Cation selectivity of and cation binding to the cGMP-dependent channel in bovine rod outer segment membranes

The properties of the cGMP-dependent channel present in membrane vesicles prepared from intact isolated bovine rod outer segments (ROS) were investigated with the optical probe neutral red. The binding of neutral red is sensitive to transport of cations across vesicular membranes by the effect of the translocated cations on the surface potential at the intravesicular membrane/water interface (Schnetkamp, P. P. M. J. Membr. Biol. 88: 249-262). Only 20-25% of ROS membrane vesicles exhibited cGMP-dependent cation fluxes. The cGMP-dependent channel in bovine ROS carried currents of alkali and earth alkali cations, but not of organic cations such as choline and tetramethylammonium; little discrimination among alkali cations (K greater than Na = Li greater than Cs) or among earth alkali cations (Ca greater than Mn greater than Sr greater than Ba = Mg) was observed. The cation dependence of cGMP-induced cation fluxes could be reasonably well described by a Michaelis-Menten equation with a dissociation constant for alkali cations of about 100 mM, and a dissociation constant for Ca2+ of 2 mM. cGMP-induced Na+ fluxes were blocked by Mg2+, but not by Ca2+, when the cations were applied to the cytoplasmic side of the channel. cGMP-dependent cation fluxes showed a sigmoidal dependence on the cGMP concentration with a Hill coefficient of 2.1 and a dissociation constant for cGMP of 92 microM. cGMP-induced cation fluxes showed two pharmacologically distinct components; one component was blocked by both tetracaine and L-cis diltiazem, whereas the other component was only blocked by tetracaine.     

Phosphorylation by cyclic GMP-dependent protein kinase of a synthetic peptide corresponding to the autophosphorylation site in the enzyme

The substrate specificity of cGMP-dependent protein kinase has been investigated by examining the ability of the enzyme to phosphorylate a series of synthetic peptides that correspond to the amino acid sequence at its site of autophosphorylation. The undecapeptide Ile53-Gly-Pro-Arg-Thr-Thr58-Arg-Ala-Gln-Gly-Ile63 which corresponds to the sequence around threonine-58 in cGMP-dependent protein kinase (Takio, K., Smith, S.B., Walsh, K.A., Krebs, E.G., and Titani, K. (1983) J. Biol. Chem. 258, 5531-5536) was synthesized and tested as a substrate for that enzyme. It was phosphorylated to the extent of 1.0 mol of phosphate/mol of peptide. Analysis of the products of Edman degradation of the phosphopeptide indicated that only threonine-58 was phosphorylated, as is the case for the autophosphorylation reaction in the native enzyme. The peptide was phosphorylated by cGMP-dependent protein kinase with a Km value of 578 +/- 25 microM and a Vmax of 0.069 +/- 0.003 mumol/min/mg of enzyme. This low Vmax value is consistent with the relatively slow rate of the autophosphorylation reaction. An analog peptide that contained serine in place of threonine-58 was also phosphorylated to 1.0 mol of phosphate/mol of peptide. That phosphopeptide contained only phosphoserine. The serine-containing analog peptide had a Km value similar to that of the parent peptide but was phosphorylated with a 70-fold higher Vmax value. Substitution of arginine-56 in the parent peptide by an alanine residue resulted in a peptide that was essentially not a substrate. Substitution of arginine-59, COOH-terminal to the phosphorylatable threonine, yielded a peptide with a Vmax similar to that of the parent peptide but a Km value of almost 22,000 microM. These results indicate that serine is a better phosphate-accepting residue than is threonine and that both arginine residues around the site of autophosphorylation are important specificity determinants for the cGMP-dependent protein kinase.     

Properties of multiple kinetic forms of soluble cyclic nucleotide phosphodiesterase activity of rat colonic mucosa

Soluble phosphodiesterase (EC 3.1.4.1) activity is 3-5-fold lower in superficial colonic epithelial cells compared to that in cells isolated from the lower colonic crypt. Higher phosphodiesterase activity in lower crypt cells is correlated with a 5-fold higher rate of incorporation of [3H]thymidine into DNA in these cells. DEAE-cellulose chromatography of the soluble fraction of superficial and proliferative colonic epithelial cells resulted in separation of three enzyme forms: (1) fraction I, an enzyme which hydrolyzes both cAMP and cGMP with high affinity (apparent Km cAMP = 5 +/- 1 microM, Km cGMP = 2.5 +/- 0.5 microM) and is stimulated 3-6-fold by Ca2+ plus calmodulin; (2) fraction II, a form which hydrolyzes both cAMP and cGMP with low affinity (S0.5 cAMP = 52 +/- 7 microM, S0.5 cGMP = 17 +/- 4 microM), exhibits positive copperativity with respect to substrate and shows cGMP stimulation of cAMP hydrolysis and (3) fraction III, a cAMP-specific form which exhibits biphasic kinetics, a low Km for cAMP (Km cAMP = 5 +/- 1 microM) and does not hydrolyze cGMP. The pattern of distribution of phosphodiesterase activities on DEAE-cellulose was similar in superficial and proliferative colonic epithelial cells. The higher specific activity in proliferative cells was reflected in higher activities of each of the three chromatographically distinct forms of the enzyme. In contrast to epithelial cells, the soluble fraction of homogenates of the submucosa and supporting cells exhibited phosphodiesterase forms I and II and was lacking in the form corresponding to fraction III of epithelial cells.     

Protein kinase C is involved in adrenergic stimulation of pineal cGMP accumulation

The amounts of cAMP and cGMP in the rat pinealocyte are regulated by norepinephrine acting through synergistic dual receptor mechanisms involving alpha 1- and beta-adrenoceptors (Vanecek, J., Sugden, D., Weller, J.L., and Klein, D.C. (1985) Endocrinology 116, 2167-2173; Sugden, L., Sugden, D., and Klein, D.C. (1986) J. Biol. Chem. 261, 11608-11612). Based on the available evidence, it appears that Ca2+-phospholipid-dependent protein kinase is involved in the alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cAMP, but not in the stimulation of cGMP (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W.B. (1985) Nature 314, 359-361). In the present study the role of protein kinase C in the adrenergic stimulation of cGMP was reinvestigated, with the purpose of determining whether protein kinase C activators would potentiate the effects of beta-adrenergic agonists on cGMP if cells were also treated with agents known to elevate intracellular free Ca2+. The protein kinase C activator 4 beta-phorbol 12-myristate 13-acetate (PMA) markedly elevated the cGMP content of beta-adrenergically stimulated pinealocytes which had also been treated with 1 microM A23187, 15 mM K+, or 1 microM ouabain. The effects of A23187 were blocked by EGTA and those of K+ were blocked by nifedipine, establishing the involvement of Ca2+. The stimulatory effects of PMA on cGMP accumulation were mimicked by other protein kinase C activators. PMA also stimulated cGMP accumulation in cells treated with cholera toxin (1 microgram/ml) and A23187 (1 microM), but not in cells treated only with cholera toxin. These results suggest that protein kinase C, which is activated in the pinealocyte by the alpha-adrenergic agonist phenylephrine, is probably involved in the adrenergic regulation of cGMP accumulation at a step distal to receptor activation.     

A catalytically inactive mutant of type I cGMP-dependent protein kinase prevents enhancement of large conductance, calcium-sensitive K+ channels by sodium nitroprusside and cGMP

The activation of large conductance, calcium-sensitive K(+) (BK(Ca)) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BK(Ca) channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BK(Ca) channel activity 4-5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the alpha isoform of type I cGMP-dependent protein kinase (cGKI). We observed that co-transfection of cells with BK(Ca) channels and a catalytically inactive ("dead") mutant of human cGKIalpha prevented enhancement of BK(Ca) channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKIalpha supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKIalpha were found to associate with BK(Ca) channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKIalpha was able to directly phosphorylate immunoprecipitated BK(Ca) channels, suggesting that cGKIalpha-dependent phosphorylation of BK(Ca) channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKIalpha alone is sufficient to promote the enhancement of BK(Ca) channels in situ after activation of the NO/cGMP signaling pathway.     

Characterization of a novel isozyme of cGMP-dependent protein kinase from bovine aorta

Two isozymic forms of cGMP-dependent protein kinase (designated types I alpha and I beta) were purified to homogeneity from bovine aorta smooth muscle. Type I alpha was apparently the same as the well characterized bovine lung cGMP-dependent protein kinase. Type I beta had a subunit Mr = 80,000 compared with Mr = 78,000 for type I alpha, and both forms were dimeric with similar calculated native Mr (170,000-178,000). Both enzymes contained two cGMP-binding sites per subunit, exhibited similar specificities for the peptide substrates tested, photoaffinity labeled with 8-N3[32P] cAMP, and catalyzed autophosphorylation. Silver-stained peptide maps of types I alpha and I beta were similar but not identical; however, autoradiographs of peptide maps of these enzymes prelabeled by either autophosphorylation or photoaffinity labeling showed clearly different patterns. The amino-terminal sequence of a breakdown product of type I beta could not be aligned confidently with any of the published sequence of bovine lung cGMP-dependent protein kinase. [3H]cGMP dissociation curves for types I alpha and I beta were both biphasic, but the dissociation rate of the slow component of type I beta was faster than the corresponding component of type I alpha. The concentration of cGMP required for half-maximal activation (K alpha) was slightly lower for type I alpha than for type I beta (0.29 and 0.44 microM, respectively), and the two enzymes had similar K alpha values for cAMP (16 and 18 microM, respectively). Types I alpha and I beta exhibited different K alpha values for several cGMP analogs. The abundance of type I beta in specific tissues suggested that it could have an important physiological role.     

Phosphorylation of phosphodiesterase-5 by cyclic nucleotide-dependent protein kinase alters its catalytic and allosteric cGMP-binding activities.

In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem. 265, 14971-14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50-70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2-0.5 microM PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the Km value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 microM. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed.     

Solubilization and functional reconstitution of the cGMP-dependent cation channel from bovine rod outer segments

The protein(s) that constitute(s) the cGMP-regulated channel in vertebrate photoreceptors has been solubilized from rod outer segment membranes and reincorporated into the membrane of calcium-containing liposomes. The properties of the reconstituted channel protein were determined by studying the cGMP-stimulated efflux of Ca2+ from these liposomes. Among several detergents tested the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) proved to be the most suitable. Solubilization of channel activity was found to be optimal at a detergent concentration of about 18 mM. The presence of Ca2+ ions and phospholipids during solubilization greatly increased the channel stability. The reconstituted channel shared most but not all properties with the channel in situ. It is cooperatively activated by cGMP with an EC50 of 19 microM. The cooperativity as determined from Hill plots was n = 2.7. Unlike the cGMP-sensitive channel in the native membrane of isolated discs and excised patches of plasma membrane it is not blocked by l-cis-diltiazem. Reconstitution of this channel protein(s) may serve as a valuable tool for identifying the polypeptide composition and to study structural and functional aspects of the purified protein(s).     

The amino terminus regulates binding to and activation of cGMP-dependent protein kinase

The allosteric regulation of binding to and the activation of cGMP-dependent protein kinase (cGMP kinase) was studied under identical conditions at 30 degrees C using three forms of cGMP-kinase which differed in the amino-terminal segment, e.g. native cGMP kinase, phosphorylated cGMP kinase which contained 1.4 +/- 0.4 mol phosphate/subunit and constitutively active cGMP kinase which lacked the amino-terminal dimerization domain. These three enzyme forms have identical kinetic constants, e.g. number of cGMP-binding sites, Km values for MgATP and the heptapeptide kemptide, and Vmax values. In the native enzyme, MgATP decreases the affinity for binding site 1. This effect is abolished by 1 M NaCl. In contrast, high concentrations of Kemptide increase the affinity of binding site 2 about fivefold. Under the latter conditions, identical Kd values of 0.2 microM were obtained for sites 1 and 2. Salt, MgATP and Kemptide do not affect the binding kinetics of the phosphorylated or the constitutively active enzyme, suggesting that allosteric regulation depends solely on the presence of a native amino-terminal segment. Cyclic GMP activates the native enzyme at Ka values which are identical with the Kd values for both binding sites. The activation of cGMP-dependent protein kinase is noncooperative but the Ka value depends on the substrate peptide concentration. These results show that the activity of cGMP kinase is primarily regulated by conformational changes within the amino-terminal domain.     

Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases

The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.     

Mode of action of iberiotoxin, a potent blocker of the large conductance Ca(2+)-activated K+ channel.

Iberiotoxin, a toxin purified from the scorpion Buthus tamulus is a 37 amino acid peptide having 68% homology with charybdotoxin. Charybdotoxin blocks large conductance Ca(2+)-activated K+ channels at nanomolar concentrations from the external side only (Miller, C., E. Moczydlowski, R. Latorre, and M. Phillips. 1985. Nature (Lond.). 313:316-318). Like charybdotoxin, iberiotoxin is only able to block the skeletal muscle membrane Ca(2+)-activated K+ channel incorporated into neutral-planar bilayers when applied to the external side. In the presence of iberiotoxin, channel activity is interrupted by quiescent periods that can last for several minutes. From single-channel records it was possible to determine that iberiotoxin binds to Ca(2+)-activate K+ channel in a bimolecular reaction. When the solution bathing the membrane are 300 mM K+ internal and 300 mM Na+ external the toxin second order association rate constant is 3.3 x 10(6) s-1 M-1 and the first order dissociation rate constant is 3.8 x 10(-3) s-1, yielding an apparent equilibrium dissociation constant of 1.16 nM. This constant is 10-fold lower than that of charybdotoxin, and the values for the rate constants showed above indicate that this is mainly due to the very low dissociation rate constant; mean blocked time approximately 5 min. The fact that tetraethylammonium competitively inhibits the iberiotoxin binding to the channel is a strong suggestion that this toxin binds to the channel external vestibule. Increasing the external K+ concentration makes the association rate constant to decrease with no effect on the dissociation reaction indicating that the surface charges located in the external channel vestibule play an important role in modulating toxin binding.     

Diastereomers of adenosine 3',5'-monothionophosphate (cAMP[S]) antagonize the activation of cGMP-dependent protein kinase

cGMP-dependent protein kinase contains four cGMP-binding sites which are homologous to the four cAMP-binding sites of cAMP-dependent protein kinase. The interaction of the diastereomers of adenosine 3',5'-thionophosphate, (PS)-cAMP[S] and (PR)-cAMP[S], with cGMP-dependent protein kinase has been studied. Autophosphorylation of cGMP-dependent protein kinase is stimulated by cAMP and (PS)-cAMP[S] with apparent KA values of 7 microM and 94 microM, respectively. cAMP-stimulated autophosphorylation is inhibited competitively by (PR)-cAMP[S] with a Ki value of 15 microM. The phosphorylation of the peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is stimulated by cGMP (approx. KA 1 microM) and cAMP (approx. KA 98 microM) but neither by the (PR) nor (PS) stereoisomer of cAMP[S]. (PR)-cAMP[S] and (PS)-cAMP[S] inhibit competitively cAMP-or cGMP-stimulated phosphorylation of the peptide substrate with Ki values of 52 microM and 73 microM, respectively. (PS)-cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme. Binding of [3H]cGMP to cGMP-dependent protein kinase is inhibited by (PS)-cAMP[S] and (PR)-cAMP[S] with IC50 values of 200 microM and 15 microM, respectively. These results show that both diastereomers of cAMP[S] bind to cGMP-dependent protein kinase. (PR)-cAMP[S] has properties of a pure antagonist whereas (PS)-cAMP[S] has properties of a partial agonist. The results provide further evidence that autophosphorylation of the enzyme affects the interaction between the cGMP-binding sites and the catalytic center of the enzyme by facilitating the activation of the phosphotransferase reaction.     

Glutamate modulates sodium-potassium-ATPase through cyclic GMP and cyclic GMP-dependent protein kinase in rat striatum

Excessive excitatory action of glutamate and nitric oxide (NO) has been implicated in degeneration of striatal neurons. Evidence had been provided that Na+K+-ATPase might be involved in this process. Here we investigated whether glutamate-regulated messengers, such as NO and cyclic GMP, could modulate the activity of membrane Na+K+-ATPase. Our results demonstrated that NO donors sodium nitroprusside (SNP at 30 and 300 microM) and S-nitroso-N-acetylpenicillamine (SNAP at 200 microM) increased alpha2,3Na+K+-ATPase activity which was blocked by the NO chelator, haemoglobin and was independent of [Na+]. This regulation was associated with cGMP synthesis and mimicked by glutamate (300 microM) and 8-Br-cyclic GMP (4 mM). 8-Br-cGMP-induced stimulation of Na+K+-ATPase activity could be blocked by KT5823 (an inhibitor of cGMP-dependent protein kinase, PKG), but not by KT5720 (an inhibitor of cAMP-dependent protein kinase, PKA). N-Methyl-D-aspartate (NMDA) receptors appeared to be involved in the effect of glutamate, since MK-801 (NMDA receptor antagonist) produced a partial reduction in glutamate-induced activation of the enzyme. MK-801 was not synergistic to L-NAME (NOS inhibitor), suggesting that glutamate stimulates the NMDA-NOS pathway to activate alpha2,3 Na+K+-ATPase in rat striatum. This regulation was associated with cyclic GMP (but not cyclic AMP) synthesis. These data indicate the existence, in vitro, of a regulatory pathway by which glutamate, acting through NO and cGMP, can cause alterations in striatal alpha2,3 Na+K+-ATPase activity.     

Inactivation of cGMP-dependent conductance of rod outer segment plasma membrane induced by cGMP.

Integral cGMP-dependent currents as well as activity of single cGMP-activated channels in plasma membrane of rod outer segment (ROS) were recorded using the patch-clamp method. The dependence of integral currents on cGMP concentration is shown to be bell-shaped. Decrease in cGMP-dependent conductance at high cGMP concentration results from a decrease in channel opening probability. Thus, cGMP in high concentrations inactivates cGMP-dependent conductance.     

Cyclic GMP-dependent stimulation of the membrane-bound insulin-sensitive cAMP phosphodiesterase from rat adipocytes

The insulin-sensitive cAMP phosphodiesterase (phosphodiesterase) in rat adipocytes is a membrane-bound low Km enzyme that can be recovered in a crude microsomal fraction (Fraction P-2). The action of this enzyme to hydrolyze cAMP is known to be inhibited by cGMP; nevertheless, it was found in our present study that under selected conditions, the enzyme can also be stimulated by cGMP as well as some other nucleotide derivatives. The maximum cGMP-dependent stimulation was observed when the enzyme in Fraction P-2 was incubated with 10 microM cGMP for 5-20 min at 37 degrees C in the presence of Mg2+, washed, and then assayed in the absence of added cGMP. The level of this stimulation was close to, but less than, that achieved by insulin in intact cells. The actions of the cGMP- and insulin-stimulated enzymes to hydrolyze labeled cAMP were inhibited in an identical manner by cilostamide (Ki = 0.10 microM), griseolic acid (Ki = 0.19 microM), unlabeled cAMP (Km = 0.20 microM), and cGMP (Ki = 0.16 microM), all added to the assay system. Also, the basal, insulin-stimulated, and cGMP-activated enzymes were identically inhibited by a polyclonal antibody raised against a purified membrane-bound low Km phosphodiesterase from bovine adipose tissue. When the same antibody was used for the Western blot analysis of Fraction P-2, it immunoreacted with a single band of protein (165 kDa). These observations indicate that the insulin-sensitive phosphodiesterase in rat adipocytes can be stimulated with 10 microM cGMP and that this stimulation is detectable only after the nucleotide has been eliminated since the enzyme would be strongly inhibited by the nucleotide if the latter exists in the assay system. It is proposed that the insulin-sensitive phosphodiesterase, which is often referred to as a Type IV enzyme, is functionally similar to the Type II enzymes that are known to be stimulated by a low concentration of cGMP and inhibited by higher concentrations of the same nucleotide.     

Sarcoballs: direct access to sarcoplasmic reticulum Ca2+-channels in skinned frog muscle fibers.

In skeletal muscle, twitch contraction is caused by the rapid release of Ca2+ from the sarcoplasmic reticulum (SR) (Endo, M. 1977. Physiol. Rev. 57:71-108) via Ca2+ conducting channels in the SR membrane (Smith, J. S., R. Coronado, and G. Meissner, 1985. Nature (Lond.). 316:446-449; Suarez-Isla, B. A., G. Orozco, P. F. Heller, and J. P. Froehlich. 1986. Proc. Natl. Acad. Sci. USA. 83:7741-7745). To facilitate study of these and other intracellular channels, we have developed a method which allows direct patch-clamp recording of currents through SR channels in native membrane. The Ca2+-release channel studied using this method exhibits two predominant conductance levels (80-100 pS and 120-160 pS), conducts Ca2+ preferentially over K+ (PCa/Pk = 6.5), is highly voltage sensitive, blocked on one side by ruthenium red (1 microM), and displays enhanced activity in the presence of caffeine (5 mM). Studied in skinned fibers, this channel appears fundamentally similar to homologous channels from isolated rabbit SR incorporated into bilayers, with some distinct differences.     

Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

An insulin-stimulated cation channel in skeletal muscle. Inhibition by calcium causes oscillation.

A cation channel has been identified in the plasma membrane of skeletal muscle that oscillates open and closed in a regular manner. In an experimental system of patch-clamped reconstituted plasma membrane in phospholipid bilayers, the oscillations are calcium-dependent and constitute regular closing events due to inhibition of the channel by calcium with a Ki of 2.2 +/- 1 x 10(-6) M, followed by reopening. There are 3.7 +/- 1 calcium binding sites/channel. With sodium as the current vehicle, conductance is increased by voltage, insulin (Km = 5 +/- 0.6 x 10(-9) M), and hydrolyzable guanine nucleotides. Cyclic GMP alone with increase the conductance with a Km of 3.7 +/- 0.6 x 10(-7) M. In the absence of calcium, the unitary conductance with insulin + GTP or cGMP at 150 mM NaCl is 153 picosiemens. Sodium current is insensitive to 10(-5) M tetrodotoxin but inhibited by mu-conotoxin (Ki = 5 x 10(-8) M). These findings in the reconstituted system were verified in patch-clamped whole muscle cells where an insulin and cGMP-dependent sodium current inhibited by mu-conotoxin could be demonstrated. In the whole cell experiments, slow calcium-dependent oscillations of the sodium current were also detected.     

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 alpha-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 microM 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 microM 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 alpha-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.     

Activation of a small-conductance Ca(2+)-dependent K+ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells.

Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.