Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: Role of inactivation

The role of channel inactivation in the molecular mechanism of calcium (Ca2+) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (IBa) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed IBa recovery after maintained membrane depolarization (1–3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of IBa recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca2+ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca2+ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.

Vibrational population relaxation of carbon monoxide in the heme pocket of photolyzed carbonmonoxy myoglobin: Comparison of time-resolved mid-IR absorbance experiments and molecular dynamics simulations

The vibrational energy relaxation of carbon monoxide in the heme pocket of sperm whale myoglobin was studied by using molecular dynamics simulation and normal mode analysis methods. Molecular dynamics trajectories of solvated myoglobin were run at 300 K for both the δ- and ɛ-tautomers of the distal His-64. Vibrational population relaxation times of 335 ± 115 ps for the δ-tautomer and 640 ± 185 ps for the ɛ-tautomer were estimated by using the Landau–Teller model. Normal mode analysis was used to identify those protein residues that act as the primary “doorway” modes in the vibrational relaxation of the oscillator. Although the CO relaxation rates in both the ɛ- and δ-tautomers are similar in magnitude, the simulations predict that the vibrational relaxation of the CO is faster in the δ-tautomer with the distal His playing an important role in the energy relaxation mechanism. Time-resolved mid-IR absorbance measurements were performed on photolyzed carbonmonoxy hemoglobin (Hb13CO). From these measurements, a T1 time of 600 ± 150 ps was determined. The simulation and experimental estimates are compared and discussed.

Rapid evolution in plant chitinases: Molecular targets of selection in plant-pathogen coevolution

Many pathogen recognition genes, such as plant R-genes, undergo rapid adaptive evolution, providing evidence that these genes play a critical role in plant-pathogen coevolution. Surprisingly, whether rapid adaptive evolution also occurs in genes encoding other kinds of plant defense proteins is unknown. Unlike recognition proteins, plant chitinases attack pathogens directly, conferring disease resistance by degrading chitin, a component of fungal cell walls. Here, we show that nonsynonymous substitution rates in plant class I chitinase often exceed synonymous rates in the plant genus Arabis (Cruciferae) and in other dicots, indicating a succession of adaptively driven amino acid replacements. We identify individual residues that are likely subject to positive selection by using codon substitution models and determine the location of these residues on the three-dimensional structure of class I chitinase. In contrast to primate lysozymes and plant class III chitinases, structural and functional relatives of class I chitinase, the adaptive replacements of class I chitinase occur disproportionately in the active site cleft. This highly unusual pattern of replacements suggests that fungi directly defend against chitinolytic activity through enzymatic inhibition or other forms of chemical resistance and identifies target residues for manipulating chitinolytic activity. These data also provide empirical evidence that plant defense proteins not involved in pathogen recognition also evolve in a manner consistent with rapid coevolutionary interactions.

Vagaries of the molecular clock

The hypothesis of the molecular evolutionary clock asserts that informational macromolecules (i.e., proteins and nucleic acids) evolve at rates that are constant through time and for different lineages. The clock hypothesis has been extremely powerful for determining evolutionary events of the remote past for which the fossil and other evidence is lacking or insufficient. I review the evolution of two genes, Gpdh and Sod. In fruit flies, the encoded glycerol-3-phosphate dehydrogenase (GPDH) protein evolves at a rate of 1.1 × 10−10 amino acid replacements per site per year when Drosophila species are compared that diverged within the last 55 million years (My), but a much faster rate of ≈4.5 × 10−10 replacements per site per year when comparisons are made between mammals (≈70 My) or Dipteran families (≈100 My), animal phyla (≈650 My), or multicellular kingdoms (≈1100 My). The rate of superoxide dismutase (SOD) evolution is very fast between Drosophila species (16.2 × 10−10 replacements per site per year) and remains the same between mammals (17.2) or Dipteran families (15.9), but it becomes much slower between animal phyla (5.3) and still slower between the three kingdoms (3.3). If we assume a molecular clock and use the Drosophila rate for estimating the divergence of remote organisms, GPDH yields estimates of 2,500 My for the divergence between the animal phyla (occurred ≈650 My) and 3,990 My for the divergence of the kingdoms (occurred ≈1,100 My). At the other extreme, SOD yields divergence times of 211 My and 224 My for the animal phyla and the kingdoms, respectively. It remains unsettled how often proteins evolve in such erratic fashion as GPDH and SOD.

Molecular mechanisms of sound amplification in the mammalian cochlea

Mammalian hearing depends on the enhanced mechanical properties of the basilar membrane within the cochlear duct. The enhancement arises through the action of outer hair cells that act like force generators within the organ of Corti. Simple considerations show that underlying mechanism of somatic motility depends on local area changes within the lateral membrane of the cell. The molecular basis for this phenomenon is a dense array of particles that are inserted into the basolateral membrane and that are capable of sensing membrane potential field. We show here that outer hair cells selectively take up fructose, at rates high enough to suggest that a sugar transporter may be part of the motor complex. The relation of these findings to a recent candidate for the molecular motor is also discussed.

Rates of nucleotide substitution in sexual and anciently asexual rotifers

The class Bdelloidea of the phylum Rotifera is the largest well studied eukaryotic taxon in which males and meiosis are unknown, and the only one for which these indications of ancient asexuality are supported by cytological and molecular genetic evidence. We estimated the rates of synonymous and nonsynonymous substitutions in the hsp82 heat shock gene in bdelloids and in facultatively sexual rotifers of the class Monogononta, employing distance based and maximum likelihood methods. Relative-rate tests, using acanthocephalan rotifers as an outgroup, showed slightly higher rates of nonsynonymous substitution and slightly lower rates of synonymous substitution in bdelloids as compared with monogononts. The opposite trend, however, was seen in intraclass pairwise comparisons. If, as it seems, bdelloids have evolved asexually, an equality of bdelloid and monogonont substitution rates would suggest that the maintenance of sexual reproduction in monogononts is not attributable to an effect of sexual reproduction in limiting the load of deleterious nucleotide substitutions.

Molecular phylogeny analysis of fiddler crabs: test of the hypothesis of increasing behavioral complexity in evolution.

The current phylogenetic hypothesis for the evolution and biogeography of fiddler crabs relies on the assumption that complex behavioral traits are assumed to also be evolutionary derived. Indo-west Pacific fiddler crabs have simpler reproductive social behavior and are more marine and were thought to be ancestral to the more behaviorally complex and more terrestrial American species. It was also hypothesized that the evolution of more complex social and reproductive behavior was associated with the colonization of the higher intertidal zones. Our phylogenetic analysis, based upon a set of independent molecular characters, however, demonstrates how widely entrenched ideas about evolution and biogeography led to a reasonable, but apparently incorrect, conclusion about the evolutionary trends within this pantropical group of crustaceans. Species bearing the set of "derived traits" are phylogenetically ancestral, suggesting an alternative evolutionary scenario: the evolution of reproductive behavioral complexity in fiddler crabs may have arisen multiple times during their evolution. The evolution of behavioral complexity may have arisen by coopting of a series of other adaptations for high intertidal living and antipredator escape. A calibration of rates of molecular evolution from populations on either side of the Isthmus of Panama suggest a sequence divergence rate for 16S rRNA of 0.9% per million years. The divergence between the ancestral clade and derived forms is estimated to be approximately 22 million years ago, whereas the divergence between the American and Indo-west Pacific is estimated to be approximately 17 million years ago.

Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack.

We develop a unifying theory of hypoxia tolerance based on information from two cell level models (brain cortical cells and isolated hepatocytes) from the highly anoxia tolerant aquatic turtle and from other more hypoxia sensitive systems. We propose that the response of hypoxia tolerant systems to oxygen lack occurs in two phases (defense and rescue). The first lines of defense against hypoxia include a balanced suppression of ATP-demand and ATP-supply pathways; this regulation stabilizes (adenylates) at new steady-state levels even while ATP turnover rates greatly decline. The ATP demands of ion pumping are down-regulated by generalized "channel" arrest in hepatocytes and by "spike" arrest in neurons. Hypoxic ATP demands of protein synthesis are down-regulated probably by translational arrest. In hypoxia sensitive cells this translational arrest seems irreversible, but hypoxia-tolerant systems activate "rescue" mechanisms if the period of oxygen lack is extended by preferentially regulating the expression of several proteins. In these cells, a cascade of processes underpinning hypoxia rescue and defense begins with an oxygen sensor (a heme protein) and a signal-transduction pathway, which leads to significant gene-based metabolic reprogramming-the rescue process-with maintained down-regulation of energy-demand and energy-supply pathways in metabolism throughout the hypoxic period. This recent work begins to clarify how normoxic maintenance ATP turnover rates can be drastically (10-fold) down-regulated to a new hypometabolic steady state, which is prerequisite for surviving prolonged hypoxia or anoxia. The implications of these developments are extensive in biology and medicine.

Myc and Max: molecular evolution of a family of proto-oncogene products and their dimerization partner.

The myc gene family encodes a group of transcription factors that regulate cell proliferation and differentiation. These genes are widely studied because of their importance as proto-oncogenes. Phylogenetic analyses are described here for 45 Myc protein sequences representing c-, N-, L-, S-, and B-myc genes. A gene duplication early in vertebrate evolution produced the c-myc lineage and another lineage that later gave rise to the N- and L-myc lineages by another gene duplication. Evolutionary divergence in the myc gene family corresponds closely to the known branching order of the major vertebrate groups. The patterns of sequence evolution are described for five separate highly conserved regions, and these analyses show that differential rates of sequence divergence (= mosaic evolution) have occurred among conserved motifs. Further, the closely related dimerization partner protein Max exhibits significantly less sequence variability than Myc. It is suggested that the reduced variability in max stems from natural selection acting to preserve dimerization capability with products of myc and related genes.

Evidence from molecular systematics for decreased avian diversification in the pleistocene Epoch.

Pleistocene glaciations have been suggested as major events influencing speciation rates in vertebrates. Avian paleontological studies suggest that most extant species evolved in the Pleistocene Epoch and that species' durations decreased through the Pleistocene because of heightened speciation rates. Molecular systematic studies provide another data base for testing these predictions. In particular, rates of diversification can be determined from molecular phylogenetic trees. For example, an increasing rate of speciation (but constant extinction) requires shorter intervals between successive speciation events on a phylogenetic tree. Examination of the cumulative distribution of reconstructed speciation events in mtDNA phylogenies of 11 avian genera, however, reveals longer intervals between successive speciation events as the present time is approached, suggesting a decrease in net diversification rate through the Pleistocene Epoch. Thus, molecular systematic studies do not indicate a pulse of Pleistocene diversification in passerine birds but suggest, instead, that diversification rates were lower in the Pleistocene than for the preceding period. Documented habitat shifts likely led to the decreased rate of diversification, although from molecular evidence we cannot discern whether speciation rates decreased or extinction rates increased.