Inhibition of isoflurane-induced increase of cell-surface redistribution and activity of glutamate transporter type 3 by serine 465 sequence-specific peptides.

Excitatory amino acid transporters (EAAT) transport glutamate into cells to regulate glutamate neurotransmission and to maintain nontoxic extracellular glutamate levels for neurons. We showed previously that the commonly used volatile anesthetic isoflurane increases the transporting activity of EAAT3, the major neuronal EAAT. This effect requires a protein kinase C (PKC) α-mediated and S465-dependent EAAT3 redistribution to the plasma membrane. Thus, we hypothesize that specific peptides can be designed to block this effect. We conjugated a 10-amino acid synthetic peptide with a sequence identical to that of EAAT3 around the S465 to a peptide that can facilitate permeation of the plasma membrane. This fusion peptide inhibited the isoflurane-increased EAAT3 activity and redistribution to the plasma membrane in C6 cells and hippocampus. It did not affect the basal EAAT3 activity. This peptide also attenuated isoflurane-induced increase of PKCα in the immunoprecipitates produced by an anti-EAAT3 antibody. A scrambled peptide that has the same amino acid composition as the S465 sequence-specific peptide but has a random sequence did not change the effects of isoflurane on EAAT3. The S465 sequence-specific peptide, but not the scrambled peptide, is a good PKCα substrate in in vitro assay. These peptides did not affect cell viability. These results, along with our previous findings, strongly suggest that PKCα interacts with EAAT3 to regulate its functions. The S465 sequence-specific peptide may interrupt this interaction and is an effective inhibitor for the regulation of EAAT3 activity and trafficking by PKCα and isoflurane.

The importance of serine 161 in the sodium channel beta3 subunit for modulation of Na(V)1.2 gating.

Voltage-gated sodium (Na) channels contribute to the regulation of cellular excitability due to their role in the generation and propagation of action potentials. They are composed of a pore-forming alpha subunit and are modulated by at least two of four distinct beta subunits (beta1-4). Recent studies have implicated a role for the intracellular domain of beta subunits in modulating Na channel gating and trafficking. In beta3, the intracellular domain contains a serine residue at position 161 that is replaced by an alanine in beta1. In this study, we have probed the functional importance of beta3S161 for modulating Na channel gating. Wild-type beta3 and point mutations beta3S161A or beta3S161E were individually co-expressed in HEK 293 cells stably expressing human Na(v)1.2. WTbeta3 expression increased Na current density, shifted steady-state inactivation in a depolarized direction, and accelerated the kinetics of recovery from inactivation of the Na current. Analogous effects were observed with beta3S161E co-expression. In contrast, beta3S161A abolished the shifts in steady-state inactivation and recovery from inactivation of the Na current, but did increase Na current density. Immunocytochemistry and Western blot experiments demonstrate membrane expression of WTbeta3, beta3S161E, and beta3S161A, suggesting that the differences in Na channel gating were not due to disruptions in beta subunit trafficking. These studies suggest that modification of beta3S161 may be important in modulating Na-channel gating.

Protein kinase C isozyme-specific potentiation of expressed Ca v 2.3 currents by acetyl-beta-methylcholine and phorbol-12-myristate, 13-acetate.

Protein kinase C (PKC) is implicated in the potentiation of Ca v 2.3 currents by acetyl-beta-methylcholine (MCh), a muscarinic M1 receptor agonist or phorbol-12-myristate, 13-acetate (PMA). The PKC isozymes responsible for the action of MCh and PMA were investigated using translocation as a measure of activation and with isozyme-selective antagonists and siRNA. Ca v channels were expressed with alpha1 2.3, beta1b and alpha2delta subunits and muscarinic M1 receptors in the Xenopus oocytes and the expressed currents (I Ba) were studied using Ba2+ as the charge carrier. Translocation of PKC isozymes to the membrane studied by Western blot revealed that all eleven known PKC isozymes are present in the Xenopus oocytes. Exposure of the oocytes to MCh led to the translocation of PKC alpha whereas PMA activated PKC betaII and epsilon isozymes. The action of MCh was inhibited by Go 6976, an inhibitor of cPKC isozymes or PKC alpha siRNA. PMA-induced potentiation of Ca v 2.3 currents was inhibited by CG533 53, a PKC betaII antagonist, betaIIV5.3, a peptide translocation inhibitor of PKC betaII or PKC betaII siRNA. Similarly, epsilonV1.2, a peptide translocation inhibitor of PKC epsilon or PKC epsilon siRNA inhibited PMA action. The inhibitors of PKC increased the basal I Ba slightly. It is possible that some PKC isozymes have negative control over the I Ba. Our results implicate PKC alpha in the potentiation of Ca v 2.3 currents by MCh and PKC betaII and epsilon in the potentiation of Ca v 2.3 currents by PMA.

Regulation of ghrelin structure and membrane binding by phosphorylation.

The peptide hormone ghrelin requires Ser-3 acylation for receptor binding, orexigenic and anti-inflammatory effects. Functions of desacylghrelin are less well understood. In vitro kinase assays reveal that the evolutionarily conserved Ser-18 in the basic C-terminus is an excellent substrate for protein kinase C. Circular dichroism reveals that desacylghrelin is approximately 12% helical in aqueous solution and approximately 50% helical in trifluoroethanol. Ser-18-phosphorylation, Ser-18-Ala substitution, or Ser-3-acylation reduces the helical character in trifluoroethanol to approximately 24%. Both ghrelin and desacylghrelin bind to phosphatidylcholine:phosphatidylserine sucrose-loaded vesicles in a phosphatidylserine-dependent manner. Phosphoghrelin and phosphodesacylghrelin show greatly diminished phosphatidylserine-dependent binding. These results are consistent with binding of ghrelin and desacylghrelin to acidic lipids via the basic face of an amphipathic helix with Ser-18 phosphorylation disrupting both helical character and membrane binding.

Initial three-dimensional reconstructions of protein kinase C delta from two-dimensional crystals on lipid monolayers.

Two-dimensional crystals of protein kinase C delta (PKCdelta) and of its regulatory domain (RDdelta) were grown on lipid monolayers and analyzed by electron microscopy at tilt angles varying from -50 degrees to +55 degrees. Although the crystals exhibit pseudo-3-fold symmetry, analysis of difference phase residuals indicates that there is only one way to align the crystals for merging so the data were processed in plane group P1. Three-dimensional reconstructions generated for several two-dimensional crystals each of PKCdelta and RDdelta show good agreement and are consistent with membrane attachment via a single C1 subdomain, a small surface contact by one or two loops from the C2 domain, and, in intact PKCdelta, a small appendage from the catalytic domain, probably V5. Two-dimensional crystallography with three-dimensional reconstruction should be suitable for examination of additional PKC isozymes and for analysis of the enzymes bound to substrates and other proteins.

Critical role of serine 465 in isoflurane-induced increase of cell-surface redistribution and activity of glutamate transporter type 3.

Glutamate transporters (also called excitatory amino acid transporters, EAATs) bind extracellular glutamate and transport it to intracellular space to regulate glutamate neurotransmission and to maintain extracellular glutamate concentrations below neurotoxic levels. We previously showed that isoflurane, a commonly used anesthetic, enhanced the activity of EAAT3, a major neuronal EAAT. This effect required a protein kinase C (PKC) alpha-dependent EAAT3 redistribution to the plasma membrane. In this study, we prepared COS7 cells stably expressing EAAT3 with or without mutations of potential PKC phosphorylation sites in the putative intracellular domains. Here we report that mutation of threonine 5 or threonine 498 to alanine did not affect the isoflurane effects on EAAT3. However, the mutation of serine 465 to alanine abolished isoflurane-induced increase of EAAT3 activity and redistribution to the plasma membrane. The mutation of serine 465 to aspartic acid increased the expression of EAAT3 in the plasma membrane and also abolished the isoflurane effects on EAAT3. These results suggest an essential role of serine 465 in the isoflurane-increased EAAT3 activity and redistribution and a direct effect of PKC on EAAT3. Consistent with these results, isoflurane induced an increase in phosphorylation of wild type, T5A, and T498A EAAT3, and this increase was absent in S465A and S465D. Our current results, together with our previous data that showed the involvement of PKCalpha in the isoflurane effects on EAAT3, suggest that the phosphorylation of serine 465 in EAAT3 by PKCalpha mediates the increased EAAT3 activity and redistribution to plasma membrane after isoflurane exposure.

Inhibitory role of Ser-425 of the alpha1 2.2 subunit in the enhancement of Cav 2.2 currents by phorbol-12-myristate, 13-acetate.

Voltage-gated calcium channels (Ca(v)) 2.2 currents are potentiated by phorbol-12-myristate, 13-acetate (PMA), whereas Ca(v) 2.3 currents are increased by both PMA and acetyl-beta-methylcholine (MCh). MCh-selective sites were identified in the alpha(1) 2.3 subunit, whereas the identified PMA sites responded to both PMA and MCh (Kamatchi, G. L., Franke, R., Lynch, C., III, and Sando, J. J. (2004) J. Biol. Chem. 279, 4102-4109; Fang, H., Franke, R., Patanavanich, S., Lalvani, A., Powell, N. K., Sando, J. J., and Kamatchi, G. L. (2005) J. Biol. Chem. 280, 23559-23565). The hypothesis that PMA sites in the alpha(1) 2.2 subunit are homologous to the PMA-responsive sites in alpha(1) 2.3 subunit was tested with Ser/Thr --> Ala mutations in the alpha(1) 2.2 subunit. WT alpha(1) 2.2 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits. Inward current (I(Ba)) was recorded using Ba(2+) as the charge carrier. T422A, S1757A, S2108A, or S2132A decreased the PMA response. In contrast, S425A increased the response to PMA, and thus, it was considered an inhibitory site. Replacement of each of the identified stimulatory Ser/Thr sites with Asp increased the basal current and decreased the PMA-induced enhancement, consistent with regulation by phosphorylation at these sites. Multiple mutant combinations showed (i) greater inhibition than that caused by the single Ala mutations; (ii) that enhancement observed when Thr-422 and Ser-2108 are available may be inhibited by the presence of Ser-425; and (iii) that the combination of Thr-422, Ser-2108, and either Ser-1757 or Ser-2132 can provide a greater response to PMA when Ser-425 is replaced with Ala. The homologous sites in alpha(1) 2.2 and alpha(1) 2.3 subunits seem to be functionally different. The existence of an inhibitory phosphorylation site in the I-II linker seems to be unique to the alpha(1) 2.2 subunit.

Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels.

Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K+ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of G alpha(q)-coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((R(p))-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.

Role of alpha1 2.3 subunit I-II linker sites in the enhancement of Ca(v) 2.3 current by phorbol 12-myristate 13-acetate and acetyl-beta-methylcholine.

Potentiation of Ca(v) 2.3 currents by phorbol 12-myristate 13-acetate (PMA) or acetyl-beta-methylcholine (MCh) may be due to protein kinase C (PKC)-mediated phosphorylation of the alpha1 2.3 subunit. Mutational analysis of potential PKC sites unique to the alpha1 2.3 subunit revealed several sites in the II-III linker that are specific to MCh (Kamatchi, G., Franke, R., Lynch, C., III, and Sando, J. (2004) J. Biol. Chem. 279, 4102-4109). To identify sites responsive to PMA, Ser/Thr --> Ala mutations were made in potential PKC sites homologous to the alpha1 2.3 and 2.2 subunits, both of which respond to PMA. Wild type alpha1 2.3 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits and muscarinic M1 receptors. Inward current (I(Ba)) was recorded using Ba2+ as the charge carrier. Thr-365 of the I-II linker was identified as the primary site of PMA action, and this site also was required, along with the previously identified MCh-selective sites, for the MCh response. Ser-369 and Ser-1995 contributed to current enhancement only if Thr-365 also was available. Mutation of the essential sites to Asp increased the basal I(Ba) and caused a corresponding decrease in the PMA or MCh responses, consistent with possible regulation of these sites by phosphorylation. These results suggest that PMA and MCh both activate a pathway that can regulate the common PMA-sensitive sites in the I-II linker but that MCh also activates an additional pathway required for regulation of the MCh-unique sites, especially in the II-III linker.

Identification of sites responsible for potentiation of type 2.3 calcium currents by acetyl-beta-methylcholine.

To address mechanisms for the differential sensitivity of voltage-gated Ca2+ channels (Cav) to agonists, channel activity was compared in Xenopus oocytes coexpressing muscarinic M(1) receptors and different Cav alpha1 subunits, all with beta1B,alpha2/delta subunits. Acetyl-beta-methylcholine (MCh) decreased Cav 1.2c currents, did not affect 2.1 or 2.2 currents, but potentiated Cav 2.3 currents. Phorbol 12-myristate 13-acetate (PMA) did not affect Cav 1.2c or 2.1 currents but potentiated 2.2 and 2.3 currents. Comparison of the amino acid sequences of the alpha1 subunits revealed a set of potential protein kinase C phosphorylation sites in common between the 2.2 and 2.3 channels that respond to PMA and a set of potential sites unique to the alpha1 2.3 subunits that respond to MCh. Quadruple Ser --> Ala mutation of the predicted MCh sites in the alpha1 2.3 subunit (Ser-888, Ser-892, and Ser-894 in the II-III linker and Ser-1987 in the C terminus) caused loss of the MCh response but not the PMA response. Triple Ser --> Ala mutation of just the II-III linker sites gave similar results. Ser-888 or Ser-892 was sufficient for the MCh responsiveness, whereas Ser-894 required the presence of Ser-1987. Ser --> Asp substitution of Ser-888, Ser-892, Ser-1987, and Ser-892/Ser-1987 increased the basal current and decreased the MCh response but did not alter the PMA response. These results reveal that sites unique to the II-III linker of alpha1 2.3 subunits mediate the responsiveness of Cav 2.3 channels to MCh. Because Cav 2.3 channels contribute to action potential-induced Ca2+ influx, these sites may account for M1 receptor-mediated regulation of neurotransmission at some synapses.

Two-dimensional crystal structures of protein kinase C-delta, its regulatory domain, and the enzyme complexed with myelin basic protein.

Two-dimensional crystals of protein kinase C (PKC) delta, its regulatory domain (RDdelta), and the enzyme complexed with the substrate myelin basic protein have been grown on lipid monolayers composed of phosphatidylcholine: phosphatidylserine: diolein (45:50:5, molar ratio). Images have been reconstructed to 10-A resolution. The unit cells of all three proteins have cell edges a = b and interedge angle gamma = 60 degrees. RDdelta has an edge length of 33 +/- 1 A, and its reconstruction is donut shaped. The three-dimensional reconstructions from the PKCdelta C1b crystal structure () can be accommodated in this two-dimensional projection. Intact PKCdelta has an edge length of 46 +/- 1 A in the presence or absence of a nonhydrolyzable ATP analog, AMP-PnP. Its reconstruction has a similar donut shape, which can accommodate the C1b domain, but the spacing between donuts is greater than that in RDdelta; some additional structure is visible between the donuts. The complex of PKCdelta and myelin basic protein, with or without AMP-PnP, has an edge length of 43 +/- 1 A and a distinct structure. These results indicate that the C1 domains of RDdelta are tightly packed in the plane of the membrane in the two-dimensional crystals, that there is a single molecule of PKCdelta in the unit cell, and that its interaction with myelin basic protein induces a shift in conformation and/or packing of the enzyme.

Protein kinase C-eta regulates resistance to UV- and gamma-irradiation-induced apoptosis in glioblastoma cells by preventing caspase-9 activation.

Both increased cell proliferation and apoptosis play important roles in the malignant growth of glioblastomas. We have demonstrated recently that the differential expression of protein kinase C (PKC)-eta increases the proliferative capacity of glioblastoma cells in culture; however, specific functions for this novel PKC isozyme in the regulation of apoptosis in these tumors has not been defined. In the present study of several glioblastoma cell lines, we investigated the role of PKC-eta in preventing UV- and gamma-irradiation-induced apoptosis and in caspase-dependent signaling pathways that mediate cell death. Exposure to UV or gamma irradiation killed 80% to 100% of PKC-eta-deficient nonneoplastic human astrocytes and U-1242 MG cells, but had little effect on the PKC-eta-expressing U-251 MG and U-373 MG cells. PKC-eta appears to mediate resistance to irradiation specifically such that when PKC-eta was stably expressed in U-1242 MG cells, more than 80% of these cells developed resistance to irradiation-induced apoptosis. Reducing PKC-eta expression by transient and stable expression of antisense PKC-eta in wild-type U-251 MG cells results in increased sensitivity to UV irradiation in a fashion similar to U-1242 MG cells and nonneoplastic astrocytes. Irradiation of PKC-eta-deficient glioblastoma cells resulted in the activation of caspase-9 and caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and a substantial increase in subdiploid DNA content that did not occur in PKC-eta-expressing tumor cells. A specific inhibitor (Ac-DEVD-CHO) of caspase-3 blocked apoptosis in PKC-eta-deficient U-1242 MG cells. The data demonstrate that resistance to UV and gamma irradiation in glioblastoma cell lines is modified significantly by PKC-eta expression and that PKC-eta appears to block the apoptotic cascade at caspase-9 activation.