Microwave enhancement of membrane conductance: calmodulin hypothesis

It has been found that 2450 MHz microwave radiation increases membrane conductance in molluscan neurons. Analysis of this effect points to the important role of Ca++ in the mechanism of neuron microwave response. However, regulation of many intracellular processes is not a direct Ca++ effect, but is mediated through calmodulin, a Ca++-binding multifunctional protein. Furthermore, there is some evidence showing that Ca++ regulation of a Ca pump, endoplasmic reticulum Ca++ buffering, and Ca++-activated K+ conductance are mediated via calmodulin. Based on that, calmodulin is hypothesized to be a microwave susceptible protein, and a qualitative model of microwave enhancement of membrane conductance is suggested.     

Synthetic fragments of calmodulin calcium-binding site III. A test of the acid pair hypothesis

The acid pair hypothesis describing the interaction of calcium with the helix-loop-helix conformation of EF hands in calmodulin and related proteins predicts that these calcium-binding sites will have increased affinity for calcium if the anionic amino acid dentates in the loop region which interact directly with the cation are paired on the axial vertices of the resulting octahedral arrangement of chelating residues about the cation. As a test of this hypothesis, synthetic 33 residue analogs of bovine brain calmodulin calcium-binding site III have been prepared by the solid-phase method and analyzed for calcium affinity. The native sequence has a Kd of 735 microM for calcium and contains three anionic ligands which assume the +x, +y, and -z coordinates of the octahedral arrangement about the cation, thus precluding any pairing of the anionic ligands. This dissociation constant is 26 times weaker than that obtained from a synthetic analog of the sequentially homologous calcium-binding site III of rabbit skeletal TnC (Kd = 28 microM) which has four anionic ligands paired on the x and z axes. An analog of calmodulin site III with substitutions in the chelating residues at positions 1, 3, 5, 7, 9, and 12 of the 12-residue loop region to make these positions identical to those of rabbit skeletal troponin C site III decreased the calcium dissociation constant of the calmodulin peptide to 19 microM, similar to the troponin C peptide. Two synthetic analogs of calmodulin site III which contain three anionic ligands with two ligands paired on the x axis and two on the z axis have a Kd for calcium of 524 and 59 microM, respectively. This study provides strong support for and a better definition of the acid pair hypothesis and further demonstrates the usefulness of synthetic calcium-binding fragments in delineating the mechanism of calcium regulation of calmodulin and related proteins.     

The interaction of calmodulin with melittin

Studies utilizing the interaction of melittin with the 1-106 fragment of calmodulin, the protection of calmodulin from tryptic digestion by melittin, and the interaction of the carbocyanine dye Stains-all with the calmodulin-melittin complex have indicated that complex formation of calmodulin with melittin involves the alpha-helical connecting bridge joining the N- and C-terminal lobes of calmodulin.     

The phosphorylation of calmodulin and calmodulin fragments by kinase fractions from bovine brain

The phosphorylation of intact calmodulin and of fragments obtained by trypsin digestion was studied, using a protein kinase partially purified from bovine brain. Brain extracts were made in the presence of the detergent CHAPS (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). The protein kinase catalyzed the incorporation of nearly 1 mol of 32P from [gamma-32P]ATP into calmodulin fragment 1-106. Incorporation was exclusively into serine 101. With fragment 78-148, the extent of phosphorylation was somewhat less and 32P appeared mainly in threonine residues. Fragment 1-90 was also a fairly good substrate, but the phosphorylation of intact calmodulin never exceeded 0.01 mol per mol. Little or no phosphorylation was seen with parvalbumin, the brain Ca2+-binding protein (CBP-18) and intestinal calcium-binding protein. The protein kinase had no requirement for cAMP or phospholipids. High levels of Mg2+ (60-70 mM) stimulated phosphorylation of the fragments 20-fold. Millimolar concentrations of Ca2+ were inhibitory. It is suggested that the calmodulin fragments were in a conformation more favorable for phosphorylation than intact soluble calmodulin.     

The interaction of cyclosporin and calmodulin

The interaction of cyclosporin A and dansyl cyclosporin A with bovine and wheat germ calmodulin has been monitored by measurements of induced changes in dansyl and bound toluidinyl naphthalene sulfonate fluorescence. The interaction is Ca2(+)-dependent and 1:1. Measurements of the efficiency of radiationless energy transfer from bound dansyl cyclosporin A to an acceptor group located on Cys-27 of wheat germ calmodulin suggest that the primary binding site is not located on the N-terminal lobe (residues 1-65). However, studies with proteolytic fragments of calmodulin indicate that elements of the N-terminal half-molecule (residues 1-77) may be involved in the stabilization of the binding site. The binding of cyclosporin alters the physical properties of calmodulin and, in particular, reduces the localized rotational mobility of a fluorescent probe.     

The early adaptive evolution of calmodulin

Interaction between gene duplication and natural selection in molecular evolution was investigated utilizing a phylogenetic tree constructed by the parsimony procedure from amino acid sequences of 50 calmodulin- family protein members. The 50 sequences, belonging to seven protein lineages related by gene duplication (calmodulin itself, troponin-C, alkali and regulatory light chains of myosin, parvalbumin, intestinal calcium-binding protein, and glial S-100 phenylalanine-rich protein), came from a wide range of eukaryotic taxa and yielded a denser tree (more branch points within each lineage) than in earlier studies. Evidence obtained from the reconstructed pattern of base substitutions and deletions in these ancestral loci suggests that, during the early history of the family, selection acted as a transforming force on expressed genes among the duplicates to encode molecular sites with new or modified functions. In later stages of descent, however, selection was a conserving force that preserved the structures of many coadapted functional sites. Each branch of the family was found to have a unique average tempo of evolutionary change, apparently regulated through functional constraints. Proteins whose functions dictate multiple interaction with several other macromolecules evolved more slowly than those which display fewer protein-protein and protein-ion interactions, e.g., calmodulin and next troponin-C evolved at the slowest average rates, whereas parvalbumin evolved at the fastest. The history of all lineages, however, appears to be characterized by rapid rates of evolutionary change in earlier periods, followed by slower rates in more recent periods. A particularly sharp contrast between such fast and slow rates is found in the evolution of calmodulin, whose rate of change in earlier eukaryotes was manyfold faster than the average rate over the past 1 billion years. In fact, the amino acid replacements in the nascent calmodulin lineage occurred at residue positions that in extant metazoans are largely invariable, lending further support to the Darwinian hypothesis that natural selection is both a creative and a conserving force in molecular evolution.      

The interaction of delta-hemolysin with calmodulin

Delta-Hemolysin forms a 1:1 complex with Ca2+ -liganded calmodulin. Probably because of the pronounced tendency of delta-hemolysin to self-associate, the apparent binding affinity is much less than that for melittin. Complex formation is reflected by an increase in quantum yield of Trp-15 of delta-hemolysin and by increased shielding from acrylamide quenching. There is, however, no indication of a change in peptide molecular ellipticity. The binding of 2-toluidinyl-naphthalene-6-sulfonate is reduced by complex formation, suggesting the involvement of a hydrophobic region. Complex formation also blocks the proteolysis by trypsin of the bond between residues 77 and 78. The time decays of fluorescence intensity and anisotropy for tryptophan are multiexponential for both free and complexed delta-hemolysin; the average decay time for intensity is substantially increased for the complex. The localized mobility of tryptophan is greatly reduced in the complex. Complex formation appears to involve both the C-terminal lobe and the connecting strand of calmodulin.     

The patriarch hypothesis

Menopause is puzzling because life-history theory predicts there should be no selection for outliving one’s reproductive capacity. Adaptive explanations of menopause offered thus far turn on women’s long-term investment in offspring and grandoffspring, all variations on the grandmother hypothesis. Here, I offer a very different explanation. The patriarch hypothesis proposes that once males became capable of maintaining high status and reproductive access beyond their peak physical condition, selection favored the extension of maximum life span in males. Because the relevant genes were not on the Y chromosome, life span increased in females as well. However, the female reproductive span was constrained by the depletion of viable oocytes, which resulted in menopause. Frank Marlowe, Ph.D., is Assistant Professor of Anthropology at Harvard University. He conducts research with the Hadza and his interests include the behavioral ecology of mating systems, life-history theory, and cooperation.     

The nubility hypothesis

A new hypothesis is proposed to explain the perennially enlarged breasts of human females. The nubility hypothesis proposes that hominid females evolved protruding breasts because the size and shape of breasts function as an honest signal of residual reproductive value. Hominid females with greater residual reproductive value were preferred by males once reliable cues to ovulation were lost and long-term bonding evolved. This adaptation was favored because female-female competition for investing males increased once hominid males began to provide valuable resources.     

The keystone-pathogen hypothesis

Recent studies have highlighted the importance of the human microbiome in health and disease. However, for the most part the mechanisms by which the microbiome mediates disease, or protection from it, remain poorly understood. The keystone-pathogen hypothesis holds that certain low-abundance microbial pathogens can orchestrate inflammatory disease by remodelling a normally benign microbiota into a dysbiotic one. In this Opinion article, we critically assess the available literature that supports this hypothesis, which may provide a novel conceptual basis for the development of targeted diagnostics and treatments for complex dysbiotic diseases.     

The properties of calmodulin at physiological temperature

Several of the physical properties of calmodulin are changed significantly upon increasing the temperature from 25 to 37°C. Among the observed changes are a minor increase in intrinsic viscosity, a decrease in molecular ellipticity in the peptide region, the development of a difference spectrum in the aromatic region, and an increase in the mobility of extrinsic fluorescent labels. Except for the latter case, the magnitudes of the changes are substantially reduced in the presence of Ca²⁺. The above observations are consistent with an expanded and less tightly organized structure of calmodulin at the higher temperature.     

The undertow of sulfur metabolism on glutamatergic neurotransmission

Metabolic interdependence between specialized cells in an organ represents a strategy for energy economy by requiring expression of only a subset of pathway genes in a given cell type. In brain, sulfur metabolism exemplifies this principle of metabolic cooperation between glial and neuronal cells and furnishes three key reagents: S-adenosylmethionine, glutathione and taurine. The pathways for glutathione and taurine syntheses depend on metabolic integration between astrocytes and neurons and intersect with the glutamine-glutamate cycle, which underlies glutamatergic synaptic transmission and requires cooperation between these cell types. We propose that underlying waves of glutamate clearance by astrocytes are activation of cystine import and taurine efflux that result, respectively, from a shared transporter and an increase in solute concentration that triggers osmoregulatory responses.     

The role of calmodulin in cell transformation

Two cell lines transformed with temperature sensitive retroviruses were examined for: their ability to grow in low Ca²⁺ medium, their calmodulin levels and changes in calmodulin acceptor proteins. Both cell lines grow in low Ca²⁺ medium at the permissive temperature 34°C while both lines did not replicate at the non-permissive temperature 39°C. The NRKLA23 cells have nearly twice as much calmodulin at the permissive temperature than they do at the non-permissive temperature while the 6M2 cells have an equal amount of calmodulin at both temperatures. Both cell lines exhibit changes in the calmodulin acceptor proteins going from the permissive to the non-permissive temperature. We suspect that the changes in the calmodulin acceptor proteins may be involved in the altered Ca²⁺-sensitivity of growth in the cells going from the permissive to non-permissive temperature.