Identification of a novel calcium binding protein from bovine brain

A novel Ca²⁺ binding protein, named caligulin, was extracted from the heat-treated 100 000 × g supernatant of bovine brain and purified to electrophoretic homogeneity. The apparent M_r of caligulin determined on sodium dodecyl sulfate polyacrylamide gels was 24 000. Analysis by gel filtration chromatography indicated an apparent M_r of 33 000, suggesting a monomeric protein. Amino acid composition data demonstrated the presence of 25% acidic residues, 12% basic residues and 10% leucine. In the presence of 1 mM MgCl₂ and 0.15 M KCl, caligulin bound 1 mol Ca²⁺/mol protein with half-maximal binding at about 0.2 μM Ca²⁺.     

Blockers of Ca2+ channels in the plasmalemma of perfused Characeae cells

Ionic currents in the plasmalemma of perfused Nitella syncarpa cells identified as currents through Ca²⁺ channels were registered for the first time. The effect of 1,4-dihydropyridine derivatives (nifedipine, nitredipine, riodipine) and phenylalkylamines (verapamil, D600) as well as the agonist CGP-28392 on the Ca²⁺ channels in the plasmelemma of perfused cells of Nitellopsis obtusa and Nitella syncarpa have been studied. A blocking effect of 1,4-dihydropyridine derivatives and phenylalkylamines on the plasmalemma Ca²⁺ channels has been detected. Phenylalkylamines have been found to block both inward and outward Ca²⁺ currents. The activating effect of the agonist CGP-28392 on the Ca²⁺ channels of plasmalemma has been shown.     

The separation and characterization of two forms ofTorpedo electric organ calelectrin

Two methods for extracting calelectrin, a Ca²⁺-regulated membrane-binding protein from the electric organ ofTorpedo marmorata, have been compared and the more promising one was modified to increase the yield to 7–8 mg · kg⁻¹ wet weight of tissue, that is 4–5-times greater than the original method. The calelectrin so obtained could be resolved into a minor component (designated L-calelectrin) eluted from an anion-exchange column at relatively low ionic strength (100 mM NaCl) and a major component (H-calelectrin) eluted at higher ionic strength (300 mM NaCl). The two forms were also separated by chromatography on a hydrophobic resin. Electrophoresis on cellulose acetate indicated that L-calelectrin had a lower mean isoelectric point that the H-form and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that under reducing conditions (presence of 5% β-mercaptoethanol) both forms migrated as single species, the L-form having a lower apparent relative molecular mass (M_r 32 000) than the H-form (34 000). Under non-reducing conditions, there was no change in the migration of L-calelectrin but the H-form was resolved into two components of M_r 34 000 and 32 000. The addition of 2 mM Ca²⁺ had no effect on the migration of either form. Both forms were equally recognized by an anti-calelectrin antiserum and were microheterogeneous with respect to their isoelectric points (pH 4.3–5.5) in two-dimensional gel electrophoresis. Physical measurements were carried out on the major H-form. The Stokes radius was estimated to be 3 nm, corresponding to an apparent M_r of 44 000. It was unaffected by changes in ionic strength, pH or Ca²⁺ concentration. Analytical ultracentrifugation gave a sedimentation constant of 2.9 S and an apparent M_r of 36 000. Measurements of circular dichroism indicated that 78% of the molecule was in the α-helix configuration and 22% in random coil. Ca²⁺ had no significant effect on the conformation.     

Inhibition of the Ca2+-and phospholipid-dependent protein kinase by a novel Mr 17,000 Ca2+-binding protein

A novel M_r 17,000 Ca²⁺-binding protein isolated from bovine brain was found to be a potent inhibitor of the Ca²⁺- and phospholipid-dependent protein kinase (protein kinase C), also isolated from bovine brain. Halfmaximal inhibition by this calciprotein of the initial rate of phosphorylation of histone III-S by protein kinase C occurred at a calciprotein concentration of 2.2 μM under standard conditions. Comparison of the effects of a number of Ca²⁺-binding proteins on protein kinase C activity indicated that the M_r 17,000 Ca²⁺-binding protein was the most potent inhibitor, followed by the intestinal Ca²⁺-binding protein and calcineurin. Calmodulin, troponin C, S-100 protein and a M_r 21,000 Ca²⁺-binding protein of bovine brain were relatively weak inhibitors of protein kinase C. The inhibitory effect of the M_r 17,000 Ca²⁺-binding protein was apparently not due to its interaction with phospholipid or the basic protein substrate and therefore appears to be due to a direct effect on the protein kinase C. These observations suggest that the novel M_r 17,000 Ca²⁺-binding protein, and possibly other Ca²⁺-binding proteins, may play a physiological role in regulating the activity of protein kinase C.     

Purification and characterization of the inactive Ca2+, Mg2+-activated adenosine triphosphatase of the unc A- mutant Escherichia coli AN120.

Resealing of erythrocyte ghosts in the presence of 4.5 mm Ca²⁺ induces the segregation of small membrane vesicles with a very high phospholipid:protein ratio and a high lysolecithin content. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that the vesicles consist mainly of the high molecular spectrin peptides, the Ca²⁺-induced increase of band IIa (M_r 198,000) which is not extractable at low ionic strength, and a weak peptide band in the 72,000 M_r region. Ca²⁺ ghosts and vesicles show significant differences with regard to the specific activities of several membrane-associated enzymes. The segregated vesicles dispose of an efficient outwarddirected Ca²⁺-transport system.     

Immunolocalization of the 110,000 molecular weight cytoskeletal protein of intestinal microvilli

The core structures of microvilli from absorptive cells of the intestinal epithelium are primarily composed of calmodulin (M_r 16,000), actin (M_r 43,000), villin (M_r 95,000) and a protein of M_r 110,000. We have isolated this protein and raised antibodies against it. The antibodies interact specifically with villin and M_r 110,000 polypeptides present in isolated microvilli or brush borders. However, after absorption on an immobilized villin preparation, these antibodies still immunoprecipitate the M_r 110,000 protein but not villin. Thus, these two proteins appear to share some antigenic determinants but also contain other determinants specific for each protein. Immunolocalization studies have been performed using specific antibodies against the M_r 110,000 protein. Immunofluorescent studies on thin frozen sections of intestinal cells show that this protein is located in the brush border and at the basolateral faces of these polarized cells. Immunoferritin studies on rat brush borders demembranated with the detergent Triton X-100 show the association of the M_r 110,000 protein with core filaments of microvilli, as well as with some filaments localized in the terminal web network.Using sealed, right-side-out vesicles prepared from pig intestinal mucosa in the presence of Ca²⁺ and Mg²⁺, a polypeptide of M_r 140,000 was found to be a major component of the Triton X-100 insoluble pellet. This protein is a minor component of an equivalent pellet obtained from isolated microvilli prepared in the presence of EDTA. The significance of this M_r 140,000 polypeptide associated with the core residue of intestinal microvilli is discussed.     

D2O-induced ion channel activation in Characeae at low ionic strength

Effects of D₂O were studied on internodal cells of the freshwater alga Nitellopsis obtusa under plasmalemma perfusion (tonoplast-free cells) with voltage clamp, and on Ca²⁺ channels isolated from the alga and reconstituted in bilayer lipid membranes (BLM). External application of artificial pond water (APW) with D₂O as the solvent to the perfused plasmalemma preparation led to an abrupt drop of membrane resistance (R _m = 0.12 ±0.03 kΩ · cm²), thus preventing further voltage clamping. APW with 25% D₂O caused a two-step reduction of R _m : first, down to 2.0 ± 0.8 kΩ · cm², and then further to 200 Ω · cm², in 2 min. It was shown that in the first stage, Ca²⁺ channels are activated, and then, Ca²⁺ ions entering through them activate the Cl^? channels. The Ca²⁺ channels are activated irreversibly. If 100 mm CsCl was substituted for 200 mm sucrose (introduced for isoosmoticity), no effect of D₂O on R _m was observed. Intracellular H₂O/D₂O substitution also did not change R _m . In experiments on single Ca²⁺ channels in BLM H₂O/ D₂O substitution in a solution containing 100 mm KCl (trans side) produced no effect on channel activity, while in 10 mm KCl, at negative voltage, the open channel probability sharply increased. This effect was irreversible. The single channel conductance was not altered after the H₂O/D₂O substitution. The discussion of the possible mechanism of D₂O action on Ca²⁺ and Cl^? channels was based on an osmotic-like stress effect and the phenomenon of higher D-bond energy compared to the H-bond.     

Regulation of rat cardiac nuclei-associated Mg2+-NTPase by phosphorylation

A nucleoside triphosphatase (NTPase) activity appeared to be associated with a highly purified nuclear preparation from rat cardiac ventricles. Different nucleoside triphosphates (UTP > GTP > ITP > CTP) supported this enzymic activity, which was stimulated by Mg` but not by Call. The nuclear NTPase activity could be down regulated by endogenous phosphorylation of a 55,000 M_r protein. Maximal phosphorylation of the 55,000 M_r protein occurred in the presence of Mg²⁺-ATP. Addition of cAMP, cGMP, Ca²⁺, Ca²⁺/phospholipid, Ca²⁺/calmodulin, and catalytic subunit of cAMP-dependent protein kinase was not associated with any further phosphorylation of the 55,000 M_r protein. However, in the presence of Ca²⁺/calmodulin or the catalytic subunit of the cAMP-dependent protein kinase additional proteins became phosphorylated, but these had no effect on the Mg²⁺-NTPase activity. These results indicate that a protein with M_r 55,000 may be involved in the regulation the Mg²⁺-NTPase activity associated with rat cardiac nuclei.Abbreviations Hg Hemoglobin - GAR Goat Anti-Rabbit antibody - SR Sarcoplasmic Reticulum - NTP Nucleoside Triphosphate - TCA Trichloroacetic acid - PAGE Polyacrylamide gel electrophoresis     

Phospholipid-sensitive calcium-dependent protein kinase and its endogenous substrate proteins in rat pancreatic acinar cells

Phospholipid-sensitive Ca²⁺-dependent protein kinase and its endogenous substrate proteins were examined in acinar cells from rat pancreas. The enzyme was clearly demonstrable by DEAE-cellulose chromatography of acinar cell extract. At least four endogenous substrate proteins (M_r = 38K, 30K, 22K and 15K) for this Ca²⁺-activated kinase were found in the acinar cell extract. These substrate proteins were maximally phosphorylated in the combined presence of Ca²⁺ and phosphatidylserine. Calmodulin was partially effective as a cofactor for phosphorylation of the 38K substrate protein, but ineffective for the other three. A slight Ca²⁺/phospholipid-dependent phosphorylation of 38K and 30K proteins, but not of 22K and 15K proteins was seen in extract of isolated pancreatic islets. The K_a for Ca²⁺ for phosphorylation of the endogenous acinar cell proteins was decreased more than ten-fold in the combined presence of phosphatidylserine and unsaturated diacylglycerol. The presence of this Ca²⁺/phospholipid-dependent protein kinase/ protein phosphorylation system provides a potential mechanism of action for Ca²⁺ as a regulator of exocrine pancreatic function.     

Regulation of potential-dependent L-type Ca2+ currents by agmatine. Imidazoline receptors in isolated cardiomyocytes

The main goal of the present work was to study the mechanisms of voltage-gated L-type Ca²⁺ currents regulation by agmatine in isolated cardiomyocytes and to determine whether agmatine is involved in mediating the “arginine paradox”. It was shown that agmatine at concentrations from 200 μM to 15 mM inhibited L-type Ca²⁺ currents in isolated cardiomyocytes in a dose-dependent manner. The selective antagonists of α₂-adrenoceptors (α₂-ARs), yohimbine and rauwolscine, did not modulate the effect of agmatine. In contrast, efaroxan and idazoxan known to antagonize both α₂-ARs and type 1 imidazoline receptors (I₁Rs) decreased the efficiency of agmatine almost twofold. The NO synthase inhibitor 7NI insignificantly influenced the suppressive action of agmatine on L-type Ca²⁺ currents, whereas the protein kinase C inhibitor, calphostin C, markedly reduced the effects of agmatine. Arginine did not affect L-type Ca²⁺ currents in the presence of agmatine and vice versa. These data suggest that agmatine is not involved in mediating the “arginine paradox” and that its effects are not due to the activation of endothelial NO synthase (eNOS) followed by cGMP-dependent inhibition of L-type Ca²⁺ current. Most likely, agmatine acts via I₁Rs coupled with the signaling pathway that involves the activation of protein kinase C. Previously nothing was known about possible localization of I₁Rs in isolated cardiomyocytes. Consistently, we have shown that single cardiomyocytes express the nischarin genes homologous to the IRAS gene, which is considered in the modern literature as the major candidate for the gene encoding I₁Rs. To the best our knowledge, this is the first demonstration of I₁Rs expression at the level of individual cells, including cardiomyocytes.     

RyR2 Modulates a Ca2+-Activated K+ Current in Mouse Cardiac Myocytes

In cardiomyocytes, Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCCs) binds to and activates RyR2 channels, resulting in subsequent Ca²⁺ release from the sarcoplasmic reticulum (SR) and cardiac contraction. Previous research has documented the molecular coupling of small-conductance Ca²⁺-activated K⁺ channels (SK channels) to VDCCs in mouse cardiac muscle. Little is known regarding the role of RyRs-sensitive Ca²⁺ release in the SK channels in cardiac muscle. In this study, using whole-cell patch clamp techniques, we observed that a Ca²⁺-activated K+ current (I_K,Ca) recorded from isolated adult C57B/L mouse atrial myocytes was significantly decreased by ryanodine, an inhibitor of ryanodine receptor type 2 (RyR2), or by the co-application of ryanodine and thapsigargin, an inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA) (p<0.05, p<0.01, respectively). The activation of RyR2 by caffeine increased the I_K,Ca in the cardiac cells (p<0.05, p<0.01, respectively). We further analyzed the effect of RyR2 knockdown on I_K,Ca and Ca²⁺ in isolated adult mouse cardiomyocytes using a whole-cell patch clamp technique and confocal imaging. RyR2 knockdown in mouse atrial cells transduced with lentivirus-mediated small hairpin interference RNA (shRNA) exhibited a significant decrease in I_K,Ca (p<0.05) and [Ca²⁺]i fluorescence intensity (p<0.01). An immunoprecipitated complex of SK2 and RyR2 was identified in native cardiac tissue by co-immunoprecipitation assays. Our findings indicate that RyR2-mediated Ca²⁺ release is responsible for the activation and modulation of SK channels in cardiac myocytes.     

The association of phosphorylase kinase with rabbit muscle T-tubules

Evidence is presented for the association of a phosphorylase kinase activity with transverse tubules as well as terminal cisternae in triads isolated from rabbit skeletal muscle. This activity remained associated with T-tubules throughout the purification of triad junctions by one cycle of dissociation and reassociation. The possibility that the presence of phosphorylase kinase in these highly purified membrane vesicle preparations was due to its association with glycogen was eliminated by digestion of the latter with α-amylase. The phosphorylase kinase activity associated with the T-tubule membranes was similar to that reported for other membrane-bound phosphorylase kinases. The enzyme had a high $\text{pH}\text{6.8}\text{pH}\text{8.2}$ activity ratio (0.4 – 0.7) and a high level of Ca²⁺ independent activity $\text{(EGTA}\text{Ca}{}^{\mathrm{2+}}\text{= 0.3?0.5)}$. The kinase activated and phosphorylated exogenous phosphorylase b with identical time courses. When mechanically disrupted triads were centrifuged on continuous sucrose gradients, the distribution of phosphorylase kinase activity was correlated with the distribution of a M_r 128,000 polypeptide in the gradients. This polypeptide and a M_r 143,000 polypeptide were labeled with ³²P by endogenous and exogenous protein kinases. These findings suggest that the membrane-associated phosphorylase kinase may be similar to the cytosolic enzyme. Markers employed for the isolated organelles included a M_r 102,000 membrane polypeptide which followed the distribution of Ca²⁺-stimulated 3-O-methylfluorescein phosphatase activity, which is specific for the sarcoplasmic reticulum. A M_r 72,000 polypeptide was confirmed to be a T-tubule-specific protein. Several proteins of the triad component organelle were phosphorylated by the endogenous kinase in a Ca²⁺/calmodulin-stimulated manner, including a M_r ca. 72,000 polypeptide found only in the transverse tubule.     

The relationship between single-channel and whole-cell conductance in the T-type Ca2+ channel CaV3.1

In T-type Ca²⁺ channels, macroscopic I_Ba is usually smaller than I_Ca, but at high Ca²⁺ and Ba²⁺, single-channel conductance (γ) is equal. We investigated γ as a function of divalent concentration and compared it to macroscopic currents using Ca_V3.1 channels studied under similar experimental conditions (TEA_o and K_i). Single-channel current-voltage relationships were nonlinear in a way similar to macroscopic open-channel I/Vs, so divalent γ was underestimated at depolarized voltages. To estimate divalent γ, concentration dependence, i_Div, was measured at voltages <−50 mV. Data were well described by Langmuir isotherms with γ_max(Ca²⁺) of 9.5 ± 0.4 pS and γ_max(Ba²⁺) of 10.3 ± 0.5 pS. Apparent K_M was lower for Ca²⁺ (2.3 ± 0.7 mM) than for Ba²⁺ (7.9 ± 1.3 mM). A subconductance state with an amplitude 70% that of the main state was observed, the relative occupancy of which increased with increasing Ca²⁺. As predicted by γ, macroscopic G_maxCa was larger than G_maxBa at 5 mM (G_maxCa²⁺/Ba:²⁺1.43 ± 0.14) and similar at 60 mM (G_maxCa²⁺/Ba:²⁺1.10 ± 0.02). However, over the range of activation, I_Ca was larger than I_Ba under both conditions. This was a consequence of the fact that V_rev was more negative for I_Ba than for I_Ca, so that the driving force determining I_Ba was smaller than that determining I_Ca over the range of potentials in standard current-voltage relationships.     

Low voltage-activated Ca2+ conductances in thalamic neurons

Low voltage-activated (LVA) Ca²⁺ conductances were characterized in the neurons of the associative laterodorsal (LD) thalamic nucleus in rat brain slices and in enzymatically isolated thalamic units using electrophysiological techniques. Voltage dependence, kinetics of inactivation, pharmacology, and selectivity of the LVA current in the thalamic neurons from animals older than 14 postnatal days were consistent with the existence of two, “fast” and “slow,” subtypes of LVA Ca²⁺ channels. “Slow” LVA current in enzymatically isolated thalamic neurons was much less prominent, compared with that in slice neurons, suggesting that respective channels are predominatly located on the distal dendrites. “Fast” Ca²⁺ channels were sensitive to nifedipine (K _d−2.6 μM) and La³⁺ (K _d−1.0 mM), whereas “slow” Ca²⁺ channels were sensitive to Ni²⁺ (25 μM). Selectivity of the “fast” Ca²⁺ channels was similar to that found for the LVA Ca²⁺ channels in other preparations (I _Ca:I _Sr:I _Ba−1.0: 1.23: 0.94), while selectivity of the “slow” Ca²⁺ channels more resembled selectivity of the HVA Ca²⁺ channels (I _Ca:I _Sr:I _Ba−1.0: 2.5: 3.4).     

Developmental changes in the endogenous Ca2+-stimulated proteolysis of mouse lung cAMP-dependent protein kinases

Regulatory subunits (R subunits) of mouse lung cAMP-dependent protein kinases undergo age-dependent changes in endogenous proteolysis, with the greatest amount of the major M_r = 37,000 proteolytic fragment detectable during fetal and neonatal development. Homogenization of lung in the presence of various protease inhibitors does not affect this age-related difference, suggesting that the observed quantitative change in R subunit proteolysis occurs in vivo. Mechanisms were sought to account for this age-dependent change. The production of a M_r = 37,000 proteolytic fragment can be stimulated in lung extracts by the addition of exogenous calcium and is due to the action of an endogenous Ca²⁺-stimulated protease. Neonatal lung extracts show more Ca²⁺-stimulated proteolysis of R subunits than adult extracts, although only slight agerelated differences in either the Ca²⁺-stimulated protease or its specific endogenous inhibitor were observed. Age-dependent differences in R subunits which may affect sensitivity to proteases were also examined. Analysis of the two-dimensional patterns of adult and neonatal 8-N₃-[³²P]cAMP-labeled R subunits before or after limited proteolysis with trypsin suggests that the R subunits are structurally similar. Differences are found, however, in the relative proportions of adult and neonatal Type I R subunits (R_I) in the holoenzyme or dissociated forms. An increased proportion of neonatal R subunits exist in the dissociated state, whereas adult R subunits exist primarily in the holoenzyme form. Dissociated R subunits from mouse lung are more susceptible than the holoenzyme to limited proteolysis by the partially purified lung Ca²⁺-stimulated protease. Dissociation of the holoenzyme in vivo may be a major factor in the age-dependent proteolytic changes observed in mouse lung protein kinases.     

Studies on the structure of the calcium-dependent adenosine triphosphatase from rabbit skeletal muscle sarcoplasmic reticulum

The linear arrangement of the three fragments of Ca²⁺-ATPase from rabbit skeletal muscle sarcoplasmic reticulum with molecular weights of 20,000, 30,000, and 45,000 obtained by limited tryptic hydrolysis was determined by locating the NH₂-terminal acetylated methionyl residue of the original peptide in the M_r = 20,000 fragment. Since both the M_r = 20,000 and 30,000 polypeptides originate from a M_r = 55,000 fragment which is distinct from the M_r = 45,000 polypeptide, the sequence of these three fragments was determined to be 20,000, 30,000, and 45,000. The M_r = 20,000 fragment was further cleaved by cyanogen bromide to yield a M_r = 7,000 COOH-terminal fragment which is relatively hydrophilic. The NH₂-terminal portion is rich in glutamyl residues. The COOH-terminus of the M_r = 30,000 fragment was determined by both digestion with carboxypeptidases and cyanogen bromide cleavage. Using the partial amino acid sequence of the Ca²⁺-ATPase, it was deduced that the active site phosphoaspartyl residue is 154 amino acids from the COOH-terminus of the M_r = 30,000 fragment and hence approximately 35,000 M_r from the NH₂-terminus of the original Ca²⁺-ATPase molecule. Furthermore, it was shown that the two tryptic cleavages of the Ca²⁺-ATPase generating these three large fragments were both single hydrolyses of arginylalanine peptide bonds.     

Perspective: The List of Potential Volume-sensitive Chloride Currents Continues to Swell (and Shrink)

The channel of the glutamate N-methyl-d-aspartate receptor (NMDAR) transports Ca²⁺ approximately four times more efficiently than that of Ca²⁺-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPAR). To investigate the basis of this difference in these glutamate receptors (GluRs), we measured the ratio of Cs⁺ efflux and Ca²⁺ influx in recombinant NMDAR and Ca²⁺-permeable AMPAR channels expressed in human embryonic kidney 293 (HEK 293) cells over a wide voltage range. At any one potential, this biionic flux ratio was measured by quantifying the total charge and the charge carried by Ca²⁺ using whole-cell currents and fluorometric techniques (dye overload) with Cs⁺ internally and Ca²⁺ externally (1.8 or 10 mM) as the only permeant ions. In AMPAR channels, composed of either GluR-A(Q) or GluR-B(Q) subunits, the biionic flux ratio had a biionic flux-ratio exponent of 1, consistent with the prediction of the Goldman-Hodgkin-Katz current equation. In contrast, for NMDAR channels composed of NR1 and NR2A subunits, the biionic flux-ratio exponent was ∼2, indicating a deviation from Goldman-Hodgkin-Katz. Consistent with these results, in NMDAR channels under biionic conditions with high external Ca²⁺ and Cs⁺ as the reference ions, Ca²⁺ permeability (P_Ca/P_Cs) was concentration dependent, being highest around physiological concentrations (1–1.8 mM; P_Ca/P_Cs ≈ 6.1) and reduced at both higher (110 mM; P_Ca/P_Cs ≈ 2.6) and lower (0.18 mM; P_Ca/P_Cs ≈ 2.2) concentrations. P_Ca/P_Cs in AMPAR channels was not concentration dependent, being around 1.65 in 0.3–110 mM Ca²⁺. In AMPAR and NMDAR channels, the Q/R/N site is a critical determinant of Ca²⁺ permeability. However, mutant AMPAR channels, which had an asparagine substituted at the Q/R site, also showed a biionic flux-ratio exponent of 1 and concentration-independent permeability ratios, indicating that the difference in Ca²⁺ transport is not due to the amino acid residue located at the Q/R/N site. We suggest that the difference in Ca²⁺ transport properties between the glutamate receptor subtypes reflects that the pore of NMDAR channels has multiple sites for Ca²⁺, whereas that of AMPAR channels only a single site.     

Effect of Introducing a Lactone Group into Typhasterol and Teasterone to Promote Rice Lamina Inclination

Protein phosphorylation may be required for plant cell response to phytohormones and other extracellular signals. Protein phosphorylation and protein kinase activity in the culm of heading time of rice (Oryza sativa L.) were studied. Before heading, protein kinase activity was increased by Ca²⁺ in the membrane fraction of the panicle and culm. The protein kinases with M_r of 51,900, 49,200, and 45,500 isolated from the membrane fraction of culm increased the protein phosphorylation of M_r and pI of 40,000/7.5 and 40,000/7.6 in the culm extract. The activation of protein kinases, associated with membrane and subsequent protein phosphorylation, thus appears to be involved in the regulation of heading time in rice.     

Nonselective Suppression of Voltage-gated Currents by Odorants in the Newt Olfactory Receptor Cells

Effects of odorants on voltage-gated ionic channels were investigated in isolated newt olfactory receptor cells by using the whole cell version of the patch–clamp technique. Under voltage clamp, membrane depolarization to voltages between −90 mV and +40 mV from a holding potential (V_h) of −100 mV generated time- and voltage-dependent current responses; a rapidly (< 15 ms) decaying initial inward current and a late outward current. When odorants (1 mM amyl acetate, 1 mM acetophenone, and 1 mM limonene) were applied to the recorded cell, the voltage-gated currents were significantly reduced. The dose-suppression relations of amyl acetate for individual current components (Na⁺ current: I_Na, T-type Ca²⁺ current: I_Ca,T, L-type Ca²⁺ current: I_Ca,L, delayed rectifier K⁺ current: I_Kv and Ca²⁺-activated K⁺ current: I_K(Ca)) could be fitted by the Hill equation. Half-blocking concentrations for each current were 0.11 mM (I_Na), 0.15 mM (I_Ca,T), 0.14 mM (I_Ca,L), 1.7 mM (I_Kv), and 0.17 mM (I_K(Ca)), and Hill coefficient was 1.4 (I_Na), 1.0 (I_Ca,T), 1.1 (I_Ca,L), 1.0 (I_Kv), and 1.1 (I_K(Ca)), suggesting that the inward current is affected more strongly than the outward current. The activation curve of I_Na was not changed significantly by amyl acetate, while the inactivation curve was shifted to negative voltages; half-activation voltages were −53 mV at control, −66 mV at 0.01 mM, and −84 mV at 0.1 mM. These phenomena are similar to the suppressive effects of local anesthetics (lidocaine and benzocaine) on I_Na in various preparations, suggesting that both types of suppression are caused by the same mechanism. The nonselective blockage of ionic channels observed here is consistent with the previous notion that the suppression of the transduction current by odorants is due to the direst blockage of transduction channels.     

Adenosine Triphosphate Activates a Noninactivating K+ Current in Adrenal Cortical Cells through Nonhydrolytic Binding

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K⁺ current (I_AC) that is inhibited by adrenocorticotropic hormone and angiotensin II at subnanomolar concentrations. Since I_AC appears to set the membrane potential of AZF cells, these channels may function critically in coupling peptide receptors to membrane depolarization, Ca²⁺ entry, and cortisol secretion. I_AC channel activity may be tightly linked to the metabolic state of the cell. In whole cell patch clamp recordings, MgATP applied intracellularly through the patch electrode at concentrations above 1 mM dramatically enhanced the expression of I_AC K⁺ current. The maximum I_AC current density varied from a low of 8.45 ± 2.74 pA/pF (n = 17) to a high of 109.2 ± 26.3 pA/pF (n = 6) at pipette MgATP concentrations of 0.1 and 10 mM, respectively. In the presence of 5 mM MgATP, I_AC K⁺ channels were tonically active over a wide range of membrane potentials, and voltage-dependent open probability increased by only ∼30% between −40 and +40 mV. ATP (5 mM) in the absence of Mg²⁺ and the nonhydrolyzable ATP analog AMP-PNP (5 mM) were also effective at enhancing the expression of I_AC, from a control value of 3.7 ± 0.1 pA/pF (n = 3) to maximum values of 48.5 ± 9.8 pA/pF (n = 11) and 67.3 ± 23.2 pA/pF (n = 6), respectively. At the single channel level, the unitary I_AC current amplitude did not vary with the ATP concentration or substitution with AMP-PNP. In addition to ATP and AMP-PNP, a number of other nucleotides including GTP, UTP, GDP, and UDP all increased the outwardly rectifying I_AC current with an apparent order of effectiveness: MgATP > ATP = AMP-PNP > GTP = UTP > ADP >> GDP > AMP and ATP-γ-S. Although ATP, GTP, and UTP all enhanced I_AC amplitude with similar effectiveness, inhibition of I_AC by ACTH (200 pM) occurred only in the presence of ATP. As little as 50 μM MgATP restored complete inhibition of I_AC, which had been activated by 5 mM UTP. Although the opening of I_AC channels may require only ATP binding, its inhibition by ACTH appears to involve a mechanism other than hydrolysis of this nucleotide. These findings describe a novel form of K⁺ channel modulation by which I_AC channels are activated through the nonhydrolytic binding of ATP. Because they are activated rather than inhibited by ATP binding, I_AC K⁺ channels may represent a distinctive new variety of K⁺ channel. The combined features of I_AC channels that allow it to sense and respond to changing ATP levels and to set the resting potential of AZF cells, suggest a mechanism where membrane potential, Ca²⁺ entry, and cortisol secretion could be tightly coupled to the metabolic state of the cell through the activity of I_AC K⁺ channels.