On the Natural and Unnatural History of the Voltage-Gated Na(+) Channel.

This review glances at the voltage-gated sodium (Na(+)) channel (NaV) from the skewed perspective of natural history and the history of ideas. Beginning with the earliest natural philosophers, the objective of biological science and physiology was to understand the basis of life and discover its intimate secrets. The idea that the living state of matter differs from inanimate matter by an incorporeal spirit or mystical force was central to vitalism, a doctrine based on ancient beliefs that persisted until the last century.

Interaction of the BKCa channel gating ring with dendrotoxins.

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca(2+)-activated K(+) channel (BKCa, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BKCa channel gating.

Interaction of local anesthetics with the K (+) channel pore domain: KcsA as a model for drug-dependent tetramer stability.

Local anesthetics and related drugs block ionic currents of Na (+) , K (+) and Ca ( 2+) 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.

The molecular mystique of tetrodotoxin.

In many respects tetrodotoxin (TTX) is the quintessential natural toxin. It is unequivocally toxic to mammals with LD(50) values for mice in the range of 10 μg/kg (intraperitoneal), 16 μg/kg (subcutaneous), and 332 μg/kg (oral) (Kao, 1966). Its biothreat status is recognized by its listing as a "Select Agent" by the US Department of Health and Human Services which includes regulated agents "determined to have the potential to pose a severe threat to both human and animal health" (http://www.

Use-dependent block of the voltage-gated Na(+) channel by tetrodotoxin and saxitoxin: effect of pore mutations that change ionic selectivity.

Voltage-gated Na(+) channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism.

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 KIR K (+) channels. We previously described block of heterologously expressed voltage-gated Na (+) channels (NaV) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal NaV channels.

Synthesis of an iberiotoxin derivative by chemical ligation: a method for improved yields of cysteine-rich scorpion toxin peptides.

Automated and manual solid phase peptide synthesis techniques were combined with chemical ligation to produce a 37-residue peptide toxin derivative of iberiotoxin which contained: (i) substitution of Val(16) to Ala, to facilitate kinetic feasibility of native chemical ligation, and; (ii) substitution of Asp(19) to orthogonally protected Cys-4-MeOBzl for chemical conjugate derivatization following peptide folding and oxidation. This peptide ligation approach increased synthetic yields approximately 12-fold compared to standard linear peptide synthesis. In a functional inhibition assay, the ligated scorpion toxin derivative, iberiotoxin V16A/D19-Cys-4-MeOBzl, exhibited 'native-like' affinity (K(d)=1.

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.

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.

Beta-subunit-dependent modulation of hSlo BK current by arachidonic acid.

In this study, we examined the effect of arachidonic acid (AA) on the BK alpha-subunit with or without beta-subunits expressed in Xenopus oocytes. In excised patches, AA potentiated the hSlo-alpha current and slowed inactivation only when beta2/3 subunit was co-expressed. The beta2-subunit-dependent modulation by AA persisted in the presence of either superoxide dismutase or inhibitors of AA metabolism such as nordihydroguaiaretic acid and eicosatetraynoic acid, suggesting that AA acts directly rather than through its metabolites.

Synthesis of a biotin derivative of iberiotoxin: binding interactions with streptavidin and the BK Ca2+-activated K+ channel expressed in a human cell line.

Iberiotoxin (IbTx) is a scorpion venom peptide that inhibits BK Ca2+-activated K+ channels with high affinity and specificity. Automated solid-phase synthesis was used to prepare a biotin-labeled derivative (IbTx-LC-biotin) of IbTx by substitution of Asp19 of the native 37-residue peptide with N--(D-biotin-6-amidocaproate)-L-lysine. Both IbTx-LC-biotin and its complex with streptavidin (StrAv) block single BK channels from rat skeletal muscle with nanomolar affinity, indicating that the biotin-labeled residue, either alone or in complex with StrAv, does not obstruct the toxin binding interaction with the BK channel.

The calcium-sensitive large-conductance potassium channel (BK/MAXI K) is present in the inner mitochondrial membrane of rat brain.

Large-conductance voltage- and calcium-sensitive channels are known to be expressed in the plasmalemma of central neurons; however, recent data suggest that large-conductance voltage- and calcium-sensitive channels may also be present in mitochondrial membranes. To determine the subcellular localization and distribution of large-conductance voltage- and calcium-sensitive channels, rat brain fractions obtained by Ficoll-sucrose density gradient centrifugation were examined by Western blotting, immunocytochemistry and immuno-gold electron microscopy. Immunoblotting studies demonstrated the presence of a consistent signal for the alpha subunit of the large-conductance voltage- and calcium-sensitive channel in the mitochondrial fraction.

Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel.

Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE.

Optimizing the connectivity in disulfide-rich peptides: alpha-conotoxin SII as a case study.

We describe a strategy for the efficient, unambiguous assignment of disulfide connectivities in alpha-conotoxin SII, of which approximately 30% of its mass is cysteine, as an example of a generalizable technique for investigation of cysteine-rich peptides. alpha-Conotoxin SII was shown to possess 3-8, 2-18, and 4-14 disulfide bond connectivity. Sequential disulfide bond connectivity analysis was performed by partial reduction with Tris(2-carboxyethyl)phosphine and real-time mass monitoring by direct-infusion electrospray mass spectrometry (ESMS).

Block of inactivation-deficient Na+ channels by local anesthetics in stably transfected mammalian cells: evidence for drug binding along the activation pathway.

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na(+) channels with higher affinities. However, an alternative view suggests that activation of Na(+) channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na(+) channels (rNav1.

Inactivation-deficient human skeletal muscle Na+ channels (hNav1.4-L443C/A444W) in stably transfected HEK-293 cells.

After transient transfection of an hNav1.4-L443C/A444W mutant clone, HEK-293 cells exhibited large inactivation-deficient Na+currents. We subsequently established a stable cell line expressing robust inactivation-deficient Na+currents.

Effect of phosphatidylserine on unitary conductance and Ba2+ block of the BK Ca2+-activated K+ channel: re-examination of the surface charge hypothesis.

Incorporation of BK Ca2+-activated K+ channels into planar bilayers composed of negatively charged phospholipids such as phosphatidylserine (PS) or phosphatidylinositol (PI) results in a large enhancement of unitary conductance (gch) in comparison to BK channels in bilayers formed from the neutral zwitterionic lipid, phospatidylethanolamine (PE). Enhancement of gch by PS or PI is inversely dependent on KCl concentration, decreasing from 70% at 10 mM KCl to 8% at 1,000 mM KCl. This effect was explained previously by a surface charge hypothesis (Moczydlowski, E.

A lithium-induced conformational change in serotonin transporter alters cocaine binding, ion conductance, and reactivity of Cys-109.

Inactivation of serotonin transporter (SERT) expressed in HeLa cells by [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) occurred much more readily when Na(+) in the reaction medium was replaced with Li(+). This did not result from a protective effect of Na(+) but rather from a Li(+)-specific increase in the reactivity of Cys-109 in the first external loop of the transporter. Li(+) alone of the alkali cations caused this increase in reactivity.

Ca2+-binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca2+-dependent activation.

Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca(2+)-activated K(+) channel (BK channel) contains Ca(2+)-binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca(2+) binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG- and His(6)-tagged protein in Escherichia coli.

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.

Expression and characterization of Flag-epitope- and hexahistidine-tagged derivatives of saxiphilin for use in detection and assay of saxitoxin.

Saxiphilin is a plasma protein from the bullfrog (Rana catesbiana) that binds saxitoxin (STX), a causative agent of paralytic shellfish poisoning. Saxiphilin is homologous to transferrin and consists of two internally homologous domains called the N-lobe and the C-lobe. STX binds to a single site in the C-lobe of saxiphilin.

Saxiphilin, a saxitoxin-binding protein with two thyroglobulin type 1 domains, is an inhibitor of papain-like cysteine proteinases.

The type 1 domain of thyroglobulin is a protein module (Thyr-1) that occurs in a variety of secreted and membrane proteins. Several examples of Thyr-1 modules have been previously identified as inhibitors of the papain family of cysteine proteinases. Saxiphilin is a neurotoxin-binding protein from bullfrog and a homolog of transferrin with a pair of such Thyr-1 modules located in the N-lobe.

Permeation of large tetra-alkylammonium cations through mutant and wild-type voltage-gated sodium channels as revealed by relief of block at high voltage.

Many large organic cations are potent blockers of K(+) channels and other cation-selective channels belonging to the P-region superfamily. However, the mechanism by which large hydrophobic cations enter and exit the narrow pores of these proteins is obscure. Previous work has shown that a conserved Lys residue in the DEKA locus of voltage-gated Na(+) channels is an important determinant of Na(+)/K(+) discrimination, exclusion of Ca(2+), and molecular sieving of organic cations.