Comparative morphology and phylogeny of the family Thompsoniidae (Cirripedia, Rhizocephala, Akentrogonida), with descriptions of three new genera and seven new species

Akentrogonid rhizocephalans morphologically resembling the genus Thompsonia are revised as a result of examination of new material. The species concerned are all obligatorily colonial and have ovoid or cylindrically shaped externae with a terminal stalk and a much reduced anatomy. A numerical cladistic analysis of all Rhizocephala Akentrogonida using the Hennig 86 program leads to a redefinition of the Thompsoniidae HOeg and Rybakov, 1992. Autapomorphies for the Thompsoniidae are primarily the morphology of the attachment to the host and the total absence of a mesentery. The cladistic analysis refutes that the Thompsoniidae should have a plesiomorphic morphology and branch off very low on the rhizocephalan phylogeny. The family now comprises four genera: Pottsia gen. n. (monotypic), Diplothylacus gen. n. with two species, Thompsonia Kossmann with five species. A revived and redefined Thylacoplethus Coutière includes eight species. The genera are distinguished by the location of the spermatogenic tissue, the site where the eggs are fertilized, the presence or absence of a mantle pore and the way it is formed, the number or absence of oviducts, and the number of cuticular annuli on the stalk. All 16 species, of which six are new to science, are described when necessary, and, if possible, illustrated. A phylogeny for the redefined family is proposed. Thylacoplethus is morphologically closest to the hypothetically ancestral thompsoniid and is likely paraphyletic. The new genus Polysaccus with two species, one of them new to science, and the monotypic genus Pirusaccus Lützen resemble thompsoniids in externa morphology and in being obligatorily colonial.     

Interaction of local anesthetics with the K+ channel pore domain

Local anesthetics and related drugs block ionic currents of Na⁺, K⁺ and Ca²⁺ conducted across the cell membrane by voltage-dependent ion channels. Many of these drugs bind in the permeation pathway, occlude the pore and stop ion movement. However channel-blocking drugs have also been associated with decreased membrane stability of certain tetrameric K⁺ channels, similar to the destabilization of channel function observed at low extracellular K⁺ concentration. Such drug-dependent stability may result from electrostatic repulsion of K⁺ from the selectivity filter by a cationic drug molecule bound in the central cavity of the channel. In this study we used the pore domain of the KcsA K⁺ channel protein to test this hypothesis experimentally with a biochemical assay of tetramer stability and theoretically by computational simulation of local anesthetic docking to the central cavity. We find that two common local anesthetics, lidocaine and tetracaine, promote thermal dissociation of the KcsA tetramer in a K⁺-dependent fashion. Docking simulations of these drugs with open, open-inactivated and closed crystal structures of KcsA yield many energetically favorable drug-channel complexes characterized by nonbonded attraction to pore-lining residues and electrostatic repulsion of K⁺. The results suggest that binding of cationic drugs to the inner cavity can reduce tetramer stability of K⁺ channels.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines: A possible basis for modulation of excitability in vivo

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Thermal stability of the K+ channel tetramer: cation interactions and the conserved threonine residue at the innermost site (S4) of the KcsA selectivity filter

The selectivity filter of most K+ channels contains a highly conserved Thr residue that uniquely forms the S4 binding site for K+ by dual coordination with the backbone carbonyl oxygen and side chain hydroxyl of the same residue. This study examines the effect of mutations of Thr75 in the S4 site of theKcsA K+ channel on the cation dependence of the thermal stability of the tetramer, a phenomenon that reflects the structural role of cations in the filter. Conservative mutations of Thr75 destabilize the tetramer and alter its temperature dependence. Replacement of Thr with Ala or Cys lowers the apparent affinity ofK+, Rb+, and Cs+ for tetramer stabilization by factors ranging from 4- to 14-fold. These same mutations lower the apparent affinity of Ba2+ by approximately 10(3)- or approximately 10(4)-fold for Ala and Cys substitution, respectively,consistent with the known preference of the S4 site for Ba2+. In contrast, substitution of Ala or Cys at T75 anomalously enhances the ability of Na+ to stabilize the tetramer, suggesting that the native Thr residue at S4 is important for ultrahigh K+/Na+ selectivity of K+ channel pores. Elevated temperature orCu2+ cation catalyzes formation of covalent dimers of the T75C mutant of KcsA via formation of disulfide bonds between Cys residues of adjacent subunits. Thiophilic cations such as Hg2+ and Ag+ specifically protect the T75C tetramer against heat-induced dimer formation, demonstrating the contribution of cation interactions to tetramer stability in a channel with a non-native S4 site engineered to bind foreign cations.     

Tarantula huwentoxin-IV inhibits neuronal sodium channels by binding to receptor site 4 and trapping the domain ii voltage sensor in the closed configuration

Peptide toxins with high affinity, divergent pharmacological functions, and isoform-specific selectivity are powerful tools for investigating the structure-function relationships of voltage-gated sodium channels (VGSCs). Although a number of interesting inhibitors have been reported from tarantula venoms, little is known about the mechanism for their interaction with VGSCs. We show that huwentoxin-IV (HWTX-IV), a 35-residue peptide from tarantula Ornithoctonus huwena venom, preferentially inhibits neuronal VGSC subtypes rNav1.2, rNav1.3, and hNav1.7 compared with muscle subtypes rNav1.4 and hNav1.5. Of the five VGSCs examined, hNav1.7 was most sensitive to HWTX-IV (IC(50) approximately 26 nM). Following application of 1 microm HWTX-IV, hNav1.7 currents could only be elicited with extreme depolarizations (>+100 mV). Recovery of hNav1.7 channels from HWTX-IV inhibition could be induced by extreme depolarizations or moderate depolarizations lasting several minutes. Site-directed mutagenesis analysis indicated that the toxin docked at neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II. Mutations E818Q and D816N in hNav1.7 decreased toxin affinity for hNav1.7 by approximately 300-fold, whereas the reverse mutations in rNav1.4 (N655D/Q657E) and the corresponding mutations in hNav1.5 (R812D/S814E) greatly increased the sensitivity of the muscle VGSCs to HWTX-IV. Our data identify a novel mechanism for sodium channel inhibition by tarantula toxins involving binding to neurotoxin receptor site 4. In contrast to scorpion beta-toxins that trap the IIS4 voltage sensor in an outward configuration, we propose that HWTX-IV traps the voltage sensor of domain II in the inward, closed configuration.     

hybridlab (version 1.0): a program for generating simulated hybrids from population samples

We present the computer program hybridlab 1.0 for simulating intraspecific hybrids from population samples of nuclear genetic markers such as microsatellites, allozymes or SNPs (single nucleotide polymorphisms). The program generates a user‐specified number of multilocus F1 hybrid genotypes between any pair of potentially hybridizing populations included in a standard input‐file of multilocus genotypes for population genetic analysis. This simple, user‐friendly program has a wide range of applications for studying natural and artificial hybridization; in particular, for evaluating the statistical power for individual assignment of parental and hybrid individuals. An example of application for Atlantic cod populations is given.     

Cytoplasmic polyamines as permeant blockers and modulators of the voltage-gated sodium channel

We report that voltage-gated Na+ channels (Na(V)) from rat muscle (mu1) expressed in HEK293 cells exhibit anomalous rectification of whole-cell outward current under conditions of symmetrical Na+. This behavior gradually fades with time after membrane break-in, as if a diffusible blocking substance in the cytoplasm is slowly diluted by the pipette solution. The degree of such block and rectification is markedly altered by various mutations of the conserved Lys(III) residue in Domain III of the Na(V) channel selectivity filter (DEKA locus), a principal determinant of inorganic ion selectivity and organic cation permeation. Using whole-cell and macropatch recording techniques, we show that two ubiquitous polyamines, spermine and spermidine, are potent voltage-dependent cytoplasmic blockers of mu1 Na(V) current that exhibit relief of block at high positive voltage, a phenomenon that is also enhanced by certain mutations of the Lys(III) residue. In addition, we find that polyamines alter the apparent rate of macroscopic inactivation and exhibit a use-dependent blocking phenomenon reminiscent of the action of local anesthetics. In the presence of a physiological Na+/K+ gradient, spermine also inhibits inward Na(V) current and shifts the voltage dependence of activation and inactivation. Similarities between the endogenous blocking phenomenon observed in whole cells and polyamine block characterized in excised patches suggest that polyamines or related metabolites may function as endogenous modulators of Na(V) channel activity.     

Blocking mechanisms of batrachotoxin-activated Na channels in artificial bilayers

The effects of various pharmacological agents that block single batrachotoxin-activated Na channels from rat muscle can be described in terms of three modes of action that correspond to at least three different binding sites. Guanidinium toxins such as tetrodotoxin, saxitoxin, and a novel polypeptide, mu-conotoxin GIIIA, act only from the extra-cellular side and induce discrete blocked states that correspond to residence times of individual toxin molecules. Such toxins apparently do not deeply penetrate the channel pore since the voltage dependence of block is insensitive to toxin charge and block is not relieved by internal Na+. Many nonspecific organic cations, including charged anesthetics, exhibit a voltage-dependent block that is enhanced by depolarization when present on the inside of the channel. This site is probably within the pore, but binding to this site is weak, as indicated by fast blockade that often appears as lowered channel conductance. A separate class of neutral and tertiary amine anesthetics such as benzocaine and procaine induce discrete closed states when added to either side of the membrane. This blocking effect can be explained by preferential binding to closed states of the channel and appears to be due to a modulation of channel gating.     

Influence of negative surface charge on toxin binding to canine heart Na channels in planar bilayers.

The presence of negative surface charge near the tetrodotoxin/saxitoxin binding site of canine heart Na channels was revealed by analysis of the kinetics of toxin block of single batrachotoxin-activated Na channels in planar bilayers as a function of [NaCl]. The voltage-dependence of toxin binding and the toxin dissociation rate are nearly constant as [NaCl] is varied from 0.05 to 3 M. In contrast, the association rate constant of the toxins is inversely dependent on [NaCl], with the rate for the divalent toxin, saxitoxin2+, affected more steeply than that of the monovalent toxin, tetrodotoxin1+. These results for toxin-insensitive Na channels from canine heart parallel previous findings for toxin-sensitive Na channels from canine brain. The model of Green et al. (Green, W. N., L. B. Weiss, and O. S. Anderson. 1987. J. Gen. Physiol. 89:873-903), which includes Na+ competition and Gouy-Chapman screening of surface charge, provided an excellent fit to the data. The results suggest that the two canine Na channel subtypes have a similar density of negative surface charge (1 e-/400 A2) and a similar dissociation constant for Na+ competition (0.5 M) at the toxin binding site. Thus, negative surface charge is a conserved feature of channel function of these two subtypes. The difference in toxin binding affinities arises from small differences in intrinsic association and dissociation rates.