Calmodulin-dependent cyclic nucleotide phosphodiesterase activity is altered by 20 microT magnetostatic fields

Absorbance measurements at 660 nm of calmodulin (CaM) dependent cyclic nucleotide phosphodiesterase activity under cell free conditions indicate that 30-min exposures to weak magnetostatic field intensities alters this activity, compared to zero magnetic field exposures. This effect depends nonlinearly on the concentration of free calcium, with maximum magnetic interaction apparently occurring at an optimal Ca(2+) concentration corresponding to 50% activation (EC(50)). If one regards Ca(2+)/CaM activation as a switching process, then increasing the magnetic field at Ca(2+) levels in excess of optimal acts to bias this switch towards lower calcium concentrations. A magnetic dependence has been previously reported by others in an homologous system, CaM dependent myosin light chain phosphorylation, implying that there may be an underlying magnetic interaction that involves the initial Ca(2+)/CaM binding process common to both enzymatic pathways. The level of magnetostatic intensity at which this effect is observed ( approximately 20 microT) implies that CaM activation may be functionally sensitive to the geomagnetic field.     

The effects of weak extremely low frequency magnetic fields on calcium/calmodulin interactions.

Mechanisms by which weak electromagnetic fields may affect biological systems are of current interest because of their potential health effects. Lednev has proposed an ion parametric resonance hypothesis (Lednev, 1991, Bioelectromagnetics, 12:71-75), which predicts that when the ac, frequency of a combined dc-ac magnetic field equals the cyclotron frequency of calcium, the affinity of calcium for calcium-binding proteins such as calmodulin will be markedly affected. The present study evaluated Lednev's theory using two independent systems, each sensitive to changes in the affinity of calcium for calmodulin. One of the systems used was the calcium/calmodulin-dependent activation of myosin light chain kinase, a system similar to that previously used by Lednev. The other system monitored optical changes in the binding of a fluorescent peptide to the calcium/calmodulin complex. Each system was exposed to a 20.9 microT static field superimposed on a 20.9 microT sinusoidal field over a narrow frequency range centered at 16 Hz, the cyclotron frequency of the unhydrated calcium ion. In contrast to Lednev's predictions, no significant effect of combined dc-ac magnetic fields on calcium/calmodulin interactions was indicated in either experimental system.     

Effects of 50 Hz magnetic fields on fMLP-induced shape changes in invertebrate immunocytes: The role of calcium ion channels

Plasma membrane Ca(2+) channels in immunocytes from the mussel Mytilus galloprovincialis exposed to 50 Hz sine wave magnetic fields (MFs) of various strengths were studied. At levels of 300 microT and above, MFs reduce shape changes in immunocytes induced by the chemotactic substance N-formyl-Meth-Leu-Phe, and this effect involves L-type Ca(2+) channels. Upon the addition of the Ca(2+) blocker verapamil to molluscan immunocytes exposed to MFs results in a synergistic cytotoxic action, while in the presence of the Ca(2+) opener SDZ-202, 791, a reactivation of the cells is observed. This suggests that, as previously reported for potassium channels, the damage to Ca(2+) channels induced by short exposure to MF at appropriate intensities is not permanent.     

ELF magnetic fields increase amino acid uptake into Vicia faba L. roots and alter ion movement across the plasma membrane

Vicia faba seedlings, subjected to a 10 microT 50 Hz square wave magnetic field for 40 min together with a radioactive pulse, showed a marked increase in amino acid uptake into intact roots. A more modest increase was observed with a 100 microT 50 Hz square wave. An increase in media conductivity at low field intensities from 10 microT 50 Hz square wave, 100 microT 50 Hz sine wave, and 100 microT 60 Hz square wave fields, indicated an alteration in the movement of ions across the plasma membrane, most likely due to an increase in net outflow of ions from the root cells. Similarly, marked elevation in media pH, indicating increased alkalinity, was observed at 10 and 100 microT for both square and sine waves at both 50 and 60 Hz. Our data would indicate that low magnetic field intensities of 10 and 100 microT at 50 or 60 Hz can alter membrane transport processes in root tips.     

Calcium uptake by leukemic and normal T-lymphocytes exposed to low frequency magnetic fields

Calcium-ion uptake by normal and leukemia lymphocytes increased during a 30-min exposure to a 13.6 Hz, sinusoidal magnetic field at 20 microT peak. The time-varying field was horizontal and parallel to a 16.5 microT component of the ambient static magnetic field. The uptake of 45Ca2+ increased 102% in a line of murine, cytotoxic T-lymphocytes (C57B1/6-derived CTLL-1), increased 126% in freshly-isolated spleen lymphocytes (C57B1/6 mice), and increased 75% in a line of lymphoma cells (C57B1/6-derived EL4). In contrast, there was no effect when the same field was applied for 30 min immediately before--as opposed to during--incorporation of calcium ions. When spleen lymphocytes were exposed during incubation with 45Ca2+ to a 60 Hz magnetic field at 20 microT peak, a small but statistically significant increase (37%) in uptake of the labeled ions occurred. These results indicate that weak, alternating magnetic fields might affect calcium-dependent functions of normal and leukemic lymphocytes.     

Calmodulin modulates initiation but not termination of spontaneous Ca2+ sparks in frog skeletal muscle

Calmodulin is a ubiquitous Ca(2+) sensing protein that binds to and modulates the sarcoplasmic reticulum Ca(2+) release channel, ryanodine receptor (RYR). Here we assessed the effects of calmodulin on the local Ca(2+) release properties of RYR in permeabilized frog skeletal muscle fibers. Fluorescently labeled recombinant calmodulin in the internal solution localized at the Z-line/triad region. Calmodulin (0.05-5.0 micro M) in the internal solution (free [Ca(2+)](i) approximately 50-100 nM) initiated a highly cooperative dose-dependent increase in Ca(2+) spark frequency, with a half-maximal activation (K) of 1.1 micro M, a Hill coefficient (n) of 4.2 and a fractional maximal increase in frequency (R) of 17-fold. A non-Ca(2+) binding mutant of calmodulin elicited a similar highly cooperative dose-dependent increase in spark frequency (K = 1.0 micro M; n = 3.7; R = 12-fold). Spatiotemporal properties of Ca(2+) sparks were essentially unaffected by either wild-type or mutant calmodulin. An N-terminal extension of calmodulin, (N+3)calmodulin, that binds to but does not activate RYR at nM [Ca(2+)] in sarcoplasmic reticulum vesicles, prevented the calmodulin-induced increase in spark frequency. These data suggest that exogenous Ca(2+)-free calmodulin cooperatively sensitizes the Ca(2+) release channel to open, but that Ca(2+) binding to the added calmodulin does not play a significant role in the termination of Ca(2+) sparks.     

Effect of treated with weak magnetic field aqueous salt solutions on the intrinsic fluorescence of bovine serum albumin. Isolation from solutions and partial characterization of the biologically active fluorescing fraction

It was shown that water with additions of Ca2+, Na+, K+ and Cl- ions preliminarily treated with weak combined constant (42 microT) and low-frequency alternating (0.06 microT) magnetic fields affects the intrinsic fluorescence of bovine serum albumin, the magnitude of the effect being dependent on the frequency of the alternating field and ionic composition of the aqueous salt solution. A practically complete transfer of the effect through a small portion of the solution treated with magnetic fields was revealed. It was also found that after magnetic treatment, the solution contains a rather large (molecular mass 700-900 D) and stable molecular associate, which possesses, at least partially, the properties and characteristics inherent in the whole solution that were as acquired as a result of magnetic treatment.     

Regulation of RYR1 activity by Ca(2+) and calmodulin

The skeletal muscle calcium release channel (RYR1) is a Ca(2+)-binding protein that is regulated by another Ca(2+)-binding protein, calmodulin. The functional consequences of calmodulin's interaction with RYR1 are dependent on Ca(2+) concentration. At nanomolar Ca(2+) concentrations, calmodulin is an activator, but at micromolar Ca(2+) concentrations, calmodulin is an inhibitor of RYR1. This raises the question of whether the Ca(2+)-dependent effects of calmodulin on RYR1 function are due to Ca(2+) binding to calmodulin, RYR1, or both. To distinguish the effects of Ca(2+) binding to calmodulin from those of Ca(2+) binding to RYR1, a mutant calmodulin that cannot bind Ca(2+) was used to evaluate the effects of Ca(2+)-free calmodulin on Ca(2+)-bound RYR1. We demonstrate that Ca(2+)-free calmodulin enhances the affinity of RYR1 for Ca(2+) while Ca(2+) binding to calmodulin converts calmodulin from an activator to an inhibitor. Furthermore, Ca(2+) binding to RYR1 enhances its affinity for both Ca(2+)-free and Ca(2+)-bound calmodulin.     

RC3/Neurogranin and Ca2+/calmodulin-dependent protein kinase II produce opposing effects on the affinity of calmodulin for calcium

The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca(2+) binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca(2+)-calmodulin should increase the affinity of calmodulin for Ca(2+), while proteins that bind to apo-calmodulin should decrease its affinity for Ca(2+). We quantified the effects on Ca(2+)-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca(2+)- and apo-calmodulin, and alphaCaM kinase II, which binds selectively to Ca(2+)-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca(2+), whereas CaM kinase II increases the calmodulin affinity for Ca(2+). Specifically, RC3 increases the rate of Ca(2+) dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca(2+) association. Conversely, CaM kinase II decreases the rates of dissociation of Ca(2+) from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr(286) induces a further decrease in the rates of Ca(2+) dissociation. RC3 dampens the effects of CaM kinase II on Ca(2+) dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca(2+)-calmodulin target complex. This work indicates that the Ca(2+) binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca(2+)-calmodulin target complexes at the end of a Ca(2+) signal.     

Evaluation of changes in diatom mobility after exposure to 16-Hz electromagnetic fields

The effect of a 16-Hz electromagnetic field on the mobility of the diatom Amphora coffeaformis was examined on agar plates that contained no added calcium and also on agar plates containing 0.25 or 2.5 mM exogenous Ca2+. Exposure conditions consisted of an ac field of 16 Hz with an amplitude of 20.9 microT parallel to the horizontal component of the dc field (BH = 20.9 microT, where BV = 0). To assess results, the percentage of diatoms that moved a distance greater than their body length was determined. We observed the field-associated increase in diatom motion at 0.25 mM Ca++, which was previously reported in the literature. Although the magnitude of the effect at 16 Hz was significant, the percentage of cells that moved was not sufficiently reproducible to allow examination for frequency dependence.     

Fluorescence studies on calmodulin binding to erythrocyte Ca2(+)-ATPase in different oligomerization states

The fluorescent spinach calmodulin derivative 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin (MIANS-CaM) was used to investigate calmodulin interaction with the purified, detergent-solubilized erythrocyte Ca2(+)-ATPase. Previous studies have shown that the Ca2(+)-ATPase exists in equilibria between monomeric and oligomeric forms. We report here that MIANS-CaM binds to both enzyme forms in a Ca2(+)-dependent manner, with a approximately 50% fluorescence enhancement. These findings confirm our previous observation that enzyme oligomers retain their ability to bind calmodulin, even though they are fully activated in the absence of calmodulin. The Ca2+ dependence of MIANS-CaM binding to monomeric Ca2(+)-ATPase is of higher affinity (K 1/2 = 0.09 microM Ca2+) and less cooperative (nH = 1.1) than the Ca2+ dependence of enzyme activation by MIANS-CaM (K 1/2 = 0.26 microM Ca2+, nH = 2.8). These Ca2+ dependences and the order of events, in which calmodulin binding precedes enzyme activation, demonstrate that calmodulin indeed could be a physiological activator of the monomeric enzyme. The calcium dependence of calmodulin binding to oligomeric Ca2(+)-ATPase occurs at even lower levels of Ca2+ (K 1/2 = 0.04 microM Ca2+), in a highly cooperative fashion (nH = 2.3), and essentially in parallel with enzyme activation (K 1/2 = 0.05 microM Ca2+, nH = 2.9). The observed differences between monomers and oligomers suggest that the oligomerized Ca2(+)-ATPase is in a conformation necessary for efficient, cooperative calcium binding at nanomolar Ca2+, which the monomeric enzyme acquires only upon interaction with calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium negatively modulates calmodulin interaction with IQGAP1

IQGAP1 regulates cytoskeletal dynamics through interactions with the Rho family GTPases Rac1 and Cdc42, F-actin, and beta-catenin. Calmodulin interaction with IQ motifs of IQGAP1 negatively influences these IQGAP1 interactions. Although, calmodulin interacts with IQGAP1 in the absence of Ca(2+) and was suggested to exhibit reduced binding when Ca(2+) bound, recent reports show substantially greater binding when Ca(2+) is present. Binding evaluations have primarily relied on IQGAP1 interaction with calmodulin conjugated to Sepharose 4B. In this study we evaluated the Ca(2+)-dependence of calmodulin interaction with native IQGAP1 using a series of independent biochemical approaches. We found the apparent binding of calmodulin to IQGAP1 was Ca(2+)-independent, being between 5- and 20-fold greater in the absence than in the presence of Ca(2+). In addition, calmodulin interaction with IQGAP1 was negatively regulated by buffer [Ca(2+)] (IC(50)=3.4x10(-7)M). Regulation was specific to Ca(2+), as Ba(2+) was approximately 400-fold less effective than Ca(2+) at modulating the interaction. Moreover, testing of calmodulin mutants demonstrated that apocalmodulin tightly binds IQGAP1 and that the N- and C-terminal pair of EF hands are important for Ca(2+) sensitivity. These data indicate that calmodulin may disassemble from IQGAP1 to facilitate IQGAP1 interaction with effectors of cytoskeletal reorganization during conditions of cell activation that promote increased cytosolic [Ca(2+)].     

Molecular mechanisms of calmodulin action on TRPV5 and modulation by parathyroid hormone

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry gate for active Ca(2+) reabsorption in the kidney. Ca(2+) influx through TRPV5 induces rapid channel inactivation, preventing excessive Ca(2+) influx. This inactivation is mediated by the last ～30 residues of the carboxy (C) terminus of the channel. Since the Ca(2+)-sensing protein calmodulin has been implicated in Ca(2+)-dependent regulation of several TRP channels, the potential role of calmodulin in TRPV5 function was investigated. High-resolution nuclear magnetic resonance (NMR) spectroscopy revealed a Ca(2+)-dependent interaction between calmodulin and a C-terminal fragment of TRPV5 (residues 696 to 729) in which one calmodulin binds two TRPV5 C termini. The TRPV5 residues involved in calmodulin binding were mutated to study the functional consequence of releasing calmodulin from the C terminus. The point mutants TRPV5-W702A and TRPV5-R706E, lacking calmodulin binding, displayed a strongly diminished Ca(2+)-dependent inactivation compared to wild-type TRPV5, as demonstrated by patch clamp analysis. Finally, parathyroid hormone (PTH) induced protein kinase A (PKA)-dependent phosphorylation of residue T709, which diminished calmodulin binding to TRPV5 and thereby enhanced channel open probability. The TRPV5-W702A mutant exhibited a significantly increased channel open probability and was not further stimulated by PTH. Thus, calmodulin negatively modulates TRPV5 activity, which is reversed by PTH-mediated channel phosphorylation.     

Investigation of AC-DC magnetic field effects in planar phospholipid bilayers.

Observations recently reported by others indicate that a combination of a weak dc magnetic field and extremely-low-frequency ac magnetic field can produce resonant effects in biological systems. We report measurements of the effects of combined dc and ac magnetic fields on the dc current through channel-free planar phospholipid membranes. The combined dc-ac magnetic fields did affect the dc current through planar phospholipid membranes, but not in every membrane, and not consistently at the same values of magnetic flux density and frequency. None of our measurements showed resonant response akin to the cyclotron-like resonance reported in diatoms [Smith et al., 1987] and lymphocytes [Liboff et al., 1987].     

The effect of static magnetic fields on the rate of calcium/calmodulin-dependent phosphorylation of myosin light chain

This study reports an attempt to confirm a published and well-defined biological effect of magnetic fields. The biological model investigated was the phosphorylation of myosin light chain in a cell free system. The rate of phosphorylation has been reported to be affected in an approximately linear manner by static magnetic field strengths in the range 0-200 microT. We performed three series of experiments, two to test the general hypothesis and a third that was a direct replication of published work. We found no effect of static magnetic field strength on the rate of phosphorylation. Hence, we were unable to confirm that weak static magnetic fields affect the binding of calcium to calmodulin. In view of the difficulty we and other authors have had making independent verifications of claimed biological effects of magnetic fields, we would urge caution in the interpretation of published data until they have been independently confirmed. There are still few well defined biological effects of low level magnetic fields that have been successfully transferred to an independent laboratory.     

The carboxy-terminal calcium binding sites of calmodulin control calmodulin's switch from an activator to an inhibitor of RYR1

Calcium and calmodulin both regulate the skeletal muscle calcium release channel, also known as the ryanodine receptor, RYR1. Ca(2+)-free calmodulin (apocalmodulin) activates and Ca(2+)-calmodulin inhibits the ryanodine receptor. The conversion of calmodulin from an activator to an inhibitor is due to Ca(2+) binding to calmodulin. We have previously shown that the binding sites for apocalmodulin and Ca(2+)-calmodulin on RYR1 are overlapping with the Ca(2+)-calmodulin site located slightly N-terminal to the apocalmodulin binding site. We now show that mutations of the calcium binding sites in either the N-terminal or the C-terminal lobes of calmodulin decrease the affinity of calmodulin for the ryanodine receptor, suggesting that both lobes interact with RYR1. Mutation of the two C-terminal Ca(2+) binding sites of calmodulin destroys calmodulin's ability to inhibit ryanodine receptor activity at high calcium concentrations. The mutated calmodulin, however, can still bind to RYR1 at both nanomolar and micromolar Ca(2+) concentrations. Mutating the two N-terminal calcium binding sites of calmodulin does not significantly alter calmodulin's ability to inhibit ryanodine receptor activity. These data suggest that calcium binding to the two C-terminal calcium binding sites within calmodulin is responsible for the switching of calmodulin from an activator to an inhibitor of the ryanodine receptor.     

Permeability changes of connexin32 hemi channels reconstituted in liposomes induced by extremely low frequency, low amplitude magnetic fields

The effect of extremely low frequency and low amplitude magnetic fields on gap junctional permeability was investigated by using reconstituted connexin32 hemi channel in liposomes. Cytochrome c was loaded inside these proteoliposomes and its reduction upon addition of ascorbate in the bulk aqueous phase was adopted as the index of hemi channel permeability. The permeability rate of the hemi channels, expressed as DeltaA/min, was dependent on the incubation temperature of proteoliposomes. The effect of exposures to magnetic fields at different frequencies (7, 13 and 18 Hz) and amplitudes (50, 50 and 70 microT, respectively), and at different temperatures (16, 18 and 24 degrees C) was studied. Only the exposure of proteoliposomes to 18-Hz (B(acpeak) and B(dc)=70 microT) magnetic field for 60 min at 16+/-0.4 degrees C resulted in a significant enhancement of the hemi channel permeability from DeltaA/min=0.0007+/-0.0002 to DeltaA/min=0.0010+/-0.0001 (P=0.030). This enhancement was not found for magnetic field exposures of liposomes kept at the higher temperatures tested. Temperature appears to influence lipid bilayer arrangement in such a way as being capable to mask possible effects induced by the magnetic field. Although the observed effect was very low, it seems to confirm the applicability of our model previously proposed for the interaction of low frequency electromagnetic fields with lipid membrane.     

Single, brief exposure to a 50 Hz magnetic field does not affect the performance of an object recognition task in adult mice

A number of studies have shown that power frequency magnetic fields may affect spatial memory functions in rodents. An experiment was performed using a spontaneous object recognition task to investigate if nonspatial working memory was similarly affected. Memory changes in adult, male C57BL/6J mice were assessed by measuring the relative time within which the animals explored familiar or novel stimulus objects. Between initial testing and retesting, the animals were exposed for 45 min to a 50 Hz magnetic field at either 7.5 microT, 75 microT or 0.75 mT. Other animals were sham-exposed with ambient fields of less than 50 nT. No significant field-dependent effects on the performance of the task were observed at any flux density (for all measures, P > 0.05). These data provide no evidence to suggest that nonspatial working memory was affected in mice by acute exposure to an intense 50 Hz magnetic field.     

Lobe-dependent regulation of ryanodine receptor type 1 by calmodulin

Calmodulin activates the skeletal muscle Ca(2+) release channel RYR1 at nm Ca(2+) concentrations and inhibits the channel at microm Ca(2+) concentrations. Using a deletion mutant of calmodulin, we demonstrate that amino acids 2-8 are required for high affinity binding of calmodulin to RYR1 at both nm and microm Ca(2+) concentrations and are required for maximum inhibition of the channel at microm Ca(2+) concentrations. In contrast, the addition of three amino acids to the N terminus of calmodulin increased the affinity for RYR1 at both nm and microm Ca(2+) concentrations, but destroyed its functional effects on RYR1 at nm Ca(2+). Using both full-length RYR1 and synthetic peptides, we demonstrate that the calmodulin-binding site on RYR1 is likely to be noncontiguous, with the C-terminal lobe of both apocalmodulin and Ca(2+)-calmodulin binding to amino acids between positions 3614 and 3643 and the N-terminal lobe binding at sites that are not proximal in the primary sequence. Ca(2+) binding to the C-terminal lobe of calmodulin converted it from an activator to an inhibitor, but an interaction with the N-terminal lobe was required for a maximum effect on RYR1. This interaction apparently depends on the native sequence or structure of the first few amino acids at the N terminus of calmodulin.     

High-intensity electrostatic-field exposure system for cultured biological cells.

We describe a new system for exposing cultured biological cells that have been plated on coverslips to strong electrostatic fields at magnitudes greater than 10(3) V/cm. Techniques are described that make use of mineral oil to render insignificant electrical conduction currents (total leakage current is less than 1.0 nA or less than 0.1 nA/coverslip), joule heating (less than 10(-6) W), or current-induced magnetic fields (less than 10(-13) T) in regions inhabited by cells. The mineral oil also eliminates a reduction in the strength of the applied field, which otherwise can occur from increased electrode-to-medium impedance at the site of application. Thus the applied field is reliably specified in the vicinity of a cell membrane. Control and electrostatic field chambers are housed in a grounded metal incubator. Cylindrical mu-metal shields can be used to reduce background magnetic fields in each chamber from 40 microT static and approximately 1 microT ac to, respectively, less than 3 microT static and approximately 100 nT ac. Contamination of cells by impurity atoms that may leach from electrodes was measured by atomic-absorption spectrophotometry and found to be negligible. Stray magnetic- and electric-field components within the incubator were measured, as were background fields around the laboratory.