Molecular determinants of high affinity binding of alpha-scorpion toxin and sea anemone toxin in the S3-S4 extracellular loop in domain IV of the Na+ channel alpha subunit

alpha-Scorpion toxins and sea anemone toxins bind to a common extracellular site on the Na+ channel and inhibit fast inactivation. Basic amino acids of the toxins and domains I and IV of the Na+ channel alpha subunit have been previously implicated in toxin binding. To identify acidic residues required for toxin binding, extracellular acidic amino acids in domains I and IV of the type IIa Na+ channel alpha subunit were converted to neutral or basic amino acids using site-directed mutagenesis, and altered channels were transiently expressed in tsA-201 cells and tested for 125I-alpha-scorpion toxin binding. Conversion of Glu1613 at the extracellular end of transmembrane segment IVS3 to Arg or His blocked measurable alpha-scorpion toxin binding, but did not affect the level of expression or saxitoxin binding affinity. Conversion of individual residues in the IVS3-S4 extracellular loop to differently charged residues or to Ala identified seven additional residues whose mutation caused significant effects on binding of alpha-scorpion toxin or sea anemone toxin. Moreover, chimeric Na+ channels in which amino acid residues at the extracellular end of segment IVS3 of the alpha subunit of cardiac Na+ channels were substituted into the type IIa channel sequence had reduced affinity for alpha-scorpion toxin characteristic of cardiac Na+ channels. Electrophysiological analysis showed that E1613R has 62- and 82-fold lower affinities for alpha-scorpion and sea anemone toxins, respectively. Dissociation of alpha-scorpion toxin is substantially accelerated at all potentials compared to wild-type channels. alpha-Scorpion toxin binding to wild type and E1613R had similar voltage dependence, which was slightly more positive and steeper than the voltage dependence of steady-state inactivation. These results indicate that nonidentical amino acids of the IVS3-S4 loop participate in alpha-scorpion toxin and sea anemone toxin binding to overlapping sites and that neighboring amino acid residues in the IVS3 segment contribute to the difference in alpha-scorpion toxin binding affinity between cardiac and neuronal Na+ channels. The results also support the hypothesis that this region of the Na+ channel is important for coupling channel activation to fast inactivation.     

Electrical properties of Y-79 cells, a multipotent line of human retinoblastoma

1. Whole-cell and perforated-patch tight-seal recording techniques were used to characterize the voltage-dependent membrane conductances of the Y-79 cells, a human retinoblastoma line composed of pluripotential retinal precursor cells. 2. Membrane resistance and capacitance were measured under current clamp, yielding approximate average values of 1.8 G omega and 26 pF, respectively. The cells are electrically excitable, and depolarization above -20 mV triggers slow action potentials. 3. Step depolarization of the membrane under voltage clamp elicits a high-threshold transient inward current, followed by a sustained, larger outward current. The outward current is carried by potassium ions, as determined by its susceptibility to blockage by K-channel antagonists [tetraethylammonium (TEA), Cs, and 4-aminopyridine (4-AP)] and insensitivity to reduction of external chloride concentration. 4. The isolated inward current displayed some unusual properties: its amplitude is directly related to extracellular calcium concentration, and replacement of calcium by magnesium completely abolishes it. However, none of the calcium channel antagonists tested (cadmium, nickel, nifedipine, and amiloride) exerted a substantial blockage. In addition, removal of external sodium or superfusion with tetrodotoxin significantly reduce the size of this current. 5. A single voltage-dependent conductance appears to underlie the inward current, because a variety of manipulations, such as changes in the holding potential, in the extracellular concentration of calcium or sodium, or superfusion with tetrodotoxin, failed to reveal the presence of kinetically distinct components. 6. The results suggest that a single voltage-dependent conductance mechanism underlies the depolarization-activated inward current in Y-79 cells. This channel appears to be primarily permeable to calcium, but with a significant contribution by sodium ions. Its functioning appears to be modulated by extracellular calcium.     

Critical role of conserved proline residues in the transmembrane segment 4 voltage sensor function and in the gating of L-type calcium channels

The fourth transmembrane segment (S4) has been shown to function as a voltage sensor in voltage-gated channels. On membrane depolarization, a stretch of S4 moves outward and initiates a number of conformational changes that ultimately lead to channel opening. Conserved proline residues are in the middle of the S4 of motifs I and III in voltage-dependent Ca2+ channels. Because proline often introduces a "kink" into a helical structure of proteins, these residues might have an intrinsic function in the voltage sensor. Here, we report that the removal of S4 prolines results in a dramatic shortening of channel open time whereas the introduction of extra prolines to the corresponding positions in motif IIS4 and IVS4 lengthens channel open time. The number of S4s with a proline residue showed a clear positive correlation with the mean open time of the channel. The mean open time was >11-fold longer for a channel mutagenized to have prolines in all four S4s compared with a channel that had no prolines in the S4 region. Additionally, prolines in the S4s slowed activation kinetics and shifted the voltage dependence of activation and inactivation in a hyperpolarized direction. Our results strongly suggest that proline residues in the S4s are critical for stabilizing the open state of the channel. Moreover, it is suggested that motif IS4 and IIIS4 contribute to the channel opening more efficiently than motif IIS4 and IVS4.     

Investigation of a catalytic zinc binding site in Escherichia coli L-threonine dehydrogenase by site-directed mutagenesis of cysteine-38

We studied block of the internal pore of the ROMK1 inward-rectifier K+ channel by Mg2+ and five quaternary ammoniums (tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and tetrapentylammonium). The apparent affinity of these blockers varied as a function of membrane voltage. As a consequence, the channel conducted K+ current more efficiently in the inward than the outward direction; i.e., inward rectification. Although the size of some monovalent quaternary ammoniums is rather large, the zdelta values (which measure voltage dependence of their binding to the pore) were near unity in symmetric 100 mM K+. Furthermore, we observed that not only the apparent affinities of the blockers themselves, but also their dependence on membrane voltage (or zdelta), varied as a function of the concentration of extracellular K+. These results suggest that there is energetic coupling between the binding of blocking and permeating (K+) ions, and that the voltage dependence of channel blockade results, at least in part, from the movement of K+ ions in the electrical field. A further quantitative analysis of the results explains why the complex phenomenon of inward rectification depends on both membrane voltage and the equilibrium potential for K+.     

Agonist-induced changes in substituted cysteine accessibility reveal dynamic extracellular structure of M3-M4 loop of glutamate receptor GluR6

Recent evidence suggests that the transmembrane topology of ionotropic glutamate receptors differs from other members of the ligand-gated ion channel superfamily. However, the structure of the segment linking membrane domains M3 and M4 (the M3-M4 loop) remains controversial. Although various data indicate that this loop is extracellular, other results suggest that serine residues in this segment are sites of phosphorylation and channel modulation by intracellular protein kinases. To reconcile these data, we hypothesized that the M3-M4 loop structure is dynamic and, more specifically, that the portion containing putative phosphorylation sites may be translocated across the membrane to the cytoplasmic side during agonist binding. To test this hypothesis, we mutated Ser 684, a putative cAMP-dependent protein kinase site in the kainate-type glutamate receptor GluR6, to Cys. Results of biochemical and electrophysiological experiments are consistent with Cys 684 being accessible, in the unliganded state, from the extracellular side to modification by a Cys-specific biotinylating reagent followed by streptavidin (SA). Interestingly, our data suggest that this residue becomes inaccessible to the extracellular biotinylating reagent during agonist binding. However, we find it unlikely that Cys 684 undergoes membrane translocation, because the addition of SA to Cys-biotinylated GluR6(S684C) has no effect on peak glutamate-evoked current and only a small effect on macroscopic desensitization. We conclude that residue 684 in GluR6 is extracellular in the receptor-channel's closed, unliganded state and does not cross the membrane after agonist binding. However, an agonist-induced conformational change in the receptor substantially alters accessibility of position 684 to the extracellular environment.     

Mutations in different functional domains of the human muscle acetylcholine receptor alpha subunit in patients with the slow-channel congenital myasthenic syndrome

Congenital myasthenic syndromes are a group of rare genetic disorders that compromise neuromuscular transmission. A subset of these disorders, the slow-channel congenital myasthenic syndrome (SCCMS), is dominantly inherited and has been shown to involve mutations within the muscle acetylcholine receptor (AChR). We have identified three new SCCMS mutations and a further familial case of the alpha G153S mutation. Single channel recordings from wild-type and mutant human AChR expressed in Xenopus oocytes demonstrate that each mutation prolongs channel activation episodes. The novel mutations alpha V156M, alpha T254I and alpha S269I are in different functional domains of the AChR alpha subunit. Whereas alpha T254I is in the pore-lining region, like five of six previously reported SCCMS mutations, alpha S269I and alpha V156M are in extracellular domains. alpha S269I lies within the short extracellular sequence between M2 and M3, and identifies a new region of muscle AChR involved in ACh binding/channel gating. alpha V156M, although located close to alpha G153S which has been shown to increase ACh binding affinity, appears to alter channel function through a different molecular mechanism. Our results demonstrate heterogeneity in the SCCMS, indicate new regions of the AChR involved in ACh binding/channel gating and highlight the potential role of mutations outside the pore-lining regions in altering channel function in other ion channel disorders.     

Independent versus coupled inactivation in sodium channels. Role of the domain 2 S4 segment

The voltage sensor of the sodium channel is mainly comprised of four positively charged S4 segments. Depolarization causes an outward movement of S4 segments, and this movement is coupled with opening of the channel. A mutation that substitutes a cysteine for the outermost arginine in the S4 segment of the second domain (D2:R1C) results in a channel with biophysical properties similar to those of wild-type channels. Chemical modification of this cysteine with methanethiosulfonate-ethyltrimethylammonium (MTSET) causes a hyperpolarizing shift of both the peak current-voltage relationship and the kinetics of activation, whereas the time constant of inactivation is not changed substantially. A conventional steady state inactivation protocol surprisingly produces an increase of the peak current at -20 mV when the 300-ms prepulse is depolarized from -190 to -110 mV. Further depolarization reduces the current, as expected for steady state inactivation. Recovery from inactivation in modified channels is also nonmonotonic at voltages more hyperpolarized than -100 mV. At -180 mV, for example, the amplitude of the recovering current is briefly almost twice as large as it was before the channels inactivated. These data can be explained readily if MTSET modification not only shifts the movement of D2/S4 to more hyperpolarized potentials, but also makes the movement sluggish. This behavior allows inactivation to have faster kinetics than activation, as in the HERG potassium channel. Because of the unique properties of the modified mutant, we were able to estimate the voltage dependence and kinetics of the movement of this single S4 segment. The data suggest that movement of modified D2/S4 is a first-order process and that rate constants for outward and inward movement are each exponential functions of membrane potential. Our results show that D2/S4 is intimately involved with activation but plays little role in either inactivation or recovery from inactivation.     

Astigmatism, accommodation, the oblique effect and meridional amblyopia

Agonist binding to GABAB receptors modulates the activity of the guanine nucleotide binding proteins, Go and Gi. These G proteins are ADP-ribosylated by pertussis toxin and this prevents them from coupling to the GABAB receptor resulting in a reduction in high-affinity GABAB binding. GTP, which binds to a different site on the G protein alpha subunit, also reduces the affinity of the receptor for the G protein, and this can be used as a "marker" for G protein-GABAB receptor linkage. We have examined GABAB binding site distribution in rat brain after unilateral intrahippocampal pertussis toxin injection in vivo, and after incubating brain slices in pertussis toxin in vitro, using the technique of receptor autoradiography. The effect of pertussis toxin was compared with that of GTP gamma S on GABAB binding. Intrahippocampal pertussis toxin administration reduced GABAB but not GABAA receptor binding and the effects appeared to be limited by pertussis toxin diffusion. More widespread reductions in GABAB binding were seen after incubation of brain slices in vitro but the extent varied in different brain regions. No reduction was detected in the corpus striatum. GABAB binding was also reduced in membranes prepared from cerebral cortex, hippocampus and cerebellum but there was no significant reduction in the corpus striatum after pertussis toxin treatment. GTP gamma S reduced GABAB binding to a similar extent in all areas studied irrespective of their sensitivity to pertussis toxin suggesting that while GABAB binding sites are linked to G proteins throughout the rat brain, those in the corpus striatum may be predominantly pertussis toxin insensitive.     

Selective alteration of sodium channel gating by Australian funnel-web spider toxins

The actions of potent mammalian neurotoxins isolated from the venom of two Australian funnel-web spiders were investigated using both electrophysiological and neurochemical techniques. Whole-cell patch clamp recording of sodium currents in rat dorsal root ganglion neurons revealed that versutoxin (VTX), isolated from the venom of Hadronyche versuta, produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation and a reduction in peak TTX-S sodium current. In contrast, VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. VTX also shifted the voltage dependence of sodium channel activation in the hyperpolarizing direction and increased the rate of recovery from inactivation. Ion flux studies performed in rat brain synaptosomes also revealed that robustoxin (RTX), from the venom of Atrax robustus, and VTX both produced a partial activation of 22Na+ flux and an inhibition of batrachotoxin-activated 22Na+ flux. This inhibition of flux through batrachotoxin-activated channels was not due to an interaction with neurotoxin receptor site 1 since [3H]saxitoxin binding was unaffected. In addition, the partial activation of 22Na+ flux was not enhanced in the presence of alpha-scorpion toxin and further experiments suggest that VTX also enhances [3H]batrachotoxin binding. These selective actions of funnel-web spider toxins on sodium channel function are comparable to those of alpha-scorpion and sea anemone toxins which bind to neurotoxin receptor site 3 on the channel to slow channel inactivation profoundly. Also, these modifications of sodium channel gating and kinetics are consistent with actions of the spider toxins to produce repetitive firing of action potentials.     

Analysis of local structure in the D2/S1-S2 region of the rat skeletal muscle type 1 sodium channel using insertional mutagenesis

A reporter epitope was inserted at 11 positions in a region encompassing proposed transmembrane segments S1 and S2 in the second repeat domain (D2) of the rat skeletal muscle type 1 sodium channel. All mutations produced full-length membrane-associated protein following transfection into cultured cells, although the level of expression varied with insertion position. Characterization of cognate cRNAs for each mutation in Xenopus oocytes by two-electrode voltage clamp defined a permissive region between the proposed transmembrane regions in which these large insertions did not interfere with channel function. Two of the mutations, in which the point of insertion was within the proposed S1-S2 loop, demonstrated extracellular membrane labeling when studied either by antibody binding in oocytes or by confocal analysis following transfection into primary muscle cells. Our results define the likely boundaries of an extramembrane region linking the S1 and S2 transmembrane segments in D2 and confirm the extracellular location of this S1-S2 loop predicted by current models of channel tertiary structure.     

Extracellular or intracellular application of argiotoxin-636 has inhibitory actions on membrane excitability and voltage-activated currents in cultured rat sensory neurones

The whole cell variant of the patch clamp technique was used to investigate the actions of the polyamine amide spider toxin, argiotoxin-636, on the excitability of cultured dorsal root ganglion neurones. Synthesized argiotoxin-636 (0.1-100 microM) reduced neuronal excitability when applied to the extracellular environment by low pressure ejection or to the intracellular environment via the patch pipette solution. The toxin prolonged the duration of evoked action potentials and reduced the peak amplitude of action potentials. Intracellular and extracellular application of argiotoxin-636 also decreased the number of action potentials evoked in response to 800-ms depolarizing current commands. This action of the toxin was mimicked by 100 microM tetraethylammonium. Extracellular application of argiotoxin-636 inhibited voltage-activated K currents in a dose-dependent manner over the complete voltage range. This inhibition occurred without any significant changes in the voltage dependence of activation or inactivation. Intracellular application of argiotoxin-636, during 5-10 min of whole cell recording, also inhibited voltage-activated K+ currents without changing the voltage dependence of activation or steady-state inactivation. Extracellular or intracellular spermidine (250 microM) reversibly attenuated the inhibitory actions of extracellular argiotoxin-636. Argiotoxin-636 also inhibited voltage-activated Na + currents; this effect was dependent on repeated activation of the currents and the period during which the neurones were in culture. We conclude that application of argiotoxin-636 to either the extracellular or intracellular environment reduced excitability of cultured sensory neurones from neonatal rats and that this involved inhibition of both voltage-activated K+ and Na+ currents. The data suggest that the toxin was more effective at attenuating action potentials when neurones were repeatedly excited, and that access to inhibitory sites of action on the voltage-activated ion channels can be achieved from the inside of the neurone.     

Transmembrane movement of the shaker K+ channel S4

We have probed internal and external accessibility of S4 residues to the membrane-impermeant thiol reagent methanethiosulfonate-ethyltrimethlammonium (MTSET) in both open and closed, cysteine-substituted Shaker K+ channels. Our results indicate that S4 traverses the membrane with no more than 5 amino acids in the closed state, and that the distribution of buried residues changes when channels open. This change argues for a displacement of S4 through the plane of the membrane in which an initially intracellular residue moves to within 3 amino acids of the extracellular solution. These results demonstrate that the putative voltage-sensing charges of S4 actually reside in the membrane and that they move outward when channels open. We consider constraints placed on channel structure by these results.     

Expression and initial characterization of a soluble glycine binding domain of the N-methyl-D-aspartate receptor NR1 subunit

Glycine is an essential co-agonist of the excitatory N-methyl-D-aspartate (NMDA) receptor, a subtype of the ionotropic glutamate receptor family. The glycine binding site of this hetero-oligomeric ion channel protein is formed by two distinct extracellular regions, S1 and S2, of the NR1 subunit, whereas the homologous domains of the NR2 subunit mediate glutamate binding. Here, segments S1 and S2 of the NR1 polypeptide were fused via a linker peptide followed by N- and C-terminally tagging with Flag and His6 epitopes, respectively. Infection of High Five insect cells with a recombinant baculovirus containing this glycine binding site construct resulted in efficient secretion of a soluble fusion protein of about 53 kDa. After affinity purification to near-homogeneity, the fusion protein bound the competitive glycine site antagonist [3H]MDL105,519 with high affinity (Kd = 5.22 +/- 0. 13 nM) similar to that determined with rat brain membrane fractions. This high affinity binding could be competed by the glycine site antagonist 7-chlorokynurenic acid as well as the agonists glycine and D-serine but not by L-glutamate. This indicates that the S1 and S2 domains of the NR1 subunit are sufficient for the formation of a glycine binding site that displays pharmacological properties similar to those of the NMDA receptor in vivo.     

3,4 dichlorobenzamil-sensitive, monovalent cation channel induced by palytoxin in cultured aortic myocytes

1. Smooth muscle cells were dispersed from rat aorta and then cultured. The action of palytoxin on rat aortic myocytes was analysed by measurement of 22Na+ uptake and single channel recording techniques. 2. Palytoxin induced an increase in 22Na+ uptake, with a concentration of 50 nM producing half-maximal activation. The action of palytoxin was inhibited by amiloride derivatives and by ouabain. The concentrations of inhibitor producing half-maximal inhibition were 10 microM for 3,4 dichlorobenzamil, 30 microM for benzamil, 100 microM for phenamil and 1 mM for ouabain. 3. In outside-out patches, palytoxin induced single channel currents that reversed near 0 mV with NaCl or KCl in the extracellular solution, but were outward with N-methyl-D-glucamine chloride or CaCl2 (110 mM), indicating that palytoxin induced a cation channel permeable to Na+ and K+ (PK/PNa = 1.2) but not to Ca2+ (PK/PCa > 30) or to N-methyl-D-glucamine (NMDG) (PK/PNMDG > 11) The unit channel conductance was 11-14 pS. 4. A high (> 0.1 mM) extracellular concentration of Ca2+ was necessary to observe channel activation by palytoxin. A high (150 mM) extracellular concentration of K+ partially prevented and reversed channel activation by palytoxin. 5. The channel activity was fully blocked by 3,4 dichlorobenzamil (20 microM) and partially blocked by phenamil (50 microM). It was not reduced by ouabain (200 microM).     

Block and modulation of N-methyl-D-aspartate receptors by polyamines and protons: role of amino acid residues in the transmembrane and pore-forming regions of NR1 and NR2 subunits

N-Methyl-D-aspartate (NMDA) receptors are modulated by extracellular spermine and protons and are blocked in a voltage-dependent manner by spermine and polyamine derivatives such as N1-dansyl-spermine (N1-DnsSpm). The effects of mutations in the first and third transmembrane domains (M1 and M3) and the pore-forming loop (M2) of NMDA receptor subunits were studied. Surprisingly, some mutations in M2 and M3 of the NR1 subunit, including mutations at W608 and N616 in M2, reduced spermine stimulation and proton inhibition. These mutations may have long-range allosteric effects or may change spermine- and pH-dependent gating processes rather than directly affecting the binding sites for these modulators because spermine stimulation and proton inhibition are not voltage dependent and are thought to involve binding sites outside the pore-forming regions of the receptor. A number of mutations in M1-M3, including mutations at tryptophan and tyrosine residues near the extracellular sides of M1 and M3, reduced block by spermine and N1-DnsSpm. The effects of these mutants on channel block were characterized in detail by using N1-DnsSpm, which produces block but not stimulation of NMDA receptors. Block by N1-DnsSpm was studied by using voltage ramps analyzed with the Woodhull model of channel block. Mutations at W563 (in M1) and E621 (immediately after M2) in the NR1A subunit and at Y646 (in M3) and N616 (in the M2 loop) in the NR2B subunit reduced the affinity for N1-DnsSpm without affecting the voltage dependence of block. These residues may form part of a binding site for N1-DnsSpm. Mutation of a tryptophan residue at position W607 in the M2 region of NR2B greatly reduced block by N1-DnsSpm, and N1-DnsSpm could easily permeate channels containing this mutation. The results suggest that at least parts of the M1 and M3 segments contribute to the pore or vestibule of the NMDA channel and that a tryptophan in M2 (W607 in NR2B) may contribute to the narrow constriction of the pore.     

Analysis of leak current properties in the lobster stretch receptor neurone

Experiments were performed to characterize the so-called leak current of the slowly adapting stretch receptor neurone of the European lobster with respect to its ionic basis, its kinetics and its pharmacology. Estimates of the leak current were obtained by subtraction of a Na-K pump current and of an unspecific impalement current from a non-dynamic ('instantaneous') current, recorded in a voltage range from approximately -120 to approximately -30 mV, after blockage of spike-generating currents and a hyperpolarization-activated inwardly rectifying current (Q-current). The leak current, estimated in this way, was seen to reverse direction at the cell's K+ equilibrium voltage, thus indicating that it is carried by K+ passing through channels which, also, proved to be permeable to Rb+ and NH4+, but not permeable to Na+ or Cl- to any significant extent. Kinetically, the leak current was found to be characterized by being enhanced by increases in extracellular K+ and by being subject to outward rectification, most distinctly at elevated extracellular [K+]. In quantitative terms, these kinetic properties could be accounted for by a mathematical model comprising (1) a one-site two-barrier Eyring formulation describing ion permeation through membrane channels and (2) an ordinary dose-response relationship describing the channel-opening effect of K+ at an extracellular regulatory site. Pharmacologically, the leak current proved to be distinguished by being reversibly blockable, in a non-voltage dependent manner, by CO2+ (Kd = 0.9 mM, Hill coefficient 1.1) and procaine, but not by Ba2+, Gd3+, bupivacaine (a local anesthetic), or other K+ channel blockers such as TEA, 4-AP and Cs+. It is concluded that, in native unimpaled cells, the K+ carried leak current (1) is setting the resting voltage together with the (mainly) Na(+)-carried Q-current and the Na-K pump current, (2) is determining the cell's firing threshold, together with the spike generating currents, and (3) is also stabilizing the cell's membrane excitability in conditions of varying extracellular [K+], by virtue of its K+ sensitivity.     

Genomic organization of the UBE3A/E6-AP gene and related pseudogenes

Inactivation is a widespread property of voltage-gated ion channels. Recent molecular biological advances in the potassium channel field have elucidated two mechanistically distinct types of inactivation, N-type and C-type. Both of these mechanisms are partially coupled to activation and are usually voltage insensitive once activation is complete. This study compared the effects of a hypothetical open channel blocker on macroscopic currents by using two different models of the same cardiac transient outward current channel. Model 1 is a Hodgkin-Huxley-like model in which inactivation is an independent voltage-sensitive process. Model 2 is a model in which inactivation is voltage insensitive but is partially coupled to activation. Both models have been shown to reproduce closely the experimentally observed current. However, when modelling open channel block, the two models can differ substantially in their equilibrium degree of drug binding. This difference in equilibrium can make substantial changes in the rate of current recovery in subsequent depolarizations. It is shown that, for a rapid series of depolarizations, the time course of development of block and the degree of steady state block can differ substantially between the two models. In conclusion, molecular mechanisms of inactivation must be taken into account when modelling conformation-specific drug binding and use dependence.     

Mechanism of action of neurotoxins acting on the inactivation of voltage-gated sodium channels

This review focuses on the mechanism(s) of action of neurotoxins acting on the inactivation of voltage-gated Na channels. Na channels are transmembrane proteins which are fundamental for cellular communication. These proteins form pores in the plasma membrane allowing passive ionic movements to occur. Their opening and closing are controlled by gating systems which depend on both membrane potential and time. Na channels have three functional properties, mainly studied using electrophysiological and biochemical techniques, to ensure their role in the generation and propagation of action potentials: 1) a highly selectivity for Na ions, 2) a rapid opening ("activation"), responsible for the depolarizing phase of the action potential, and 3) a late closing ("inactivation") involved in the repolarizing phase of the action potential. As an essential protein for membrane excitability, the Na channel is the specific target of a number of vegetal and animal toxins which, by binding to the channel, alter its activity by affecting one or more of its properties. At least six toxin receptor sites have been identified on the neuronal Na channel on the basis of binding studies. However, only toxins interacting with four of these sites (sites 2, 3, 5 et 6) produce alterations of channel inactivation. The maximal percentage of Na channels modified by the binding of neurotoxins to sites 2 (batrachotoxin and some alkaloids), 3 (alpha-scorpion and sea anemone toxins), 5 (brevetoxins and ciguatoxins) et 6 (delta-conotoxins) is different according to the site considered. However, in all cases, these channels do not inactivate. Moreover, Na channels modified by toxins which bind to sites 2, 5 and 6 activate at membrane potentials more negative than do unmodified channels. The physiological consequences of Na channel modifications, induced by the binding of neurotoxins to sites 2, 3, 5 and 6, are (i) an inhibition of cellular excitability due to an important membrane depolarization (site 2), (ii) a decrease of cellular excitability due to an important increase in the action potential duration (site 3) and (iii) an increase in cellular excitability which results in spontaneous and repetitive firing of action potentials (sites 5 and 6). The biochemical and electrophysiological studies performed with these toxins, as well as the determination of their molecular structure, have given basic information on the function and structure of the Na channel protein. Therefore, various models representing the different states of Na channels have been proposed to account for the neurotoxin-induced modifications of Na inactivation. Moreover, the localization of receptor binding sites 2, 3, 5 et 6 for these toxins on the neuronal Na channel has been deduced and the molecular identification of the recognition site(s) for some of them has been established on the alpha sub-unit forming the Na channel protein.     

ERP mapping in phonological and lexical semantic monitoring tasks: A study complementing previous PET results

When attached to specific sites near the S4 segment of the nonconducting (W434F) Shaker potassium channel, the fluorescent probe tetramethylrhodamine maleimide undergoes voltage-dependent changes in intensity that correlate with the movement of the voltage sensor (Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Science. 271:213-216; Cha, A., and F. Bezanilla. 1997. Neuron. 19:1127-1140). The characteristics of this voltage-dependent fluorescence quenching are different in a conducting version of the channel with a different pore substitution (T449Y). Blocking the pore of the T449Y construct with either tetraethylammonium or agitoxin removes a fluorescence component that correlates with the voltage dependence but not the kinetics of ionic activation. This pore-mediated modulation of the fluorescence quenching near the S4 segment suggests that the fluorophore is affected by the state of the external pore. In addition, this modulation may reflect conformational changes associated with channel opening that are prevented by tetraethylammonium or agitoxin. Studies of pH titration, collisional quenchers, and anisotropy indicate that fluorophores attached to residues near the S4 segment are constrained by a nearby region of protein. The mechanism of fluorescence quenching near the S4 segment does not involve either reorientation of the fluorophore or a voltage-dependent excitation shift and is different from the quenching mechanism observed at a site near the S2 segment. Taken together, these results suggest that the extracellular portion of the S4 segment resides in an aqueous protein vestibule and is influenced by the state of the external pore.     

Clonal composition of malignant fibrous histiocytoma: analysis by PCR-based assay of the human androgen receptor gene (HUMARA)

Poly(A)+ mRNA was isolated from leaves of potato plants (Solanum tuberosum L. cv. Desiree) according to standard protocols. This poly(A)+ mRNA was injected via glass microcapillaries into oocytes that were surgically removed from the African clawed toad Xenopus laevis. As a control, oocytes were either injected with H2O or remained untreated. Three days after injection the oocytes were analyzed by two electrode voltage clamping. Current voltage analysis revealed that a K+ channel from potato was functionally expressed in injected oocytes. The identity of this K+ channel was confirmed by its substrate specificity and a shift in the reversal potential. In particular, when the outside K+ concentration was increased the reversal potential of poly(A)+ injected oocytes shifted to more positive values. Furthermore, K+ outward currents declined when the outside K+ concentration was raised from 0.1 to 100 mM. Inward currents increased with an elevation of the K+ concentration. Several pharmaceuticals were tested for their potential to block this K+ channel. As a result, the channel was completely blocked by BaCl2. A three state reaction kinetic model was used to simulate the currents through the K+ transport protein as function of the extracellular K+ concentration. In particular, the simulation revealed current voltage relations that exactly matched the measured ones. Saturation of current voltage curves emerged from the simulation as a consequence of high extracellular potassium concentration.