Absence Of cGMP Research Articles

A substitution in cGMP-dependent protein kinase 1 associated with aortic disease induces an active conformation in the absence of cGMP

Type 1 cGMP-dependent protein kinases (PKGs) play important roles in human cardiovascular physiology, regulating vascular tone and smooth-muscle cell phenotype. A mutation in the human PRKG1 gene encoding cGMP-dependent protein kinase 1 (PKG1) leads to thoracic aortic aneurysms and dissections. The mutation causes an arginine-to-glutamine (RQ) substitution within the first cGMP-binding pocket in PKG1. This substitution disrupts cGMP binding to the pocket, but it also unexpectedly causes PKG1 to have high activity in the absence of cGMP via an unknown mechanism. Here, we identified the molecular mechanism whereby the RQ mutation increases basal kinase activity in the human PKG1α and PKG1β isoforms. Although we found that the RQ substitution (R177Q in PKG1α and R192Q in PKG1β) increases PKG1α and PKG1β autophosphorylation in vitro, we did not detect increased autophosphorylation of the PKG1α or PKG1β RQ variant isolated from transiently transfected 293T cells, indicating that increased basal activity of the RQ variants in cells was not driven by PKG1 autophosphorylation. Replacement of Arg-177 in PKG1α with alanine or methionine also increased basal activity. PKG1 exists as a parallel homodimer linked by an N-terminal leucine zipper, and we show that the WT chain in WT-RQ heterodimers partly reduces basal activity of the RQ chain. Using hydrogen/deuterium-exchange MS, we found that the RQ substitution causes PKG1β to adopt an active conformation in the absence of cGMP, similar to that of cGMP-bound WT enzyme. We conclude that the RQ substitution in PKG1 increases its basal activity by disrupting the formation of an inactive conformation.

Mechanism of Allosteric Inhibition of Plasmodium falciparum CGMP-Dependent Protein Kinase

Malaria is a life-threatening disease responsible for about a million fatalities per year worldwide. Most of the malaria deaths are caused by Plasmodium falciparum. An essential regulator for the development of P. falciparum is the cyclic GMP (cGMP) dependent protein kinase (PfPKG). PfPKG is composed of a regulatory domain and a catalytic domain. In the absence of cGMP, the regulatory domain inhibits the kinase domain. Upon cGMP binding to the regulatory domain, the inhibition is released and PfPKG is activated. Targeting directly the active site of PfPKG poses a major selectivity challenge, since the kinase catalytic domains are highly conserved among eukaryotes. One approach to circumvent this problem is to selectively target less conserved allosteric sitesof PfPKG, such as the cGMP-binding domains (CBDs), which can be achieved through cGMP-analogs. Here, we report the mechanism of action for cGMP-antagonists of PfPKG to gain structural and dynamical insight on the otherwise elusive bound-inhibited state of PfPKG. This will provide another avenue to rationally design PfPKG-selective inhibitors for the treatment of malaria. NMR Chemical Shift Projection Analysis (CHESPA) of PfPKG CBD-D with cGMP-analogsshows that the cGMP-antagonist inhibits PfPKG not through a simple reversal of a two-state active-inactive equilibrium, but through multi-state equilibria sampling distinct holo-inactive intermediate that combine elements of both active and inactive states in different regions of CBD-D. This intermediate state exhibits inhibitory competent characteristics and also provides the inhibitors the avenue for preserving a high affinity for CBD-D. NMR spin relaxation measurements further complement the CHESPA data by probing the dynamical changes within the CBD-D of PfPKG that occur upon inhibition.

CGMP Binding Domain D Mediates a Unique Activation Mechanism in Plasmodium falciparum PKG.

cGMP-dependent protein kinase from Plasmodium falciparum ( PfPKG) plays a crucial role in the sexual as well as the asexual proliferation of this human malaria causing parasite. However, function and regulation of PfPKG are largely unknown. Previous studies showed that the domain organization of PfPKG significantly differs from human PKG ( hPKG) and indicated a critical role of the cyclic nucleotide binding domain D (CNB-D). We identified a novel mechanism, where the CNB-D controls activation and regulation of the parasite specific protein kinase. Here, kinase activity is not dependent on a pseudosubstrate autoinhibitory sequence (IS), as reported for human PKG. A construct lacking the putative IS and containing only the CNB-D and the catalytic domain is inactive in the absence of cGMP and can efficiently be activated with cGMP. On the basis of structural evidence, we describe a regulatory mechanism, whereby cGMP binding to CNB-D induces a conformational change involving the αC-helix of the CNB-D. The inactive state is defined by a unique interaction between Asp597 of the catalytic domain and Arg528 of the αC-helix. The same arginine (R528), however, stabilizes cGMP binding by interacting with Tyr480 of the phosphate binding cassette (PBC). This represents the active state of PfPKG. Our results unveil fundamental differences in the activation mechanism between PfPKG and hPKG, building the basis for the development of strategies for targeted drug design in fighting malaria.

Balance between S-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation.

Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.

Altered Smooth Muscle Cell Force Generation as a Driver of Thoracic Aortic Aneurysms and Dissections.

The importance of maintaining contractile function in aortic smooth muscle cells (SMCs) is evident by the fact that heterozygous mutations in the major structural proteins or kinases controlling contraction lead to the formation of aneurysms of the ascending thoracic aorta that predispose to life-threatening aortic dissections. Force generation by SMC requires ATP-dependent cyclic interactions between filaments composed of SMC-specific isoforms of α-actin (encoded by ACTA2) and myosin heavy chain (MYH11). ACTA2 and MYH11 mutations are predicted or have been shown to disrupt this cyclic interaction predispose to thoracic aortic disease. Movement of the myosin motor domain is controlled by phosphorylation of the regulatory light chain on the myosin filament, and loss-of-function mutations in the dedicated kinase for this phosphorylation, myosin light chain kinase (MYLK) also predispose to thoracic aortic disease. Finally, a mutation in the cGMP-activated protein kinase (PRKG1) results in constitutive activation of the kinase in the absence of cGMP, thus driving SMC relaxation in part through increased dephosphorylation of the regulatory light chain and predisposes to thoracic aortic disease. Furthermore, SMCs cannot generate force without connections to the extracellular matrix through focal adhesions, and mutations in the major protein in the extracellular matrix, fibrillin-1, linking SMCs to the matrix also cause thoracic aortic disease in individuals with Marfan syndrome. Thus, disruption of the ability of the aortic SMC to generate force through the elastin-contractile units in response to pulsatile blood flow may be a primary driver for thoracic aortic aneurysms and dissections.

Recurrent Gain-of-Function Mutation in PRKG1 Causes Thoracic Aortic Aneurysms and Acute Aortic Dissections

Gene mutations that lead to decreased contraction of vascular smooth-muscle cells (SMCs) can cause inherited thoracic aortic aneurysms and dissections. Exome sequencing of distant relatives affected by thoracic aortic disease and subsequent Sanger sequencing of additional probands with familial thoracic aortic disease identified the same rare variant, PRKG1 c.530G>A (p.Arg177Gln), in four families. This mutation segregated with aortic disease in these families with a combined two-point LOD score of 7.88. The majority of affected individuals presented with acute aortic dissections (63%) at relatively young ages (mean 31 years, range 17-51 years). PRKG1 encodes type I cGMP-dependent protein kinase (PKG-1), which is activated upon binding of cGMP and controls SMC relaxation. Although the p.Arg177Gln alteration disrupts binding to the high-affinity cGMP binding site within the regulatory domain, the altered PKG-1 is constitutively active even in the absence of cGMP. The increased PKG-1 activity leads to decreased phosphorylation of the myosin regulatory light chain in fibroblasts and is predicted to cause decreased contraction of vascular SMCs. Thus, identification of a gain-of-function mutation in PRKG1 as a cause of thoracic aortic disease provides further evidence that proper SMC contractile function is critical for maintaining the integrity of the thoracic aorta throughout a lifetime.

Functional Proteomic Profiling of Phosphodiesterases Using SeraFILE Separations Platform

Functional proteomic profiling can help identify targets for disease diagnosis and therapy. Available methods are limited by the inability to profile many functional properties measured by enzymes kinetics. The functional proteomic profiling approach proposed here seeks to overcome such limitations. It begins with surface-based proteome separations of tissue/cell-line extracts, using SeraFILE, a proprietary protein separations platform. Enzyme kinetic properties of resulting subproteomes are then characterized, and the data integrated into proteomic profiles. As a model, SeraFILE-derived subproteomes of cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) from bovine brain homogenate (BBH) and rat brain homogenate (RBH) were characterized for cAMP hydrolysis activity in the presence (challenge condition) and absence of cGMP. Functional profiles of RBH and BBH were compiled from the enzyme activity response to the challenge condition in each of the respective subproteomes. Intersample analysis showed that comparable profiles differed in only a few data points, and that distinctive subproteomes can be generated from comparable tissue samples from different animals. These results demonstrate that the proposed methods provide a means to simplify intersample differences, and to localize proteins attributable to sample-specific responses. It can be potentially applied for disease and nondisease sample comparison in biomarker discovery and drug discovery profiling.

The Glutamic Acid-Rich Protein Is a Gating Inhibitor of Cyclic Nucleotide-Gated Channels

The cyclic nucleotide-gated (CNG) cation channel of rod photoreceptors is a heterotetramer consisting of homologous CNGA1 and CNGB1a subunits. While CNGA1 is indispensable for channel activation, the specific role of CNGB1a in this process has remained elusive. Here, we show that the N-terminal glutamic acid-rich protein (GARP) domain of CNGB1a and soluble GARP2, which corresponds to the proximal portion of the GARP domain, act as autoinhibitory domains that decrease the opening probability of the CNG channel. In the presence of mutations that structurally impair the cyclic nucleotide-binding domain (CNBD) of CNGB1a, the GARP domain completely abolishes channel activity. In agreement with an inhibitory function of GARP, the activity of mutant CNG channels could be fully restored by deletion of the GARP domain. We identified two sequences within the GARP domain that confer most of the inhibitory effect and demonstrate that the profound inhibition imposed by the GARP domain is caused by direct and autonomous protein-protein interaction with the CNG channel complex. In wild-type rod CNG channels, this inhibitory effect can be relieved by binding of cGMP to the CNBD of CNGB1a. In conclusion, we propose that the N terminus of CNGB1a and soluble GARPs act as molecular gate keepers that control the activation of heteromeric rod CNG channels. Our results suggest that the GARP domain has evolved in rod photoreceptors to reduce current noise resulting from openings of CNG channels in the absence of cGMP.

The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

Small-molecule modulators of cGMP signaling are of interest to basic and clinical research. The cGMP-dependent protein kinase type I (cGKI) is presumably a major mediator of cGMP effects, and the cGMP analogue Rp-8-Br-PET-cGMPS (Rp-PET) (chemical name: beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer) is currently considered one of the most permeable, selective, and potent cGKI inhibitors available for intact cell studies. Here, we have evaluated the properties of Rp-PET using cGKI-expressing and cGKI-deficient primary vascular smooth muscle cells (VSMCs), purified cGKI isozymes, and an engineered cGMP sensor protein. cGKI activity in intact VSMCs was monitored by cGMP/cGKI-stimulated cell growth and phosphorylation of vasodilator-stimulated phosphoprotein. Unexpectedly, Rp-PET (100 microm) did not efficiently antagonize activation of cGKI by the agonist 8-Br-cGMP (100 microm) in intact VSMCs. Moreover, in the absence of 8-Br-cGMP, Rp-PET (100 microm) stimulated cell growth in a cGKIalpha-dependent manner. Kinase assays with purified cGKI isozymes confirmed the previously reported inhibition of the cGMP-stimulated enzyme by Rp-PET in vitro. However, in the absence of the agonist cGMP, Rp-PET partially activated the cGKIalpha isoform. Experiments with a fluorescence resonance energy transfer-based construct harboring the cGMP binding sites of cGKI suggested that binding of Rp-PET induces a conformational change similar to the agonist cGMP. Together, these findings indicate that Rp-PET is a partial cGKIalpha agonist that under certain conditions stimulates rather than inhibits cGKI activity in vitro and in intact cells. Data obtained with Rp-PET as cGKI inhibitor should be interpreted with caution and not be used as sole evidence to dissect the role of cGKI in signaling processes.

Solution Structure of the cGMP Binding GAF Domain from Phosphodiesterase 5: INSIGHTS INTO NUCLEOTIDE SPECIFICITY, DIMERIZATION, AND cGMP-DEPENDENT CONFORMATIONAL CHANGE

Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular nuclear Overhauser effects. Comparison with GAF domains from PDE2A and adenylyl cyclase cyaB2 reveals a conserved overall domain fold of a six-stranded beta-sheet and four alpha-helices that form a well defined cGMP binding pocket. However, the nucleotide coordination is distinct with a series of altered binding contacts. The structure suggests that nucleotide binding specificity is provided by Asp-196, which is positioned to form two hydrogen bonds to the guanine ring of cGMP. An alanine mutation of Asp-196 disrupts cGMP binding and increases cAMP affinity in constructs containing only GAF A causing an altered cAMP-bound structural conformation. NMR studies on the tandem GAF domains reveal a flexible GAF A domain in the absence of cGMP, and indicate a large conformational change upon ligand binding. Furthermore, we identify a region of approximately 20 residues directly N-terminal of GAF A as critical for tight dimerization of the tandem GAF domains. The features of the PDE5 regulatory domain revealed here provide an initial structural basis for future investigations of the regulatory mechanism of PDE5 and the design of GAF-specific regulators of PDE5 function.

A comparison of electrophysiological properties of the CNGA1, CNGA1tandem and CNGA1cys-free Channels

Three constructs are used for the analysis of biophysical properties of CNGA1 channels: the WT CNGA1 channel, a CNGA1 channel where all endogenous cysteines were removed (CNGA1cys-free) and a construct composed of two CNGA1 subunits connected by a small linker (CNGA1tandem). So far, it has been assumed, but not proven, that the molecular structure of these ionic channels is almost identical. The I/V relations, ionic selectivity to alkali monovalent cations, blockage by tetracaine and TMA+ were not significantly different. The cGMP dose response and blockage by TEA+ and Cd2+ were instead significantly different in CNGA1 and CNGA1cys-free channels, but not in CNGA1 and CNGA1tandem channels. Cd2+ blocked irreversibly the mutant channel A406C in the absence of cGMP. By contrast, Cd2+ did not block the mutant channel A406C in the CNGA1cys-free background (A406Ccys-free), but an irreversible and almost complete blockage was observed in the presence of the cross-linker M-4-M. Results obtained with different MTS cross-linkers and reagents suggest that the 3D structure of the CNGA1cys-free differs from that of the CNGA1 channel and that the distance between homologous residues at position 406 in CNGA1cys-free is longer than in the WT CNGA1 by several Angstroms.

Conversion of Phosphodiesterase-5 (PDE5) Catalytic Site to Higher Affinity by PDE5 Inhibitors

Phosphodiesterase-5 (PDE5) specifically hydrolyzes cGMP, thereby contributing to modulation of intracellular levels of this nucleotide. In the present study, preincubation with cGMP increased PDE5 catalytic activity for cGMP degradation, and it converted the PDE5 catalytic site to a form that was more potently inhibited by each of the three PDE5 catalytic site-specific inhibitors: sildenafil, vardenafil, and tadalafil. These results implied that elevated cGMP initiates a physiological negative feedback on the cGMP pathway by increasing the affinity of the PDE5 catalytic site for cGMP. This increase in catalytic site activity or affinity for inhibitors could be caused by binding of cGMP to either the PDE5 allosteric sites, catalytic site, or both. Whether occupation of the catalytic site alone could mediate the effect was examined using radiolabeled PDE5 inhibitors in the absence of cGMP. Exchange-dissociation of [(3)H]sildenafil (Viagra), [(3)H]vardenafil (Levitra), or [(3)H]tadalafil (Cialis) from full-length PDE5 or isolated catalytic domain revealed two kinetic components (slow and fast). Extended preincubation of full-length PDE5, but not isolated catalytic domain, with (3)H inhibitors converted the biphasic pattern to a single slow (high-affinity) component. Studies of amino-terminally truncated PDE5 established that full-length mammalian GAF-B (cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases, Escherichia coli FhlA) subdomain conjoined with the catalytic domain was sufficient for this conversion. In conclusion, binding of substrate or substrate analogs such as PDE5 inhibitors to the catalytic site converts a fast (low-affinity) inhibitor dissociation component of the PDE5 catalytic site to a slow (high-affinity) inhibitor dissociation component. This effect is predicted to improve the substrate affinity or inhibitory potencies of these compounds in intact cells.

Probing noncovalent protein–ligand interactions of the cgmp-dependent protein kinase using electrospray ionization time of flight mass spectrometry

Nanoflow electrospray ionization time of flight mass spectrometry (ESI-TOF-MS) was used to study activation properties of the cGMP-dependent protein kinase (PKG). Our nanoflow ESI-TOF-MS analysis confirms that PKG mainly occurs as a 153 kDa homodimer and is able to bind four cGMP molecules, which is in agreement with the known stoichiometry. Binding order and stoichiometry of cGMP, the non-hydrolysable ATP analog beta,gamma-imidoadenosine 5'-triphosphate (AMPPNP) and Mn2+ for PKG were characterized as model for the active PKG-cGMP-ATP/Mg2+ complex. Already in the absence of cGMP, a noncovalent complex between PKG and two molecules of AMPPNP could be observed by ESI-TOF-MS. Binding of AMPPNP to PKG was strongly enhanced by the addition of MnCl2 to the spray solution. This is in agreement with binding of AMPPNP/Mn2+ in the ATP binding pocket of PKG since all protein kinases require a metal ion to accompany ATP in the ATP-binding pocket for proper positioning of the beta and gamma phosphates. Additionally, this finding could imply that within the inactive conformation of PKG, the autoinhibition-domain, when in contact with the substrate-docking domain, does not block the entrance to the ATP-binding site. In the presence of cGMP, less of the fully saturated PKG-(cGMP)4(AMPPNP/Mn2+)2 complex was observed, suggesting that the PKG-ATP interaction is weakened in the active conformation of PKG. Additionally, limited proteolysis in combination with native-ESI MS showed to be a useful tool to study the contact regions on the PKG-dimer and also allowed the rapid determination of the overall autophosphorylation status of the protein. These measurements indicated that autophosphorylation mainly occurs within the first 80 aminoterminal residues and involves in total 3-4 phosphates per subunit.

Movement of the C-Helix during the Gating of Cyclic Nucleotide-Gated Channels

Movements within the cyclic nucleotide-binding domain of cyclic nucleotide-gated channels are thought to underlie the initial phase of channel gating (Tibbs, G. R., D. T. Liu, B. G. Leypold, and S. A. Siegelbaum. 1998. J. Biol. Chem. 273:4497–4505; Zong, X., H. Zucker, F. Hofmann, and M. Biel. 1998. EMBO J. 17:353–362; Matulef, K., G. E. Flynn, and W. N. Zagotta. 1999. Neuron. 24:443–452; Paoletti, P., E. C. Young, and S. A. Siegelbaum. 1999. J. Gen. Physiol. 113:17–33; Johnson, J. P., and W. N. Zagotta. 2001. Nature. 412:917–921). To investigate these movements, cysteine mutation was performed on each of the 28 residues (Leu-583 to Asn-610), which span the agonist-binding domain of the α-subunit of the bovine rod cyclic nucleotide-gated channel. The effects of Cd 2+ ions, 2-trimethylammonioethylmethane thiosulfonate (MTSET) and copper phenanthroline (CuP) on channel activity were examined, in excised inside-out patches in the presence and in the absence of a saturating concentration of cGMP. The application of 100 μM Cd 2+ in the presence of saturating concentration of cGMP caused an irreversible and almost complete reduction of the current in mutant channels E594C, I600C, and L601C. In the absence of cGMP, the presence of 100 μM Cd 2+ caused a strong current reduction in all cysteine mutants from Asp-588 to Leu-607, with the exception of mutant channels A589C, M592C, M602C, K603C, and L606C. The selective effect of Cd 2+ ions was very similar to that observed when adding the oxidizing agent CuP to the bath medium, except for mutant channel G597C, where CuP caused a stronger current decrease (67 ± 7%) than Cd 2+ (23 ± 4%). In the absence of cGMP, MTSET caused a reduction of the current by >40% in mutant channels L607C, L601C, I600C, G597C, and E594C, whereas in the presence of cGMP only mutant channel L601C was affected. The application of MTSET protected many mutant channels from the effects of Cd 2+ and CuP. These results suggest that, when CNG channels are in the open state, residues from Asp-588 to Leu-607 are in an α-helical structure, homologous to the C-helix of the catabolite gene activator protein (Weber, I. T., and T. A. Steitz. 1987. J. Mol. Biol. 198:311–326). Furthermore, residues Glu-594, Gly-597, Ile-600, and Leu-601 of these helices belonging to two different subunits must be in close proximity. In the closed state the C-helices are in a different configuration and undergo significant fluctuations.

A Conserved Serine Juxtaposed to the Pseudosubstrate Site of Type I cGMP-dependent Protein Kinase Contributes Strongly to Autoinhibition and Lower cGMP Affinity

Serines 64 and 79 are homologous residues that are juxtaposed to the autoinhibitory pseudosubstrate site in cGMP-dependent protein kinase type Ialpha and type Ibeta (PKG-Ialpha and PKG-Ibeta), respectively. Autophosphorylation of this residue is associated with activation of type I PKGs. To determine the role of this conserved serine, point mutations have been made in PKG-Ialpha (S64A, S64T, S64D, and S64N) and PKG-Ibeta (S79A). In wild-type PKG-Ialpha, basal kinase activity ratio (-cGMP/+cGMP) is 0.11, autophosphorylation increases this ratio 3-fold, and the K(a) and K(D) values for cGMP are 127 and 36 nm, respectively. S64A PKG-Ialpha basal kinase activity ratio increases 2-fold, cGMP binding affinity increases approximately 10-fold in both K(a) and K(D), and activation by autophosphorylation is slight. S64D and S64N mutants are nearly constitutively active in the absence of cGMP, cGMP binding affinity in each increases 18-fold, and autophosphorylation does not affect the kinase activity of these mutants. Mutation of the homologous site in PKG-Ibeta (S79A) increases the basal kinase activity ratio 2-fold and cGMP binding affinity 5-fold over that of wild-type PKG-Ibeta. The combined results demonstrate that a conserved serine juxtaposed to the pseudosubstrate site in type I PKGs contributes importantly to enzyme function by increasing autoinhibition and decreasing cGMP binding affinity.

Overexpression of a Constitutively Active Protein Kinase G Mutant Reduces Neointima Formation and In-Stent Restenosis

Neointima formation after arterial injury is associated with reduced vascular cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG), a major cGMP effector in vascular smooth muscle. We tested the effect of PKG overexpression on the neointimal response to vascular injury. Methods and Results- Infection of cultured rat aortic smooth muscle cells (RASMCs) with an adenoviral vector specifying a cGMP-independent, constitutively active PKG mutant (AdPKGcat) reduced serum-induced migration by 33% and increased serum-deprivation-induced apoptosis 2-fold (P<0.05 for both). Infection with wild-type PKG (AdPKG), in the absence of cGMP, did not affect migration or apoptosis. Two weeks after balloon-injured rat carotid arteries were infected with 1x 10(10) pfu AdPKGcat (n=12), AdPKG (n=8), or a control adenovirus (n=8), intima-to-media ratio was less in AdPKGcat-infected arteries than in AdPKG- or control adenovirus-infected vessels (0.26+/-0.06 versus 0.61+/-0.12 and 0.70+/-0.12, respectively, P<0.05 for both). Two weeks after intramural administration of 1.75x10(10) pfu AdPKGcat (n=8) or a control adenovirus (n=8) into porcine coronary arteries with in-stent restenosis, luminal diameter was greater in AdPKGcat-infected arteries than in control adenovirus-infected vessels (2.32+/-0.16 versus 1.81+/-0.13 mm, P=0.028), associated with reduced neointimal area (3.30+/-0.24 versus 4.15+/-0.13 mm(2), P=0.008), neointima-to-vessel area ratio (0.42+/-0.05 versus 0.58+/-0.04, P<0.05), and percent stenosis (45+/-6% versus 70+/-4%, P<0.05). Expression of a constitutively active PKG reduces neointima formation after balloon injury in rats and reduces coronary in-stent restenosis in pigs. PKGcat gene transfer may be a promising strategy for vasculoproliferative disorders.

Involvement of cyclic GMP and potassium channels in relaxation evoked by the nitric oxide donor, diethylamine NONOate, in the rat small isolated mesenteric artery.

The relative functional importance of potassium channels and cGMP-dependent pathways in the relaxation of vascular smooth muscle to the novel nitric oxide donor, diethylamine NONOate (DEA NONOate), was investigated in a resistance artery. The contribution from cGMP-dependent signalling pathways was examined by exposing arteries to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, while the contribution through potassium channels was assessed with different sub-type-selective potassium channel blockers. DEA NONOate (3 nM-10 microM) evoked sustained relaxation in isolated segments of the rat small mesenteric artery contracted with phenylephrine (pEC50=6.7+/-0.2; n=11). The relaxation was attenuated significantly by either ODQ (10 microM; pEC50=5.8+/-0.4; n=7) or charybdotoxin (ChTX; 50 nM; pEC50=6.3+/-0.2; n=4), a peptide blocker of large conductance, calcium-activated potassium channels (BK(Ca)). The inhibitory effects of ODQ and ChTX were additive (pEC50=5.1+/-0.4; n=9). The selective inhibitor of BK(Ca) channels, iberiotoxin (IbTX; 30 nM), and 4-aminopyridine (4-AP; 1 mM), an inhibitor of voltage-gated potassium channels (Kv), failed to modify DEA NONOate-evoked relaxation. However, in the combined presence of both ODQ and either IbTX or 4-AP the relaxation was attenuated significantly (n=3). The blocker of ATP-modulated potassium channels (K(ATP)), glibenclamide (10 microM), and of small conductance calcium-activated potassium channels (SK(Ca)), apamin (30 nM), each failed to affect ODQ-sensitive or -resistant relaxations to DEA NONOate (n=3). In conclusion, relaxation to DEA NONOate in the rat isolated, small mesenteric artery can occur via both cGMP-dependent (ODQ-sensitive) and -independent (ODQ-resistant) mechanisms. However, the contribution made to relaxation by potassium channels appears to be unmasked following pharmacological attenuation of cGMP-dependent signalling pathways. The inhibitory action of ChTX suggests part of the cGMP-insensitive component involves the activation of potassium channels, a suggestion supported by the inhibitory actions of 4-AP and IbTX in the absence of cGMP.

Identification of a conserved residue responsible for the autoinhibition of cGMP-dependent protein kinase Iα and β

We isolated a constitutively active form of cGMP-dependent protein kinase Iα (cGK Iα) by PCR-driven random mutagenesis. The replacement of Ile-63 by Thr in the autoinhibitory domain results in the enhancement of autophosphorylation and the basal kinase activity in the absence of cGMP. The hydrophobicity at position 63 is essential for the inactive state of cGK Iα, and Ile-78 of cGK Iβ is also required for the autoinhibitory property. Furthermore, cGK Iα (Ile-63-Thr) is constitutively active in vivo. These findings suggest that a conserved residue in the autoinhibitory domain was involved in the autoinhibition of both cGK Is.

Cyclic nucleotide-gated channels. Pore topology studied through the accessibility of reporter cysteines.

In voltage- and cyclic nucleotide-gated ion channels, the amino-acid loop that connects the S5 and S6 transmembrane domains, is a major component of the channel pore. It determines ion selectivity and participates in gating. In the alpha subunit of cyclic nucleotide-gated channels from bovine rod, the pore loop is formed by the residues R345-S371, here called R1-S27. These 24 residues were mutated one by one into a cysteine. Mutant channels were expressed in Xenopus laevis oocytes and currents were recorded from excised membrane patches. The accessibility of the substituted cysteines from both sides of the plasma membrane was tested with the thiol-specific reagents 2-aminoethyl methanethiosulfonate (MTSEA) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Residues V4C, T20C, and P22C were accessible to MTSET only from the external side of the plasma membrane, and to MTSEA from both sides of the plasma membrane. The effect of MTSEA applied to the inner side of T20C and P22C was prevented by adding 10 mM cysteine to the external side of the plasma membrane. W9C was accessible to MTSET from the internal side only. L7C residue was accessible to internal MTSET, but the inhibition was partial, approximately 50% when the MTS compound was applied in the absence of cGMP and 25% when it was applied in the presence of cGMP, suggesting that this residue is not located inside the pore lumen and that it changes its position during gating. Currents from T15C and T16C mutants were rapidly potentiated by intracellular MTSET. In T16C, a slower partial inhibition took place after the initial potentiation. Current from I17C progressively decayed in inside-out patches. The rundown was accelerated by inwardly applied MTSET. The accessibility results of MTSET indicate a well-defined topology of the channel pore in which residues between L7 and I17 are inwardly accessible, residue G18 and E19 form the narrowest section of the pore, and T20, P21, P22 and V4 are outwardly accessible.

CGMP potentiates receptor-stimulated Ca 2+ influx in Dictyostelium discoideum

Binding of extracellular cAMP to surface receptors induces at least two responses in Dictyostelium discoideum, the G-protein-dependent activation of guanylyl cyclase, and the opening of a plasma membrane Ca 2+ channel. Some experiments suggest that intracellular cGMP opens the Ca 2+ channel, while others demonstrate that the channel can open in the absence of functional G-proteins (and thus in the absence of cGMP formation). We have analysed 45Ca 2+ uptake in three mutants with altered cGMP formation. Mutant stmF shows a prolonged cGMP response due to deletion of an intracellular phosphodiesterase. Uptake of receptor-stimulated 45Ca 2+ is enhanced about two-fold in this mutant if compared to wild-type cells, suggesting that cGMP regulates the opening of the channel. Mutant KI-7 has very low levels of surface cAMP receptors, but nevertheless an enhanced receptor-stimulated cGMP response due to a defect in the turn-off of guanylyl cyclase. This mutant shows poor receptor-stimulated 45Ca 2+ uptake, suggesting that cGMP alone is not sufficient to open the Ca 2+ channel. Finally, mutant KI-8 has no cGMP due to the absence of nearly all guanylyl cyclase activity. The mutant shows significant but reduced 45Ca 2+ uptake (19% of wild-type; 60% if corrected for the reduced level of surface cAMP receptors), suggesting that the channel can open in the absence of cGMP. Taken together, the results demonstrate that receptor-stimulated Ca 2+ influx is not directly induced by cGMP formation; it can occur in the absence of cGMP, but is potentiated two- to four-fold by cGMP.