cAMP-dependent phosphorylation of two sites in the alpha subunit of the cardiac sodium channel

The voltage-sensitive Na+ channel is responsible for generating action potentials in the heart which are critical for coordinated cardiac muscle contraction. Cardiac Na+ channels are regulated by cAMP-dependent phosphorylation, but the sites of phosphorylation are not known. Using mammalian cells expressing the rat cardiac Na+ channel (rH1) alpha subunit and site-specific antibodies, we have shown that the alpha subunit of rat heart Na+ channel is phosphorylated selectively by cAMP-dependent protein kinase (PKA) in vitro and in intact cells. Analysis of the sites of phosphorylation by two-dimensional phosphopeptide mapping and site-directed mutagenesis of fusion proteins revealed that the cardiac alpha subunit is phosphorylated selectively in vitro by PKA on Ser526 and Ser529 in the intracellular loop connecting homologous domains I and II (LI-II). These two residues were phosphorylated in intact cells expressing the rH1 alpha subunit when PKA was activated. Our results define a different pattern of phosphorylation of LI-II of cardiac and brain Na+ channels and implicate phosphorylation of Ser526 and Ser529 in the differential regulation of cardiac and brain Na+ channels by PKA.     

Sites of selective cAMP-dependent phosphorylation of the L-type calcium channel alpha 1 subunit from intact rabbit skeletal muscle myotubes

The principal (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive (L-type) calcium channels is present in full-length (212 kDa) and COOH-terminal truncated (190 kDa) forms, which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. Immunoprecipitation of the calcium channel from rabbit muscle myotubes in primary cell culture followed by phosphorylation with cA-PK, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed comparable phosphorylation of three COOH-terminal phosphopeptides found in the purified full-length alpha 1 subunit. Stimulation of muscle myotubes with a permeant cAMP analogue, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate, prior to immunoprecipitation of alpha 1 results in a 60-80% reduction of cA-PK catalyzed "back" phosphorylation of each of these sites in vitro in calcium channels purified from the cells, indicating that these sites are phosphorylated in vivo in response to increased intracellular cAMP. Serine 687, the most rapidly phosphorylated site in the truncated 190-kDa alpha 1 subunit, was observed as a minor phosphopeptide whose level of phosphorylation was not significantly affected by stimulation of endogenous cA-PK in the myotubes. The COOH-terminal sites, designated tryptic phosphopeptides 4, 5, and 6, were identified as serine 1757 (phosphopeptides 4 and 6) and 1854 (phosphopeptide 5) by a combination of protease cleavage, phosphorylation of synthetic peptides and fusion proteins, specific immunoprecipitation, and phosphopeptide mapping. Phosphorylation of serines 1757 and 1854 in the COOH-terminal region of the 212-kDa alpha 1 subunit in intact skeletal muscle cells may play a pivotal role in the regulation of calcium channel function by cA-PK.     

cAMP-dependent phosphorylation of the cardiac-type alpha 1 subunit of the voltage-dependent Ca2+ channel in a murine pancreatic beta-cell line

Two voltage-dependent calcium channels (VDCCs) have been reported in pancreatic islets: the beta-cell/endocrine-brain and cardiac subtypes. The cardiac-type alpha 1 subunit was isolated from cultured beta TC3 cells, a murine pancreatic beta-cell line, by immunoprecipitation with a specific polyclonal antibody. We have examined the effects of 1-isobutyl-3-methylxanthine (IBMX) and forskolin, agonists that elevate cAMP in these cells, on the phosphorylation of this subunit in intact beta TC3 cells using a sensitive back-phosphorylation technique. This technique allows quantitative detection of protein phosphorylation that is specifically stimulated by cAMP. The stimulation of intact beta TC3 cells with forskolin or IBMX resulted in the phosphorylation of the cardiac-type alpha 1 subunit as evidenced by a 40-60% decrease in the ability of the 257-kDa form to serve as a substrate in the in vitro back-phosphorylation reaction with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA). The effects of forskolin were time- and concentration-dependent. The concentration-dependency of forskolin-induced phosphorylation of the cardiac-type alpha 1 subunit and the potentiation of glucose-induced insulin secretion were highly correlated, a finding that is consistent with a role for such phosphorylation in mediating at least some of the effects of cAMP on secretion.     

Novel splice variants of the voltage-sensitive sodium channel alpha subunit

In this study, we have identified novel splice variants of the Na+ channel alpha subunit mRNA from cultured rat astrocytes and neuroblastoma cells. These splice variants are characterized by premature truncation or deletion of a segment in the third domain of the Na+ channel alpha subunit. The expression of three splice variants was upregulated by exposure to 1 mM dibutyryl cAMP in spinal cord astrocytes but not in cerebral astrocytes and in B50 and B104 neuroblastoma cells. The calcium ionophore 1 microM A23187, did not influence the expression of splice variants in either astrocytes or neuroblastoma cells. These findings suggest that spinal cord astrocytes may maintain a unique regulatory pathway that participates in the control of Na+ channel mRNA expression.     

In vivo phosphorylation of the epithelial sodium channel

The activity of the epithelial sodium channel (ENaC) in the distal nephron is regulated by an antidiuretic hormone, aldosterone, and insulin, but the molecular mechanisms that mediate these hormonal effects are mostly unknown. We have investigated whether aldosterone, insulin, or activation of protein kinases has an effect on the phosphorylation of the channel. Experiments were performed in an epithelial cell line generated by stable cotransfection of the three subunits (alpha, beta, and gamma) of ENaC. We found that beta and gamma, but not the alpha subunit, are phosphorylated in the basal state. Aldosterone, insulin, and protein kinases A and C increased phosphorylation of the beta and gamma subunits in their carboxyl termini, but none of these agents induced de novo phosphorylation of alpha subunits. Serines and threonines but not tyrosines were found to be phosphorylated. The results suggest that aldosterone, insulin, and protein kinases A and C modulate the activity of ENaC by phosphorylation of the carboxyl termini of the beta and gamma subunits.     

The alpha subunit of Gq contributes to muscarinic inhibition of the M-type potassium current in sympathetic neurons

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (IK(M)), which can be inhibited by activation of M1 muscarinic receptors. This inhibition occurs via pertussis toxin-insensitive G-proteins belonging to the Galphaq family (Caulfield et al., 1994 ). We have used DNA plasmids encoding antisense sequences against the 3' untranslated regions of Galpha subunits (antisense plasmids) to investigate the specific G-protein subunits involved in muscarinic inhibition of IK(M). These antisense plasmids specifically reduced levels of the target G-protein 48 hr after intranuclear injection. In cells depleted of Galphaq, muscarinic inhibition of IK(M) was attenuated compared both with uninjected neurons and with neurons injected with an inappropriate GalphaoA antisense plasmid. In contrast, depletion of Galpha11 protein did not alter IK(M) inhibition. To determine whether the alpha or beta gamma subunits of the G-protein mediated this inhibition, we have overexpressed the C terminus of beta adrenergic receptor kinase 1 (betaARK1), which binds free beta gamma subunits. betaARK1 did not reduce muscarinic inhibition of IK(M) at a concentration of plasmid that can reduce beta gamma-mediated inhibition of calcium current (). Also, expression of beta1gamma2 dimers did not alter the IK(M) density in SCG neurons. In contrast, IK(M) was virtually abolished in cells expressing GTPase-deficient, constitutively active forms of Galphaq and Galpha11. These data suggest that Galphaq is the principal mediator of muscarinic IK(M) inhibition in rat SCG neurons and that this more likely results from an effect of the alpha subunit than the beta gamma subunits of the Gq heterotrimer.     

Dopaminergic modulation of early signs of excitotoxicity in visualized rat neostriatal neurons

Cell swelling induced by activation of excitatory amino acid receptors is presumably the first step in a toxic cascade that may ultimately lead to cell death. Previously we showed that bath application of N-methyl-D-aspartate (NMDA) or kainate (KA) produces swelling of neostriatal cells. The present experiments examined modulation of NMDA and KA-induced cell swelling by dopamine (DA) and its receptor agonists. Nomarski optics and infra-red videomicroscopy were utilized to visualize neostriatal medium-sized neurons in thick slices from rat pups (12-18 postnatal days). Increase in somatic cross-sectional area served as the indicator of swelling induced by bath application of glutamate receptor agonists. NMDA induced cell swelling in a dose-dependent manner. Activation of DA receptors in the absence of NMDA did not produce swelling. DA and the D1 receptor agonist SKF 38393, increased the magnitude of swelling produced by NMDA. This effect was reduced in the presence of the D1 receptor antagonist, SCH 23390. In contrast, activation of D2 receptors by quinpirole decreased the magnitude of NMDA-induced cell swelling. DA slightly attenuated cell swelling induced by activation of KA receptors. Quinpirole produced a significant concentration-dependent reduction in KA-induced swelling while SKF38393 increased KA-induced swelling, but only at a low concentration of KA. Together, these results provide additional support for the hypothesis that the direction of DA modulation depends on the glutamate receptor subtype, as well as the DA receptor subtype activated. One possible consequence of these observations is that endogenous DA may be an important contributing factor in the mechanisms of cell death in Huntington's disease.     

Mitosis-specific phosphorylation and subcellular redistribution of the RIIalpha regulatory subunit of cAMP-dependent protein kinase

Phosphorylation of the RII regulatory subunits of cyclic AMP-dependent protein kinases (PKAs) was examined during the HeLa cell cycle. Three RIIalpha isoforms of 51, 54, and 57 kDa were identified by RIIalpha immunodetection and labeling with 8-azido[32P]cAMP in different cell cycle phases. These isoforms were characterized as different phosphorylation states by the use of selective PKA and cyclin-directed kinase inhibitors. Whereas RIIalpha autophosphorylation by PKA caused RIIalpha to shift from 51 to 54 kDa, phosphorylation of RIIalpha by one other or a combination of several kinases activated during mitosis caused RIIalpha to shift from 51 to 57 kDa. In vivo incorporation of [32P]orthophosphate into mitotic cells and RIIalpha immunoprecipitation demonstrated that RIIalpha was hyperphosphorylated on a different site than the one phosphorylated by PKA. Deletion and mutation analysis demonstrated that the cyclin B-p34(cdc2) kinase (CDK1) phosphorylated human recombinant RIIalpha in vitro on Thr54. Whereas RIIalpha was associated with the Golgi-centrosomal region during interphase, it was dissociated from its centrosomal localization at metaphase-anaphase transition. Furthermore, particulate RIIalpha from HeLa cell extracts was solubilized following incubation with CDK1 in vitro. Our results suggest that at the onset of mitosis, CDK1 phosphorylates RIIalpha, and this may alter its subcellular localization.     

cAMP-dependent phosphorylation of the cardiac L-type Ca channel: a missing link?

Cardiac inotropic effects of beta adrenergic agonists occur mainly through an increase in L-type (class C) calcium channel activity. This response has been attributed to phosphorylation of the L-type Ca channel, or a closely associated protein, by the cAMP-dependent protein kinase A (PKA). Among the three subunits forming the cardiac L-type Ca channel (alpha 1, beta and alpha 2-delta), biochemical studies have revealed that two subunits, alpha 1 and beta, are phosphorylated in vitro by protein kinase A, the alpha 1 subunit being the primary target. However, attempts to reconstitute the cAMP-dependent regulation of the expressed class C Ca channel, either in Xenopus oocytes or in cell lines, have provided contradictory results. We were unable to detect cAMP-dependent modulation of class C alpha 1 subunit Ca channels expressed in Xenopus oocytes, even when coinjected with auxiliary subunits beta and alpha 2-delta. Nevertheless, activity of Ca channels recorded from cardiac-mRNA injected oocytes was potentiated by injection of cAMP or PKA, even when expression of the beta subunit was suppressed using antisense oligonucleotide. Taken together, these results indicate that cAMP-dependent regulation does not exclusively involve the alpha 1 and the beta subunits of the Ca channel and suggest that unidentified protein(s), expressed in cardiac tissue, are most likely necessary.     

Multiple modulation pathways of calcium channel activity by a beta subunit. Direct evidence of beta subunit participation in membrane trafficking of the alpha1C subunit

In order to study the precise mechanisms of alpha1 subunit modulation by an auxiliary beta subunit of voltage-dependent calcium channels, a recombinant beta3 subunit fusion protein was produced and introduced into oocytes that express the human alpha1C subunit. Injection of the beta3 subunit protein rapidly modulated the current kinetics and voltage dependence of activation, whereas massive augmentation of peak current amplitude occurred over a longer time scale. Consistent with the latter, a severalfold increase in the amount of the alpha1C subunit in the plasma membrane was detected by quantitative confocal laser-scanning microscopy after beta3 subunit injection. Pretreatment of oocytes with bafilomycin A1, a vacuolar type H+-ATPase inhibitor, abolished the increase of the alpha1C subunit in the plasma membrane, attenuated current increase, but did not affect the modulation of current kinetics and voltage dependence by the beta3 subunit. These results provide clear evidence that the beta subunit modifies the calcium channel complex in a binary fashion; one is an allosteric modulation of the alpha1 subunit function and the other is a chaperoning of the alpha1 subunit to the plasma membrane.     

Enzymatic phosphorylation of soy globulins by the protein kinase CK2. Determination of the phosphorylation sites of beta-conglycinin alpha subunit by mass spectrometry

Being quite experienced in the field of gynaecologic surgery and hysterectomies especially, being familiar with recent innovations in laparoscopic surgery and also having some own experience in laparoscopic surgery, the authors discuss the advantages and disadvantages of all surgical methods of hysterectomy. Comparing the techniques, the duration, bearing in mind the the risks, overall costs and all other surgical details, the authors concluded that laparoscopically assisted vaginal hysterectomy is the best choice because it is the least invasive, less risky, no scars are left, the postoperative recovery is quick, there are numerous indications for it, the preparation obtained as a whole can be used for further clinical examination. The only disadvantage is it is too costly and sometimes the operation itself lasts too long, so it should not be applied in some cases.     

Differential phosphorylation of two size forms of the N-type calcium channel alpha 1 subunit which have different COOH termini

Two size forms of the class B N-type calcium channel alpha 1 subunit were recently identified with CNB1, an antipeptide antibody directed against an intracellular loop of this channel (Westenbroek, R.E., Hell, J.W., Warner, C., Dubel, S.J., Snutch, T.P., and Catterall, W.A. (1992) Neuron 9, 1099-1115). To investigate the biochemical differences between these two size forms, the antibodies CNB3 and CNB4 were raised against peptides with sequences corresponding to the COOH-terminal end of the full-length form. Immunoblot experiments demonstrated that both antibodies specifically recognize the longer form of 250 kDa, indicating that the COOH-terminal regions of the two size forms of the class B N-type channel alpha 1 subunit are different. Phosphorylation experiments with immunopurified calcium channels and different second messenger-activated protein kinases revealed that both the 220- and 250-kDa forms of the class B N-type calcium channel alpha 1 subunit are substrates for cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein kinase C. These three kinases incorporated approximately 1 mol of phosphate/mol of binding sites for omega-conotoxin (omega-CgTx) GVIA, a ligand specific for the N-type calcium channel, and may regulate the activity of both forms in vivo. In contrast, calcium- and calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated only the long form of the class B N-type calcium channel alpha 1 subunit, with a stoichiometry of 0.5 mol of phosphate/mol of total omega-CgTx GVIA binding sites. Specific phosphorylation of the long form of the class B alpha 1 subunit by CaM kinase II may differentially regulate the function of N-type calcium channels containing different size forms of their alpha 1 subunits in vivo.     

Calcium mobilization by ryanodine promotes the phosphorylation of initiation factor 2alpha subunit and inhibits protein synthesis in cultured neurons

Protein synthesis plays an important role in the viability and function of the cell. There is evidence indicating that Ca2+ may be a physiological regulator of the translational process. In the present study, the effect of agents that increase intracellular calcium levels by different mechanisms, as well as repercussion on the rate of protein synthesis, including phosphorylation of initiation factor 2alpha subunit, and double-stranded RNA-dependent eIF-2alpha kinase (PKR) activity were analyzed. Glutamate (100 microM) and K+ (60 mM), which increase intracellular calcium levels (the former mostly by the influx of extracellular calcium via voltage-sensitive calcium channels, and the latter by receptor-operated calcium channels), and carbachol (1 mM), as well as glutamate, which mobilizes intracellular calcium from the endoplasmic reticulum via activation of inositol 1,4,5-trisphosphate receptor, did not modify any of the analyzed parameters. Nevertheless, 100 nM ryanodine, which increases intracellular calcium concentration by activating the ryanodine receptor, promoted a significant decrease in the rate of protein synthesis and increased both initiation factor 2alpha subunit phosphorylation and PKR activity. From our results, we can conclude that inhibition of protein synthesis is dependent on the mobilization of intracellular calcium from internal stores. Moreover, they strongly suggest that this inhibition is only promoted when calcium is increased via ryanodine receptor, and possibly by activation of PKR activity.     

Flow-cytometric detection of the RI alpha subunit of type I cAMP-dependent protein kinase in human cells

cAMP-dependent protein kinase (PKA) is composed of two genetically distinct catalytic (C) and regulatory (R) subunits. There are two different classes of PKA, designated as type I and type II, which contain distinct R subunits (RI or RII, respectively) but share a common C subunit. Enhanced expression of type I PKA has been correlated with cell proliferation and neoplastic transformation. Detection of the different PKA subunits is usually performed by photoaffinity labeling with 8-N3-32P-cAMP or by radioimmunolabeling techniques. Both techniques are time consuming and require a high number of cells and the use of radioactive reagents. Using the MCF-10A normal human mammary cell line infected with a recombinant retroviral vector containing the human RI alpha gene (MCF-10A RI alpha), we have developed a flow-cytometric assay to detect the intracellular content of RI alpha protein in human cells. MCF-10A and MCF-10A RI alpha cells were fixed in 1.5% paraformaldehyde at 37 degrees C for 15 min and permeabilized by methanol and acetone (1:1) at -20 degrees C for 5 min before staining with a specific IgG2a MoAb followed by a FITC-conjugate rabbit-anti mouse IgG. This procedure was also successfully utilized to recognize RI alpha protein content in human peripheral blood lymphocytes. Flow-cytometric detection of the RI alpha subunit in human cells is feasible and allows the study of the role of type I PKA in cell growth and neoplastic transformation.     

Molecular diversity of the calcium channel alpha2delta subunit

Sequence database searches with the alpha2delta subunit as probe led to the identification of two new genes encoding proteins with the essential properties of this calcium channel subunit. Primary structure comparisons revealed that the novel alpha2delta-2 and alpha2delta-3 subunits share 55.6 and 30.3% identity with the alpha2delta-1 subunit, respectively. The number of putative glycosylation sites and cysteine residues, hydropathicity profiles, and electrophysiological character of the alpha2delta-3 subunit indicates that these proteins are functional calcium channel subunits. Coexpression of alpha2delta-3 with alpha1C and cardiac beta2a or alpha1E and beta3 subunits shifted the voltage dependence of channel activation and inactivation in a hyperpolarizing direction and accelerated the kinetics of current inactivation. The kinetics of current activation were altered only when alpha2delta-1 or alpha2delta-3 was expressed with alpha1C. The effects of alpha2delta-3 on alpha1C but not alpha1E are indistinguishable from the effects of alpha2delta-1. Using Northern blot analysis, it was shown that alpha2delta-3 is expressed exclusively in brain, whereas alpha2delta-2 is found in several tissues. In situ hybridization of mouse brain sections showed mRNA expression of alpha2delta-1 and alpha2delta-3 in the hippocampus, cerebellum, and cortex, with alpha2delta-1 strongly detected in the olfactory bulb and alpha2delta-3 in the caudate putamen.     

Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons

The control of branching of axons and dendrites is poorly understood. It has been hypothesized that branching may be produced by changes in the cytoskeleton [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419; P. Friedrich, A. Aszodi, MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture, FEBS Lett. 295 (1991) 5-9]. The assembly and stability of microtubules, which are prominent cytoskeletal elements in both axons and dendrites, are regulated by microtubule-associated proteins, including tau (predominantly found in axons) and MAP2 (predominantly found in dendrites). The phosphorylation state of tau and MAP2 modulates their interactions with microtubules. In their low-phosphorylation states, tau and MAP2 bind to microtubules and increase microtubule assembly and/or stability. Increased phosphorylation decreases these effects. Diez-Guerra and Avila [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419] found that protein phosphorylation correlates with neurite branching in cultured rat hippocampal neurons, and hypothesized that increased protein phosphorylation stimulates neurite branching. To test this hypothesis, we cultured rat hippocampal neurons in the presence of specific modulators of serine-threonine protein kinases and phosphatases. Inhibitors of several protein kinases, which would be expected to decrease protein phosphorylation, reduced branching. KT5720, an inhibitor of cyclic AMP-dependent protein kinase, and KN62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinases, inhibited branching of both axons and dendrites. Calphostin C and chelerythrine, inhibitors of protein kinase C, inhibited branching of axons but not dendrites. Treatments that would be expected to increase protein phosphorylation, including inhibitors of protein phosphatases (okadaic acid, cyclosporin A and FK506) and stimulators of PKA (SP-cAMPS) or PKC (phorbol 12-myristate 13-acetate), increased dendrite branching. Only FK506 and phorbol 12-myristate 13-acetate stimulated axon branching. A subset of these agents was tested to confirm their effects on protein phosphorylation in this preparation. Okadaic acid, FK506 and SP-cAMPS all increased protein phosphorylation; KT5720 and KN62 decreased protein phosphorylation. On Western blots, the position of MAP2c extracted from cultures exposed to okadaic acid was slightly shifted toward higher molecular weight, suggesting greater phosphorylation, while the position of MAP2c from cultures exposed to KT5720 and KN62 was slightly shifted toward lower molecular weight, suggesting less phosphorylation. We conclude that protein phosphorylation modulates both dendrite branching and axon branching, but with differences in sensitivity to phosphorylation and/or dephosphorylation by specific kinases and phosphatases.     

Backbone flexibility of five sites on the catalytic subunit of cAMP-dependent protein kinase in the open and closed conformations

To develop an alternative approach to measure peptidyl backbone flexibility and to expand our understanding of the segmental flexibility of cAMP-dependent protein kinase (cAPK), the effect of protein kinase inhibitor peptide, PKIalpha(5-24), and MgATP on the mobility of fluorescein selectively conjugated to five sites on the catalytic subunit of cAPK was examined. Specifically, five full-length, single-site catalytic subunit mutants (K16C, K81C, I244C, C199A, and N326C) were prepared, and fluorescein maleimide was selectively attached to the side chains of each substituted cysteine or, in the case of the C199A mutant, to the unprotected native C343. The time-resolved anisotropy decay profiles of the five fluorescein maleimide-conjugated mutants were well fit to a biexponential equation. The fast rotational correlation times of the fluorescein conjugates ranged between 1.9 and 2.8 ns and were inversely correlated (r = -0.87) to the averaged crystallographic main-chain atom B factors around each site of conjugation. The slow correlation times ranged between 25 and 28 ns and were about the same magnitude as the value of 21 ns estimated from the Stokes-Einstein equation. The presence of MgATP and PKIalpha(5-24), which induces the closed conformation of cAPK, was associated with a reduction of the fast rotational correlation time of the K81C conjugate, indicating that the peptidyl backbone around K81 is measurably less flexible when the C subunit is in the closed compared with the open conformation. The results suggest (i) that time-resolved fluorescence anisotropy can assess the nanosecond flexibility of short segments of the peptidyl backbone around each site of labeling and (ii) that the substrate/pseudosubstrate binding differentially affects the backbone flexibility of cAPK.     

Differences in the binding sites of two site-3 sodium channel toxins

Site-3 toxins from scorpion and sea anemone bind to Na channels and selectively inhibit current decay. Anthopleurins A and B (ApA and ApB, respectively), toxins found in the venom of the sea anemone Anthopleura xanthogrammica, bind to closed states of mammalian skeletal and cardiac Na channels with differing affinities which arise from differences in first-order toxin/channel dissociation rate constants, koff. Using chimera comprising domain interchanges between channel isoforms, we examined the structural basis of this differential affinity. Toxin/channel association rates, kon, were similar for both toxins and both parental channels. Domain 4 determined koff for ApA, while ApB dissociated from all tested chimera in a cardiac-like manner. To probe this surprising difference between two such closely related toxins, we examined the interaction of chimeric channels with a form of ApB in which the two nonconserved basic residues, Arg-12 and Lys-49, were converted to the corresponding neutral amino acids from ApA. In the chimera comprising domain 1 from the cardiac muscle isoform and domains 2-4 from the skeletal muscle isoform, toxin dissociated at a rate intermediate between those of the parental channels. We conclude that the differential component of ApA binding is controlled by domain 4 and that some component of ApB binding is not shared by ApA. This additional component probably binds to an interface between channel domains and is partly mediated by toxin residues Arg-12 and Lys-49.     

Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states

The catalytic subunit of mouse cAMP-dependent protein kinase expressed in Escherichia coli was separated into three distinct species using Mono-S ion exchange chromatography. These isoenzymes corresponded to three isoelectric variants with pIs of 6.4 (30%), 7.2 (60%) and 8.2 (10%). The Stokes' radius of each form was 27.7, 27.1 and 26.3 A respectively. Using electrospray mass spectroscopy the differences between the isozymes were shown to be due to phosphorylation, with each form differing by 80 mass units corresponding to a single phosphate. The fully phosphorylated recombinant enzyme contained four phosphates while the dominant isozyme contained only three. Since the enzyme is not phosphorylated when active site mutations are introduced into the C-subunit, these phosphates are incorporated in an autocatalytic mechanism and are not due to E. coli protein kinases. When the recombinant enzyme was compared with the mammalian porcine heart enzyme significant differences in post-translational modifications were observed. The mammalian enzyme could also be separated into two isozymes. However, in contrast to the recombinant enzyme, the mammalian isozymes displayed an identical mass of 40 840. This correlated with two different post-translational modifications: two phosphates and an N-terminal myristyl moiety. The importance of post-translational modifications, and in particular the phosphorylation state, for the expression of eukaryotic proteins in E. coli is discussed.