Absence Of Free Ca2 Research Articles

Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers.

The voltage-dependent gating of single, batrachotoxin-activated Na channels from rat brain was studied in planar lipid bilayers composed of negatively charged or neutral phospholipids. The relationship between the probability of finding the Na channel in the open state and the membrane potential (Po vs. Vm) was determined in symmetrical NaCl, both in the absence of free Ca2+ and after the addition of Ca2+ to the extracellular side of the channel, the intracellular side, or both. In the absence of Ca2+, neither the midpoint (V0.5) of the Po vs. Vm relation, nor the steepness of the gating curve, was affected by the charge on the bilayer lipid. The addition of 7.5 mM Ca2+ to the external side caused a depolarizing shift in V0.5. This depolarizing shift was approximately 17 mV in neutral bilayers and approximately 25 mV in negatively charged bilayers. The addition of the same concentration of Ca2+ to only the intracellular side caused hyperpolarizing shifts in V0.5 of approximately 7 mV (neutral bilayers) and approximately 14 mV (negatively charged bilayers). The symmetrical addition of Ca2+ caused a small depolarizing shift in Po vs. Vm. We conclude that: (a) the Na channel protein possesses negatively charged groups on both its inner and outer surfaces. Charges on both surfaces affect channel gating but those on the outer surface exert a stronger influence. (b) Negative surface charges on the membrane phospholipid are close enough to the channel's gating machinery to substantially affect its operation. Charges on the inner and outer surfaces of the membrane lipid affect gating symmetrically. (c) Effects on steady-state Na channel activation are consistent with a simple superposition of contributions to the local electrostatic potential from charges on the channel protein and the membrane lipid.

Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein.

Neuromodulin (formerly designated P-57) is an abundant, neural specific, calmodulin-binding protein which exhibits higher affinity for calmodulin in the absence of free Ca2+ than in the presence of free Ca2+. In this study a series of proteolytic fragments of neuromodulin were systematically screened for calmodulin-Sepharose binding activity. A 9-amino acid fragment, designated M1-C1 and having the sequence RGHITRKKL, was identified as the putative CaM-binding domain of neuromodulin. Two heptadecapeptides, designated FP57-Phe and FP57-Trp, were synthesized, each containing the M1-C1 sequence and the four flanking amino acids from each site. The FP57-Trp peptide contained a tryptophan residue in place of the native phenylalanine. Anti-FP57-Phe antibody binding to neuromodulin was inhibited by preincubation of antibodies with excess FP57-Phe. 125I-CaM gel overlay of neuromodulin was inhibited by anti-FP57-Phe antibodies. Addition of CaM to FP57-Trp increased peptide tryptophanyl fluorescence. In the presence of Ca2+, the stoichiometry of the FP57-Trp.CaM complex was 1:1, FP57-Trp binding to CaM was competitive with neuromodulin. The Ca2+-independent dissociation constant of the FP57-Phe.CaM complex was 0.41 microM. The Ca2+-dependent affinity of the complex could not be measured directly but appeared to be significantly greater than the Ca2+-independent affinity.

The interaction of Ca2+ with the calmodulin-sensitive adenylate cyclase from Bordetella pertussis.

Bordetella pertussis, the etiologic agent of whooping cough, produces a calmodulin-sensitive adenylate cyclase which elevates intracellular cAMP in a variety of eucaryotic cells. Exogenous calmodulin added to the partially purified adenylate cyclase has been shown to inhibit invasion of animal cells by this enzyme (Shattuck, R. L., and Storm, D. R. (1985) Biochemistry 24, 6323-6328). In this study, several properties of the calmodulin-sensitive adenylate cyclase are shown to be influenced by Ca2+ in the absence of calmodulin. The presence or absence of Ca2+ during QAE-Sephadex ion exchange chromatography produced two distinct chromatographic patterns of adenylate cyclase activity. Two different forms of the enzyme (Pk1 and Pk2EGTA) were isolated by this procedure. Pk1 adenylate cyclase readily elevated intracellular cAMP levels in mouse neuroblastoma cells (N1E-115) while Pk2EGTA adenylate cyclase had no effect on cAMP levels in these cells. Gel exclusion chromatography of Pk1 adenylate cyclase gave apparent Stokes radii (RS) of 43.5 A (+/- 1.3) in the presence of 2 mM CaCl2 and 33.8 A (+/- 0.94) in the presence of 2 mM EGTA [( ethylenebis (oxyethylenenitrilo)]tetraacetic acid). These Stokes radii are consistent with molecular weights of 104,000 (+/- 6,400) and 61,000 (+/- 3,600), respectively. Pk2EGTA adenylate cyclase had an apparent RS of 33.0 (+/- 1.2) (Mr = 60,600 (+/- 2,800] in the presence of Ca2+ or excess EGTA. At 60 degrees C, Pk1 adenylate cyclase exhibited a Ca2+-dependent heat stability with a half-life for loss of enzyme activity of 10.3 min in 5 mM CaCl2 and a half-life of 2.8 min in the presence of 0.1 microM CaCl2. The stability of Pk2EGTA adenylate cyclase was not affected by changes in free Ca2+. The adenylate cyclase preparations described above were submitted to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and enzyme activity was recovered from gel slices by extraction with detergent containing buffers. The catalytic subunit isolated from SDS-polyacrylamide gels was activated 7-fold in the presence of Ca2+ with maximum activity observed at 1 microM free Ca2+. With both preparations, the apparent molecular weight of the catalytic subunit on SDS gels was 51,000 in the presence of 2 mM CaCl2 and 45,000 in the presence of 2 mM EGTA. The catalytic subunit of the enzyme was purified to apparent homogeneity by preparative SDS-polyacrylamide gel electrophoresis and resubmitted to SDS gel electrophoresis in the presence or absence of free Ca2+. The purified catalytic subunit also exhibited a Ca2+-dependent shift in its mobility on SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein.

P-57 is a neural-specific calmodulin binding protein with novel calmodulin binding properties. P-57 exhibits higher affinity for calmodulin-Sepharose in the absence of free Ca2+ than in the presence of Ca2+ (Andreasen, T.J., Luetje, C.W., Heideman, W. & Storm, D.R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T.J., Andreasen, K.I. & Storm, D.R. (1985) J. Biol. Chem. 260, 10784-10788). In this study, the dissociation constants for P-57 and immunopurified 5-[[(iodoacetylamino)ethyl]-amino]-1-naphthalenesulfonic acid-labeled calmodulin (AEDANS-CaM) were determined under low and high ionic strength conditions. In the absence of added KCl, the dissociation constants for the P-57 X AEDANS-CaM complex were 2.3 X 10(-7) +/- 6 X 10(-8) M and 1.0 X 10(-6) +/- 3 X 10(-7) M in the presence and absence of excess Ca2+ chelator. The addition of KCl to 150 mM increased the Ca2+-independent and -dependent dissociation constants to 3.4 X 10(-6) +/- 9 X 10(-7) M and 3.0 X 10(-6) +/- 9 X 10(-7) M, respectively. The association of P-57 with AEDANS-CaM under low Ca2+ conditions was determined as a function of KCl concentrations. By taking into account the amount of P-57 found in brain and its affinity for calmodulin, it is concluded that most or all of the CaM would be complexed to P-57 in unstimulated cells. P-57 was phosphorylated by the Ca2+-phospholipid-dependent protein kinase (protein kinase C) with a phosphate:protein molar ratio of 1.3. Phosphoamino acid analysis demonstrated phosphorylation at a serine residue. CaM decreased the rate of phosphorylation of P-57 by protein kinase C, and phosphorylation prevented P-57 binding to calmodulin-Sepharose. P-57 was not phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase. It is proposed that P-57 binds and localizes calmodulin at specific sites within the cell and that free calmodulin is released locally in response to phosphorylation of P-57 by protein kinase C and/or to increases in intracellular free Ca2+. This regulatory mechanism, which appears to be specific to brain, would serve to decrease the response time for Ca2+-calmodulin-regulated processes.

P-57 is a neural specific calmodulin-binding protein.

P-57 is a novel calmodulin-binding protein which has recently been isolated from bovine cerebral cortex (Andreasen, T. J., Luetje, C. W., Heideman, W., and Storm, D. R. (1983) Biochemistry 22, 4615-4618). In contrast to all other calmodulin-binding proteins characterized thus far, P-57 has equivalent or higher affinity for calmodulin in the absence of free Ca2+ compared to the presence of Ca2+. In this study, the distribution of P-57 in other tissues and within brain was examined using a radioimmune assay and photoaffinity labeling with azido-125I-calmodulin. P-57 was not found in tissues other than brain, retina, and spinal cord. Within brain, P-57 levels varied from 0.1% of the total protein in white matter regions to about 0.5% in cell body-rich fractions. The protein was found in both membrane and soluble fractions. P-57 is the most abundant calmodulin-binding protein in brain and appears to be neural specific. The concentrations of P-57 in brain and its affinity for calmodulin in the absence of Ca2+ are sufficient to complex a significant fraction of the total calmodulin present.

Protein phosphorylation in permeabilized pancreatic islet cells.

A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmodulin-dependent protein phosphorylation further and to determine whether activation of this membrane-bound process was sufficient for initiation of Ca2+-stimulated insulin secretion. The efficacy of digitonin in permeabilizing the plasma membrane was assessed by Trypan Blue exclusion, by extracellular leakage of lactate dehydrogenase, and by permeability to [gamma-32P]ATP. This treatment did not detectably alter the ultrastructure of the permeabilized cells. Digitonin was equally effective when presented to islet cells that had been previously dispersed or directly to intact isolated islets. The Ca2+- and calmodulin-dependent phosphorylation of endogenous membrane-bound substrates could be demonstrated in the permeabilized cells incubated with [gamma-32P]ATP. This activity displayed characteristics that were similar to those described for the protein kinase measured in subcellular fractions and was dependent on addition of exogenous calmodulin, indicating that calmodulin had been removed from the kinase by permeabilization of the cells. Ca2+-dependent insulin release by the digitonin-permeabilized islet was demonstrated, with half-maximal release occurring at 0.1 microM-free Ca2+ and maximal secretion at 0.2 microM-free Ca2+. Under these conditions, calmodulin did not further enhance insulin release, although a stimulatory effect of calmodulin was observed in the absence of free Ca2+. These studies indicate that the permeabilized-islet model will be useful in dissecting out the factors involved in Ca2+-activated insulin secretion.

Calcium-independent stimulation of Bordetella pertussis adenylate cyclase by calmodulin.

Bordetella pertussis produces an extracellular adenylate cyclase activity that is present in the culture medium of exponentially growing cells. We have determined that calmodulin (CaM) stimulates this enzyme both in the presence and in the absence of free Ca2+. In the presence of 90 micron Ca2+ half-maximal stimulation of the enzyme occurred at 95 pM calmodulin. Comparable levels of calmodulin stimulation were observed when free Ca2+ levels were minimized by using EGTA-containing buffers. However, the concentration of CaM required for half-maximal stimulation of B. pertussis adenylate cyclase in the presence of 1 nM free Ca2+ was 24 nM. The apparent affinity of the enzyme for calmodulin was also significantly enhanced by Mn2+. In addition, troponin I inhibited calmodulin stimulation of the bacterial adenylate cyclase. Photoaffinity cross-linking experiments using azido[125I]calmodulin and B. pertussis adenylate cyclase revealed only one major cross-linked product having a molecular weight of 97000. It is proposed that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is 77000.

Extraction and some properties of the proteins, Spastin B, from the spasmoneme of Carchesium polypinum.

Proteins of the contractile spasmoneme from Carchesium polypinum were extracted in 2% SDS, 30% acetic acid, or 8 M urea. The proteins extracted in SDS had a wide molecular weight distribution when examined by SDS-polyacrylamide gel electrophoresis. On the other hand, the proteins extracted in urea and acetic acid had three major peaks with molecular weights of about 16,000, 18,000, and 22,000. Most of these proteins were soluble even in the absence of urea and furthermore were found to be monomeric, since the sedimentation coefficient, s20,w, measured by analytical ultracentrifugation was 2.0S. The electrophoretic mobility of the proteins extracted in urea or in acetic acid was examined on alkaline gels. In the presence of free Ca2+, the mobility was significantly reduced compared with that in the absence of free Ca2+. These Ca-binding proteins were heat-stable and could not interact with troponin I. The implications of these proteins and others in relation to the contractility of the spasmoneme in Carchesium stalk are discussed.

Protein from macrophages inhibiting polymerization of actin.

The crude cytoplasmic extracts of macrophages showed a low actin-polymerization activity as compared with the purified actin preparations. This suggested that an actin-polymerization inhibitor should exist in the crude cytoplasmic extracts of macrophages. Thus, the isolation of actin-polymerization inhibiting protein from the crude extract was attempted. The protein with a high actin-polymerization inhibiting activity had a molecular weight of 65,000 daltons as determined by polyacrylamide gel electrophoresis in dodecyl sulfate, a Stokes radius of 33.8 A as measured by gel filtration and a sedimentation coefficient of 4.9 S as determined by analytical centrifugation. The actin-polymerization inhibiting activity was much higher in 0.1 M KCl than in 0.6 M KCl. It was not affected by either the presence or the absence of free Ca2+. It was considered that the protein is responsible for the large proportion of unpolymerized actin in the crude extract of macrophages.

Release of Ca2+ from intracellular pools stops cytoplasmic streaming inTradescantia staminal hairs

Ionophore A 23187 reversibly stops cytoplasmic streaming inTradescantia staminal hairs. Cessation of streaming occurs even in the absence of free Ca2+ ions in the medium.

Myofilament-generated tension oscillations during partial calcium activation and activation dependence of the sarcomere length-tension relation of skinned cardiac cells.

During partial Ca2+ activation, skinned cardiac cells with sarcoplasmic reticulum destroyed by detergent developed spontaneous tension oscillations consisting of cycles (0.1-1 Hz) of rapid decrease of tension corresponding to the yield of some sarcomeres and slow redevelopment of tension corresponding to the reshortening of these sarcomeres. Such myofilament-generated tension oscillations were never observed during the full activation induced by a saturating [free Ca2+] or during the rigor tension induced by decreasing [MgATP] in the absence of free Ca2+ or when the mean sarcomere length (SL) of the preparation was greater than 3.10 microm during partial Ca2+ activation. A stiff parallel elastic element borne by a structure that could be digested by elastase hindered the study of the SL--active tension diagram in 8-13-microm-wide skinned cells from the rat ventricle, but this study was possible in 2-7-microm-wide myofibril bundles from the frog or dog ventricle. During rigor the tension decreased linearly when SL was increased from 2.35 to 3.80 microm. During full Ca2+ activation the tension decreased by less than 20% when SL was increased from 2.35 to approximately 3.10 microm. During partial Ca2+ activation the tension increased when SL was increased from 2.35 to 3.00 microm. From this observation of an apparent increase in the sensitivity of the myofilaments to Ca2+ induced by increasing SL during partial Ca2+ activation, a model was proposed that describes the tension oscillations and permits the derivation of the maximal velocity of shortening (Vmax). Vmax was increased by increasing [free Ca2+] or decreasing [free Mg2+] but not by increasing SL.

Induction of platelet aggregation by the complement-derived peptides C3a and C5a.

The effect of two complement-derived peptides, hog serum C3a and C5a, on platelet aggregation in platelet-rich plasma and suspensions in Tyrode solution was investigated. 1. Guinea-pig platelets were aggregated by both C3a and C5a; the spasmogenically inactive product of C3a, C3ai, also induced aggregation. Threshold concentrations were in the range of 10(-6)--10(-9) M depending on the peptide and platelet preparation. 2. Cat platelets were aggregated by C5a (threshold concentrations 10(-7)--10(-8) M) but not by C3a. 3. Platelets from pig, rabbit and man were not aggregated by either of the two peptides in concentrations of up to 5 X 10(-6) M. 4. When C5a was administered repeatedly in subthreshold doses guinea-pig platelets became tachyphylactic to C5a but were still aggregable by C3a or ADP. Conversely, platelets desensitized to C3a still reacted to C5a or ADP. Tachyphylaxis towards C5a developed also when platelets were incubated with C5a in the absence of free Ca2+ under which condition they do not react. The tachyphylaxis in this case became evident after recalcification of the medium. The lack of cross-desensitization indicates that C3a and C5a react via different receptors. 5. C3a and C5a were injected i.v. into guinea pigs. Histological examination of the lungs revealed that some of the smaller vessels (20-30 mu in diameter) were occluded by platelet aggregates. In addition signs of severe acute emphysema were seen in animals treated with C5a, but only slight emphysema in C3a-treated animals. Intravenous injections of C3a into guinea pigs caused but weak respiratory distress and drowsiness and never killed an animal (at doses of up to 20 mg per kg body weight), whereas C5a caused the well-known severe respiratory failure and death already at doses of 0.03 mg/kg body weight.