that caveolin, the main structural component of caveolae, specifically binds Gαq and stabilizes its activation state resulting in an enhanced Ca(2+) signal upon activation. Here, we show that osmotic stress caused by decreasing the osmolarity in half reversibly changes the configuration of caveolae without releasing a significant portion of caveolin molecules. This change in configuration due to flattening leads to a loss in Cav1-Gαq association. This loss in Gαq/Cav1 association due to osmotic stress results in a significant reduction of Gαq/phospholipase Cß-mediated Ca(2+) signals. This reduced Ca(2+) response is also seen when caveolae are reduced by treatment with siRNA(Cav1) or by dissolving them by methyl-ß-cyclodextran. No change in Ca(2+) release with osmotic swelling can be seen when growth factor pathways are activated. Taken together, these results connect the mechanical deformation of caveolae to Gαq-mediated Ca(2+) signals.

A loss in cellular protein partners promotes α-synuclein aggregation in cells resulting from oxidative stress.

There is a consensus that oxidative stress promotes neurodegeneration and may be linked to plaque formation. α-Synuclein is the main component of neurodegenerative plaques. We have found that α-synuclein binds strongly to the enzyme phospholipase Cß1 (PLCß1) in vitro and in cells affecting both its G protein activation and its degradation. Because PLCß1 binds to α-synuclein in cells, we tested whether decreasing its level would promote α-synuclein aggregation and whether overproducing PLCß1 would inhibit aggregation. By imaging fluorescent α-synuclein in living HEK293, PC12, and SK-H-SH cells, we find that α-synuclein aggregation is directly related to the level of PLCß1. Importantly, we found that oxidative stress does not affect the cellular levels of α-synuclein but results in the down-regulation of PLCß1 thereby promoting α-synuclein aggregation. A peptide that mimics part of the α-synuclein binding site to PLCß prevents aggregation. Our studies indicate that PLCß1 can reduce cell damage under oxidative stress and offers a potential site that might be exploited to prevent α-synuclein aggregation.

Phospholipase Cß1 is linked to RNA interference of specific genes through translin-associated factor X.

Phospholipase Cß1 (PLCß1) is a G-protein-regulated enzyme whose activity results in proliferative and mitogenic changes in the cell. We have previously found that in solution PLCß1 binds to the RNA processing protein translin-associated factor X (TRAX) with nanomolar affinity and that this binding competes with G proteins. Here, we show that endogenous PLCß1 and TRAX interact in SK-N-SH cells and also in HEK293 cells induced to overexpress PLCß1. In HEK293 cells, TRAX overexpression ablates Ca(2+) signals generated by G protein-PLCß1 activation. TRAX plays a key role in down-regulation of proteins by small, interfering RNA, and PLCß1 overexpression completely reverses the 2- to 4-fold down-regulation of GAPDH by siRNA in HEK293 and HeLa cells as seen by an ∼4-fold recovery in both the transcript and protein levels. Also, down-regulation of endogenous PLCß1 in HEK293 and HeLa cells allows for an ∼20% increase in siRNA(GAPDH) silencing. While PLCß1 overexpression results in a 50% reversal of cell death caused by siRNA(LDH), it does not affect cell survival or silencing of other genes (e.g., cyclophilin, Hsp90, translin). PLCß1 overexpression in HEK293 and HeLa cells causes a 30% reduction in the total amount of small RNAs. LDH and GAPDH are part of a complex that promotes H2B synthesis that allows cells to progress through the S phase. We find that PLCß1 reverses the cell death and completely rescues H2B levels caused by siRNA knockdown of LDH or GAPDH. Taken together, our study shows a novel role of PLCß1 in gene regulation through TRAX association.

Improving cardiac conduction with a skeletal muscle sodium channel by gene and cell therapy.

The voltage-gated Na+ channel is a critical determinant of the action potential (AP) upstroke. Increasing Na+ conductance may speed AP propagation. In this study, we propose use of the skeletal muscle Na+ channel SkM1 as a more favorable gene than the cardiac isoform SCN5A to enhance conduction velocity in depolarized cardiac tissue. We used cells that electrically coupled with cardiac myocytes as a delivery platform to introduce the Na+ channels. Human embryonic kidney 293 cells were stably transfected with SkM1 or SCN5A. SkM1 had a more depolarized (18 mV shift) inactivation curve than SCN5A. We also found that SkM1 recovered faster from inactivation than SCN5A. When coupled with SkM1 expressing cells, cultured myocytes showed an increase in the dV/dtmax of the AP. Expression of SCN5A had no such effect. In an in vitro cardiac syncytium, coculture of neonatal cardiac myocytes with SkM1 expressing but not SCN5A expressing cells significantly increased the conduction velocity under both normal and depolarized conditions. In an in vitro reentry model induced by high-frequency stimulation, expression of SkM1 also enhanced angular velocity of the induced reentry. These results suggest that cells carrying a Na+ channel with a more depolarized inactivation curve can improve cardiac excitability and conduction in depolarized tissues.

Modulation of Ca²+ activity in cardiomyocytes through caveolae-Gαq interactions.

Cardiomyocytes have a complex Ca(2+) behavior and changes in this behavior may underlie certain disease states. Intracellular Ca(2+) activity can be regulated by the phospholipase Cß-Gα(q) pathway localized on the plasma membrane. The plasma membranes of cardiomycoytes are rich in caveolae domains organized by caveolin proteins. Caveolae may indirectly affect cell signals by entrapping and localizing specific proteins. Recently, we found that caveolin may specifically interact with activated Gα(q), which could affect Ca(2+) signals. Here, using fluorescence imaging and correlation techniques we show that Gα(q)-Gßγ subunits localize to caveolae in adult ventricular canine cardiomyoctyes. Carbachol stimulation releases Gßγ subunits from caveolae with a concurrent stabilization of activated Gα(q) by caveolin-3 (Cav3). These cells show oscillating Ca(2+) waves that are not seen in neonatal cells that do not contain Cav3. Microinjection of a peptide that disrupts Cav3-Gα(q) association, but not a control peptide, extinguishes the waves. Furthermore, these waves are unchanged with rynaodine treatment, but not seen with treatment of a phospholipase C inhibitor, implying that Cav3-Gα(q) is responsible for this Ca(2+) activity. Taken together, these studies show that caveolae play a direct and active role in regulating basal Ca(2+) activity in cardiomyocytes.

The small G protein Rac1 activates phospholipase Cdelta1 through phospholipase Cbeta2.

Rac1, which is associated with cytoskeletal pathways, can activate phospholipase Cbeta2 (PLCbeta2) to increase intracellular Ca(2+) levels. This increased Ca(2+) can in turn activate the very robust PLCdelta1 to synergize Ca(2+) signals. We have previously found that PLCbeta2 will bind to and inhibit PLCdelta1 in solution by an unknown mechanism and that PLCbeta2.PLCdelta1 complexes can be disrupted by Gbetagamma subunits. However, because the major populations of PLCbeta2 and PLCdelta1 are cytosolic, their regulation by Gbetagamma subunits is not clear. Here, we have found that the pleckstrin homology (PH) domains of PLCbeta2 and PLCbeta3 are the regions that result in PLCdelta1 binding and inhibition. In cells, PLCbeta2.PLCdelta1 form complexes as seen by Förster resonance energy transfer and co-immunoprecipitation, and microinjection of PHbeta2 dissociates the complex. Using PHbeta2 as a tool to assess the contribution of PLCbeta inhibition of PLCdelta1 to Ca(2+) release, we found that, although PHbeta2 only results in a 25% inhibition of PLCdelta1 in solution, in cells the presence of PHbeta2 appears to eliminates Ca(2+) release suggesting a large threshold effect. We found that the small plasma membrane population of PLCbeta2.PLCdelta1 is disrupted by activation of heterotrimeric G proteins, and that the major cytosolic population of the complexes are disrupted by Rac1 activation. Thus, the activity of PLCdelta1 is controlled by the amount of bound PLCbeta2 that changes with displacement of the enzyme by heterotrimeric or small G proteins. Through PLCbeta2, PLCdelta1 activation is linked to surface receptors as well as signals that mediate cytoskeletal pathways.

Evidence for a second, high affinity Gbetagamma binding site on Galphai1(GDP) subunits.

It is well known that Galpha(i1)(GDP) binds strongly to Gbetagamma subunits to form the Galpha(i1)(GDP)-Gbetagamma heterotrimer, and that activation to Galpha(i1)(GTP) results in conformational changes that reduces its affinity for Gbetagamma subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Galpha(i1)(GDP) can bind a second Gbetagamma subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Galpha(i1) and Gbetagamma subunits. Also, we find that phospholipase Cbeta2, an effector of Gbetagamma, does not compete with the second binding site implying that effectors can be bound to the Galpha(i1)(GDP)-(Gbetagamma)(2) complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Galpha(i1) competes with binding of the second Gbetagamma subunit. Injection of this peptide into cultured cells expressing eYFP-Galpha(i1)(GDP) and eCFP-Gbetagamma reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.

Replacing damaged myocardium.

Heart failure survival after diagnosis has barely changed for more than half a century. Recently, investigation has focused on differentiation of stem cells in vitro and their delivery for use in vivo as replacement cardiac contractile elements. Here we report preliminary results using mesenchymal stem cells partially differentiated to a cardiac lineage in vitro. When delivered to the canine heart on an extracellular matrix patch to replace a full-thickness ventricular defect in vivo, they improve regional mechanical function. The delivered cells were also tracked, and some became myocytes with mature sarcomeres.

Phospholipase Cß connects G protein signaling with RNA interference.

Phosphoinositide-specific-phospholipase Cß (PLCß) is the main effector of Gαq stimulation which is coupled to receptors that bind acetylcholine, bradykinin, dopamine, angiotensin II as well as other hormones and neurotransmitters. Using a yeast two-hybrid and other approaches, we have recently found that the same region of PLCß that binds Gαq also interacts with Component 3 Promoter of RNA induced silencing complex (C3PO), which is required for efficient activity of the RNA-induced silencing complex. In purified form, C3PO competes with Gαq for PLCß binding and at high concentrations can quench PLCß activation. Additionally, we have found that the binding of PLCß to C3PO inhibits its nuclease activity leading to reversal of RNA-induced silencing of specific genes. In cells, we found that PLCß distributes between the plasma membrane where it localizes with Gαq, and in the cytosol where it localizes with C3PO. When cells are actively processing small interfering RNAs the interaction between PLCß and C3PO gets stronger and leads to changes in the cellular distribution of PLCß. The magnitude of attenuation is specific for different silencing RNAs. Our studies imply a direct link between calcium responses mediated through Gαq and post-transcriptional gene regulation through PLCß.

Development of a universal RNA beacon for exogenous gene detection.

Stem cell therapy requires a nontoxic and high-throughput method to achieve a pure cell population to prevent teratomas that can occur if even one cell in the implant has not been transformed. A promising method to detect and separate cells expressing a particular gene is RNA beacon technology. However, developing a successful, specific beacon to a particular transfected gene can take months to develop and in some cases is impossible. Here, we report on an off-the-shelf universal beacon that decreases the time and cost of applying beacon technology to select any living cell population transfected with an exogenous gene.

Multiple roles of pleckstrin homology domains in phospholipase Cbeta function.

Since their discovery almost 10 years ago pleckstrin homology (PH) domains have been identified in a wide variety of proteins. Here, we focus on two proteins whose PH domains play a defined functional role, phospholipase C (PLC)-beta(2) and PLCdelta(1). While the PH domains of both proteins are responsible for membrane targeting, their specificity of membrane binding drastically differs. However, in both these proteins the PH domains work to modulate the activity of their catalytic core upon interaction with either phosphoinositol lipids or G protein activators. These observations show that these PH domains are not simply binding sites tethered onto their host enzyme but are intimately associated with their catalytic core. This property may be true for other PH domains.

α-Synuclein increases the cellular level of phospholipase Cß1.

α-Synuclein is a conserved protein that is a key component in neurodegenerative plaques [1,2]. α-Synuclein binds strongly to phospholipase Cß (PLCß) and promotes Ca2+ release in cells. Here, we show that expression of α-synuclein increases the cellular level of PLCß1 in two neuronal cell lines: PC12 and SK-N-S-SH. The increase in PLCß1 is not accompanied by changes in the level of RNA or in ubiquitination. Instead, we find that α-synuclein protects PLCß1 from trypsin digestion and from degradation by the Ca(+2) activated protease calpain. Calpain removes the C-terminal region of the enzyme which mediates activation by Gα(q). We find that in SK-N-SH cells, α-synuclein reduced degradation of PLCß1 by calpain during Ca2+ signaling allowing the enzyme to remain sensitive to Gα(q) activation. Taken together, our studies show that α-synuclein protects the integrity of PLCß1 and its ability to be activated by Gα(q), which may in turn impact Ca2+ signaling.

γ-Synuclein interacts with phospholipase Cß2 to modulate G protein activation.

Phospholipase Cß2 (PLC ß2) is activated by G proteins and generates calcium signals in cells. PLCß2 is absent in normal breast tissue, but is highly expressed in breast tumors where its expression is correlated with the progression and migration of the tumor. This pattern of expression parallels the expression of the breast cancer specific gene protein 1 which is also known as γ-synuclein. The cellular function of γ-synuclein and the role it plays in proliferation are unknown. Here, we determined whether γ-synuclein can interact with PLCß2 and affect its activity. Using co-immunprecitation and co-immunofluorescence, we find that in both benign and aggressive breast cancer cell lines γ-synuclein and PLCß2 are associated. In solution, purified γ-synuclein binds to PLCß2 with high affinity as measured by fluorescence methods. Protease digestion and mass spectrometry studies show that γ-synuclein binds to a site on the C-terminus of PLCß2 that overlaps with the Gαq binding site. Additionally, γ-synuclein competes for Gαq association, but not for activators that bind to the N-terminus (i.e. Rac1 and Gßγ). Binding of γ-synuclein reduces the catalytic activity of PLCß2 by mechanism that involves inhibition of product release without affecting membrane interactions. Since activated Gαq binds more strongly to PLCß2 than γ-synuclein, addition of Gαq(GTPγS) to the γ-synuclein -PLCß2 complex allows for relief of enzyme inhibition along with concomitant activation. We also find that Gßγ can reverse γ-synuclein inhibition without dissociating the γ-synuclein- PLCß2- complex. These studies point to a role of γ-synuclein in promoting a more robust G protein activation of PLCß2.

Effect of skeletal muscle Na(+) channel delivered via a cell platform on cardiac conduction and arrhythmia induction.

BACKGROUND: In depolarized myocardial infarct epicardial border zones, the cardiac sodium channel is largely inactivated, contributing to slow conduction and reentry. We have demonstrated that adenoviral delivery of the skeletal muscle Na(+) channel (SkM1) to epicardial border zones normalizes conduction and reduces induction of ventricular tachycardia/ventricular fibrillation. We now studied the impact of canine mesenchymal stem cells (cMSCs) in delivering SkM1. METHODS AND RESULTS: cMSCs were isolated and transfected with SkM1. Coculture experiments showed cMSC/SkM1 but not cMSC alone and maintained fast conduction at depolarized potentials. We studied 3 groups in the canine 7d infarct: sham, cMSC, and cMSC/SkM1. In vivo epicardial border zones electrograms were broad and fragmented in sham, narrower in cMSCs, and narrow and unfragmented in cMSC/SkM1 (P<0.05). During programmed electrical stimulation of epicardial border zones, QRS duration in cMSC/SkM1 was shorter than in cMSC and sham (P<0.05). Programmed electrical stimulation-induced ventricular tachycardia/ventricular fibrillation was equivalent in all groups (P>0.05). CONCLUSION: cMSCs provide efficient delivery of SkM1 current. The interventions performed (cMSCs or cMSC/SkM1) were neither antiarrhythmic nor proarrhythmic. Comparing outcomes with cMSC/SkM1 and viral gene delivery highlights the criticality of the delivery platform to SkM1 antiarrhythmic efficacy.

Phospholipase Cbeta2 binds to and inhibits phospholipase Cdelta1.

Phospholipase Cbeta (PLCbeta) isoforms, which are under the control of Galphaq and Gbetagamma subunits, generate Ca2+ signals induced by a broad array of extracellular agonists, whereas PLCdelta isoforms depend on a rise in cytosolic Ca2+ for their activation. Here we find that PLCbeta2 binds strongly to PLCdelta1 and inhibits its catalytic activity in vitro and in living cells. In vitro, this PLC complex can be disrupted by increasing concentrations of free Gbetagamma subunits. Such competition has consequences for signaling, because in HEK293 cells PLCbeta2 suppresses elevated basal [Ca2+] and inositol phosphates levels and the sustained agonist-induced elevation of Ca2+ levels caused by PLCdelta1. Also, expression of both PLCs results in a synergistic release of [Ca2+] upon stimulation in A10 cells. These results support a model in which PLCbeta2 suppresses the basal catalytic activity of PLCdelta1, which is relieved by binding of Gbetagamma subunits to PLCbeta2 allowing for amplified calcium signals.

Fluorescence studies suggest a role for alpha-synuclein in the phosphatidylinositol lipid signaling pathway.

Alpha-synuclein plays a key role in the pathogenesis of many neurodegenerative diseases. To date, its cellular role has yet to be determined, although it has been proposed to be connected to calcium and G protein-mediated dopamine signaling. Alpha-synuclein is known to bind strongly to model membrane surfaces where it may interact with other membrane-associated proteins. Here, we find that the membrane association of alpha-synuclein is enhanced by the presence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and Ca(2+). We also find that alpha-synuclein interacts with high affinity with the G protein-regulated enzyme phospholipase Cbeta(2) (PLCbeta(2)), which catalyzes the hydrolysis of PI(4,5)P(2). Binding of alpha-synuclein to PLCbeta(2) reduces its catalytic activity by 50%, but causes its level of activation by Gbetagamma subunits to increase from 4- to 24-fold. This effect is greatly reduced for A53T alpha-synuclein, which is a mutant associated with familial Parkinson's disease. PI(4,5)P(2) hydrolysis by PLCbeta(2) results in an increase in the intracellular Ca(2+) concentration, and we find that in cultured cells the presence of alpha-synuclein results in a 6-fold enhancement in the release of Ca(2+) from intracellular stores in response to agents that release Gbetagamma subunits relative to controls. Alpha-synuclein also enhances the increase in the level of inositol phosphates seen upon G protein stimulation, suggesting that it also may interact with PLCbeta(2) in cells. Given that Ca(2+) and dopamine regulation are mediated through PLCbeta and G protein signals, our results suggest that alpha-synuclein may play a role in inositol phospholipid signaling.

The Pleckstrin homology domains of phospholipases C-beta and -delta confer activation through a common site.

Mammalian inositol-specific phospholipase C-beta2 (PLC beta 2) and PLC delta 1 differ in their cellular activators. PLC beta 2 can be activated by G beta gamma subunits, whereas PLC delta 1 can be activated by phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2). For both proteins, the N-terminal pleckstrin homology (PH) domain appears to mediate activation. Here, we have constructed a chimera in which we placed the N-terminal PH domain of PLC delta 1 into remaining C-terminal regions of PLC beta 2. The PH delta PLC beta chimera showed PI(4,5)P2-dependent membrane binding similar to PLC delta 1 and a G beta gamma interaction energy close to that of PLC delta 1. Like PLC delta 1, the chimera was activated by PI(4,5)P2 through the PH domain but not by G beta gamma. Because these and previous results indicate a common site of contact between the PH and catalytic domains in these two enzymes, we computationally docked the known structures of the PH and catalytic domains of PLC delta 1. A synthetic peptide whose sequence matches a potential interaction site between the two domains inhibited the basal activity of PLC beta 2, PLC delta 1, and a G beta gamma-activable PH beta 2-PLC delta 1 chimera. Also, the peptide was able to inhibit PI(4,5)P2 and G beta gamma activation of the PH-PLC delta 1 PH-PLC beta 2 enzymes in a concentration-dependent manner, suggesting that this is the region responsible for PH domain-mediated activation of the catalytic core.