Modulation by L- and D-isoforms of amino acids of the L-glutamate response of N-methyl-D-aspartate receptors.

N-Methyl-D-aspartate (NMDA) receptor subtypes epsilon 1 and zeta 1 were coexpressed in Xenopus oocytes for the investigation of the magnitude of augmentation of the L-glutamate response by 20 common L-amino acids and their 19 D-isoforms. Simultaneous application of L- and D-alanine, -cysteine, and -serine, or glycine and L-glutamate potentiated the glutamate-induced current. Other amino acids produced only marginal effects. Analysis of the relationship between the response and amino acid size revealed that the critical threshold size is between those of cysteine and aspartate. No amino acid alone induced a current. The effects of L- and D-alanine, -cysteine, and -serine applied with L-glutamate were concentration-dependent. Molecular modeling of these three amino acids revealed a positive relationship between the charge at an atom of the side chain and the receptor sensitivity, which may explain the efficacies of these amino acids.

Localization of GABA receptor rho 2 and rho 3 subunits in rat brain and functional expression of homooligomeric rho 3 receptors and heterooligomeric rho 2 rho 3 receptors.

Ionotropic GABA receptors that are composed of rho subunits act to gate bicuculline-insensitive Cl- currents. Reverse transcription-polymerase chain reaction analysis revealed that the expression of rho 2 mRNA in adult rat brain was approximately eight times higher than mRNA in the rat brain at embryonic day 16, while that of rho 3 in the embryonic brain was approximately six times higher than in the adult brain. In the adult rat brain the rho 3 mRNA was present in the mesencephalon, hippocampus, cerebellum, thalamus and basal ganglia. In situ hybridization has been used to demonstrate the presence of rho 2 mRNA in the hippocampal CA1 region of the 8-day-old rat, and in the CAl region of the hippocampus, lateral geniculate nucleus, superficial gray layer of the superior colliculus and the pars compacta of the substantia nigra of the adult rat. When the homooligomeric rho 3 receptors were expressed in Xenopus oocytes, applications of agonists induced ionic currents. The order of potency of the agonists was muscimol > GABA = trans-4-amino-crotonic acid > cis-4-aminocrotonic acid. The ionic currents induced by GABA were blocked by picrotoxinin and Zn2+ in dose-dependent manner. In heterooligomeric rho 2 rho 3 receptors, picrotoxinin sensitivity was significantly reduced.

Localization of gamma-aminobutyric acid (GABA) receptor rho 3 subunit in rat retina.

We used digoxigenin-labelled single strand DNA probes to examine the expression of the mRNA encoding gamma-aminobutyric acid receptor rho 3 subunit in sections of the adult rat retina. Transcript for the rho 3 subunit was found in cell somata of a portion of cells lying in the ganglion cell layer.

Functional expression of GABA rho 3 receptors in Xenopus oocytes.

Homomeric rat GABA rho 3 receptors were expressed in Xenopus oocytes, and their pharmacological profile was investigated electrophysiologically. GABA activated the rho 3 receptors with an EC50 value of 7.5 microM and a Hill coefficient of 1.6. The GABA-induced current was not antagonized by bicuculline (100 microM), but was blocked by picrotoxin (IC50: 0.68 microM for 100 microM GABA). The current was almost insensitive to pentobarbital, diazepam and a neurosteroid, 3 alpha-OH-DHP. Many of the pharmacological properties of the rho 3 subunit were similar to those of the previously reported rat rho 1 and rho 2 subunits and GABAC receptors.

Cloning of a putative gamma-aminobutyric acid (GABA) receptor subunit rho 3 cDNA.

Cloned cDNA encoding a putative member of GABA receptor rho-subunit class was isolated from rat-retina-mRNA-derived libraries. The cDNA encodes a signal peptide of 21 amino acids followed by the mature rho 3 subunit sequence of 443 amino acids. The proposed amino acid sequence exhibits 63 and 61% homology to the previously-reported human rho 1 and rat rho 2 sequences, respectively. Northern blot analysis demonstrated the expression of mRNA for rho 3 subunit in retina.

Identification of GABAA receptor subunits in rat retina: cloning of the rat GABAA receptor rho 2-subunit cDNA.

We identified GABAA receptor subunits in rat retina using PCR. The high degree of conservation among previously described members of ligand-gated anion channels in transmembrane domains was used to design degenerate sense and antisense oligonucleotides. These oligonucleotides were used as primers for PCR, which was applied to the rat retina cDNA. Analysis of clones derived from the PCR amplification identified the GABAA alpha 1, beta 1, beta 3, and gamma 2 subunits and the glycine alpha 1 subunit. In addition, two clones closely related to the human GABAA rho-subunit class were obtained. Molecular cloning revealed one of them as the rat counterpart of the human rho 2 subunit. Northern blot analysis demonstrated the expression of mRNAs for rho subunits in retina. These results further support the hypothesis that bicuculline-insensitive GABA channels in rat retina are comprised of rho subunits.

Modulation of ionic currents through GABAA receptor subtypes by endogenous steroids.

GABAA receptors were expressed in Xenopus oocytes by injecting messenger RNAs from chick retina and chick cortex, and the potency of 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP) and 5 alpha-pregnane-3 alpha, 21-diol-20-one (THDOC) was investigated by electrophysiology under voltage clamp conditions. For the receptors of chick retina, 3 alpha-OH-DHP (100 nM) and THDOC (100 nM) augmented GABA actions (peak current) 1.6 times (n = 6) and 1.5 times (n = 7), respectively. The currents induced by 0.3-10 microM GABA for the receptors of the chick retina were sensitive to bicuculline, pentobarbital, and diazepam. GABAA receptors in the chick retina in vivo as well as the receptors in the brain may be functionally modulated by endogenous steroids.

Altered agonist-induced Ca2+ mobilization in aortic smooth muscle cells from cardiomyopathic hamsters.

We compared the responses to the Ca(2+)-mobilizing effects of norepinephrine (NE), endothelin-1 (ET), and arginine vasopressin (AVP) of primary cultures of vascular smooth muscle cells (VSMCs) from aortas of young (30-40 days old) cardiomyopathic (Bio 14.6 strain) and age-matched control hamsters. In Bio 14.6 VSMCs, saturating concentrations of NE and ET elicited increases in the intracellular Ca2+ concentration ([Ca2+]i), which reached approximately 50 and 62% of those in control VSMCs. AVP elicited a significant increase in [Ca2+]i only in Bio 14.6 VSMCs. In contrast, 10 microM ionomycin induced similar Ca2+ responses in both types of cells in the absence of extracellular Ca2+. Ligand binding assay revealed that maximal binding values for alpha 1-adrenergic antagonists and ET in Bio 14.6 VSMCs were 59-64 and 85% of those in control VSMCs. Thus VSMCs from aortas of Bio 14.6 hamsters had reduced reactivity to NE and ET but increased response to AVP. The reduced Ca2+ response of Bio 14.6 VSMCs to NE appeared to be due mainly to a decrease in the receptor number.

Functional characterization of lanthanide binding sites in the sarcoplasmic reticulum Ca(2+)-ATPase: do lanthanide ions bind to the calcium transport site?

Gd3+ binding sites on the purified Ca(2+)-ATPase of sarcoplasmic reticulum were characterized at 2 and 6 degrees C and pH 7.0 under conditions in which 45Ca2+ and 54Mn2+ specifically labeled the calcium transport site and the catalytic site of the enzyme, respectively. We detected several classes of Gd3+ binding sites that affected enzyme function: (a) Gd3+ exchanged with 54Mn2+ of the 54MnATP complex bound at the catalytic site. This permitted slow phosphorylation of the enzyme when two Ca2+ ions were bound at the transport site. The Gd3+ ion bound at the catalytic site inhibited decomposition of the ADP-sensitive phosphoenzyme. (b) High-affinity binding of Gd3+ to site(s) distinct from both the transport site and the catalytic site inhibited the decomposition of the ADP-sensitive phosphoenzyme. (c) Gd3+ enhanced 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence in NBD-modified enzyme by probably binding to the Mg2+ site that is distinct from both the transport site and the catalytic site. (d) Gd3+ inhibited high-affinity binding of 45Ca2+ to the transport site not by directly competing with Ca2+ for the transport site but by occupying site(s) other than the transport site. This conclusion was based mainly on the result of kinetic analysis of displacement of the enzyme-bound 45Ca2+ ions by Gd3+ and vice versa, and the inability of Gd3+ to phosphorylate the enzyme under conditions in which GdATP served as a substrate. These results strongly suggest that Ln3+ ions cannot be used as probes to structurally and functionally characterize the calcium transport site on the Ca(2+)-ATPase.

Activation of sarcoplasmic reticulum Ca(2+)-ATPase by Mn2+: a Mn2+ binding study.

We used Mn2+ as an analogue for Mg2+ to examine the minimum requirement of divalent cations for the rapid turnover of the sarcoplasmic reticulum ATPase. We measured the binding of Ca2+ and Mn2+ to the purified Ca(2+)-ATPase during steady-state hydrolysis of MnATP at 2 degrees C and pH 7. In the presence of 20 microM Ca2+, Mn2+ was as effective as Mg2+ in stimulating ATPase activity and the maximal activation of ATP hydrolysis was observed at 0.1 mM MnCl2. Under these conditions, 2 mol of Ca2+ were bound per mol of the ADP-sensitive phosphoenzyme, whereas no Ca2+ was bound to the ADP-insensitive phosphoenzyme. On the other hand, the stoichiometry for ATP-dependent binding of Mn2+ to these intermediates was about 1. We found that Mn2+ remained bound to the ADP-insensitive phosphoenzyme even in the presence of added chelator. In the absence of ATP, we detected a low level of Mn2+ binding, which reached 0.4 mol per mol of the phosphorylation site at 0.1 mM free Mn2+. We present evidence that this extra Mn2+ binding did not affect the rate of decomposition of the ADP-sensitive phosphoenzyme, which was the rate-limiting step for ATP hydrolysis under the conditions used.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation.

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.

Participation of H+ in the Ca2(+)-induced conformational transition of 4-nitro-2,1,3-benzoxadiazole-labeled sarcoplasmic reticulum ATPase.

The binding of Ca2+ to 4-nitro-2,1,3-benzoxadiazole (NBD)-labeled sarcoplasmic reticulum Ca2(+)-ATPase was accelerated markedly when the pH was changed at 11 degrees C from 6.5 to 8.0 at the time of Ca2+ addition. We examined the effect of pH on the enzyme conformational transition by measuring the kinetics of NBD fluorescence rises induced by a pH jump under various ligand conditions. The fast fluorescence rise following a pH jump from 6.0 or 6.5 to various test pHs in the presence and absence of Ca2+ proceeded monoexponentially. The amplitude of this fluorescence rise in the presence of Ca2+ was independent of the test pH, whereas the observed rate constant (kobs) increased markedly as the test pH increased. In contrast, the amplitude of the fast fluorescence rise in the absence of Ca2+ increased with increasing test pH, whereas kobs decreased. MgATP or Mg2+ influenced the pH dependences of these parameters in a complex way except for the amplitudes measured in the presence of Ca2+. These data could be simulated by using a reaction model in which Ca2+ binding is preceded by a rate-limiting enzyme conformational transition from a low to a high NBD fluorescence state and 1 mol each of H+ is liberated before and after this conformational transition. MgATP or Mg2+ appeared to promote this conformational transition by enhancing deprotonation of the enzyme. These results suggest that deprotonation may be the primary event in the activation of the unphosphorylated enzyme by Ca2+.

Protein kinase C-dependent phosphorylation of sarcolemmal Ca2(+)-ATPase isolated from bovine aortic smooth muscle.

We examined the effect of protein kinase C (PKC)-dependent phosphorylation on Ca2+ uptake and ATP hydrolysis by microsomal as well as purified sarcolemmal Ca2(+)-ATPase preparations isolated from bovine aortic smooth muscle. The phosphorylation was performed by treating these preparations with PKC and saturating concentrations of ATP (or ATP-gamma S), Ca2+, and 12-O-tetradecanoyl phorbol-13-acetate (TPA) at 37 degrees C for 10 min. In microsomes, treatment with PKC enhanced a portion of the Ca2+ uptake activity inhibitable by 10 microM vanadate, by up to about 30%. On the other hand, Ca2(+)-dependent ATPase activity in the purified Ca2(+)-ATPase preparation was stimulated by up to twofold. Up to twofold stimulation by PKC was also observed for the Ca2+ uptake by proteoliposomes reconstituted from purified sarcolemmal Ca2(+)-ATPase and phospholipids. Since these effects were evident only at Ca2+ concentrations between 0.1 to 1.0 microM, we concluded that it was the affinity of the Ca2(+)-ATPase for Ca2+ that was increased by the PKC treatment. Under conditions in which PKC increased Ca2+ pump activity, the sarcolemmal Ca2(+)-ATPase was phosphorylated to a level of about 1 mol per mol of the enzyme. There was good parallelism between the ATPase phosphorylation and the extent of enzyme activation. These results strongly suggest that the activity of the sarcolemmal Ca2+ pump in vascular smooth muscle is regulated through its direct phosphorylation by PKC.

Protein kinase-dependent phosphorylation of cardiac sarcolemmal Ca2(+)-ATPase, as studied with a specific monoclonal antibody.

Phosphorylation of the Ca2(+)-pump ATPase of cardiac sarcolemmal vesicles by exogenously added protein kinases was examined to elucidate the molecular basis for its regulation. The Ca2(+)-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca2(+)-ATPase. Protein kinase C (C-kinase) was found to phosphorylate the Ca2(+)-ATPase. The stoichiometry of this phosphorylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca2+ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca2+ pump for Ca2+ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C kinase were also observed with a highly-purified Ca2(+)-ATPase preparation isolated from cardiac sarcolemma with calmodulin-Sepharose and a high salt-washing procedure. Thus, C-kinase appears to stimulate the activity of the sarcolemmal Ca2(+)-pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca2+/calmodulin-dependent protein kinase II phosphorylated the Ca2(+)-ATPase in the sarcolemmal membrane or the purified enzyme preparation, and also they exerted virtually no effect on Ca2+ uptake by sarcolemmal vesicles.

Guanosine triphosphate utilization by canine cardiac muscle sarcoplasmic reticulum.

We investigated the reaction mechanism for GTP-dependent Ca2+ uptake by canine cardiac microsomes enriched in fragmented sarcoplasmic reticulum (SR), because previous studies reported that GTP utilization in cardiac SR occurs via a pathway very different from that for ATP utilization (for a review, see "Entman, M.L., Bick, R., Chu, A., Van Winkle, W.B., & Tate, C.A. (1986) J. Mol. Cell. Cardiol. 18, 781-792"). In cardiac microsomes, we detected slow but distinct oxalate-dependent Ca2+ accumulation, which reached 550 nmol/mg protein in 10 min, and similarly slow Ca2+-dependent GTP hydrolysis. In 50 microM [gamma-32P]-GTP at 0 degrees C, we detected Ca2+-dependent formation of phosphoprotein whose level in the steady state was about a half of the maximum obtained with [gamma-32P]ATP. Kinetic properties of the phosphoprotein, its molecular weight and its chemical stability after the acid treatment are consistent with the conclusion that the phosphoprotein is an acylphosphate intermediate for Ca2+-dependent GTP hydrolysis catalyzed by the Ca2+-pump ATPase. Analysis of the kinetics of the turnover of phosphoprotein revealed that slow GTP hydrolysis is due to slow phosphoprotein formation; at 25 degrees C, the latter arises mainly from slow binding of Ca2+ to the dephosphorylated enzyme. These results indicate that, contrary to the previous data, the reaction pathway for GTP-dependent Ca2+ transport in cardiac SR is basically the same as that for ATP-dependent transport.

Mechanism for 3,3',4',5-tetrachlorosalicylanilide-induced activation of sarcoplasmic reticulum ATPase.

Effects of 3,3',4',5-tetrachlorosalicylanilide (TCS), a lipophilic weak acid, on Ca2+ uptake and ATP hydrolysis by the sarcoplasmic reticulum calcium pump were characterized to obtain insight into the possible role of hydrophobic portions of the Ca2+-ATPase in the catalytic mechanism of the enzyme. TCS exhibited both the stimulatory and inhibitory effects on the calcium pump activities depending on its concentration. At optimal concentrations, it increased these activities by up to 5-fold at pH 7.0 and 6 degrees C. Analysis of partial reactions of ATP hydrolysis by the purified ATPase revealed that TCS accelerated Ca2+ release from the ADP-sensitive phosphoenzyme up to 6-fold, whereas it affected other reaction steps to a much less extent, indicating that the site of the stimulatory action of TCS is rather specific in terms of the reaction sequence. These effects of TCS became less prominent at higher temperatures, although the enzyme-TCS interactions as detected in the direct binding experiment or by measurement of quenching of protein fluorescence were not affected by a similar change in temperature. The TCS effects were also dependent on pH of the 8.0 suggested that the protonated form of TCS is responsible for both the stimulatory and inhibitory effects of the drug. These results, taken together with those obtained previously with a spin-labeled probe (Barratt, M. D., and Weaver, A. C. (1979) Biochim. Biophys. Acta 555, 337-348), may suggest that TCS stimulates the calcium pump activity through its effect on the lipid bilayer, although its direct action on hydrophobic portion(s) of the ATPase protein cannot be ruled out.

Modulation of the hydrolysis rate of the ADP-insensitive phosphoenzyme of the sarcoplasmic reticulum ATPase by H+ and Mg2+.

Effects of H+ and Mg2+ on the hydrolysis rate of the ADP-insensitive phosphoenzyme intermediate (E2P) of the sarcoplasmic reticulum ATPase were investigated at 6 degrees C in the presence and absence of K+. In the absence of K+, the pH dependence of the E2P hydrolysis rate obtained in the absence of divalent cations showed a bell-shaped profile with an optimum at pH 9. At neutral pH, Mg2+ or other divalent cations accelerated the E2P hydrolysis while they strongly inhibited it at alkaline pH. The accelerating effect occurred on the cytoplasmic side of the membrane whereas the inhibitory effect occurred on the luminal side of the membrane, presumably at the low affinity calcium transport sites. The presence of Mg2+ or other divalent cations, therefore, shifted the pH activity profile to the acidic side while the magnitude of this shift and the activity obtained at the optimum pH depended on the species and the concentration of the divalent cation used. Simulation of a set of the pH activity curves obtained in 0 to 40 mM Mg2+ suggests that the marked activation of E2P hydrolysis by high Mg2+ observed at neutral pH is primarily caused by a Mg2+-induced increase in the dissociation constant of the ionizing group(s) rather than a markedly increased rate constant for E2P hydrolysis. In the presence of K+, the stimulatory effect of Mg2+ at pH 7 was less pronounced but its inhibitory effect at pH 9 was similar to that observed in the absence of K+. These effects of Mg2+ and other divalent cations should be taken into account when the role of H+ in the ATPase reaction is investigated.

Factors influencing calcium release from the ADP-sensitive phosphoenzyme intermediate of the sarcoplasmic reticulum ATPase.

Calcium release from the ADP-sensitive phosphoenzyme intermediate of the sarcoplasmic reticulum ATPase was investigated at 6 degrees C under a variety of conditions using the purified ATPase protein and the rapid membrane filtration system. The rate of calcium release measured in the presence of [ethylene bis-(oxyethylenenitrilo)]tetraacetic acid increased monotonically with increasing pH of the medium, the time at which 50% of the bound calcium was released being reduced to one third when the pH was raised from 5.5 to 9.0. Dimethyl sulfoxide at 10 or 20% (v/v) also was very effective in accelerating the calcium release. ATP at a millimolar concentration range also was stimulatory, but millimolar concentrations of Mg2+ were found to be inhibitory. Using an indirect method, i.e. by measuring the overall rate of calcium transport by the reconstituted vesicles under conditions where calcium release from the ADP-sensitive phosphoenzyme was presumably rate-limiting, the calcium release was shown to be accelerated up to 1.5-fold by the inside-negative potential imposed across the membrane using the K+-valinomycin system. As evidence was presented suggesting that the observed calcium release primarily reflects the phosphoenzyme isomerization which leads to reduction in calcium affinity of the phosphoenzyme, the results strongly suggest that this phosphoenzyme isomerization was affected significantly by each of the factors described above.

Absorption spectral properties of purified halorhodopsin.

Halorhodopsin in the membrane fragments of Halobacterium halobium Y1 showed an absorption band at 576 nm, the intensity of which decreased on irradiation with red light at 0 degrees C (Ogurusu, T., Maeda, A., Sasaki, N., & Yoshizawa, T. (1981) J. Biochem. 90, 1267-1273). Using this photobleachable property as the basis for an assay of halorhodopsin, we purified halorhodopsin by octyl-Sepharose column chromatography after extracting it from the membrane with Triton X-100. In NaDodSO4-polyacrylamide gel electrophoresis, hR appeared as a major band with an apparent molecular weight of 22,000, but the preparation still showed several other faint bands. The purified halorhodopsin showed a main absorption band at 576 nm and a small band at around 415 nm in 1 M NaCl. The photoreactions of the purified halorhodopsin at 0 degrees C and at -75 degrees C were similar to those of halorhodopsin in membrane fragments. Irradiation of the purified halorhodopsin with red light at 0 degrees C resulted in a decrease of absorbance at around 576 nm with a concomitant increase of absorbance at around 410 nm. A hypsochromic photoproduct was obtained on irradiation with 650 nm light at -75 degrees C. The dependency of the absorption spectrum of halorhodopsin on the concentration of chloride indicates that halorhodopsin has a single chloride binding site, occupation of which is responsible for modifying the spectrum.