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Cardiovascular effects induced by snake venoms, in spite of having a crucial role in the outcome of the envenomation, have been less studied than other toxic activities displayed by these venoms. In this study we evaluated acute cardiovascular responses to Bothrops leucurus venom - Bl-V - both in vivo, in anesthetized rats, and in vitro, in isolated rat mesenteric resistance arteries. Bl-V (10-100 μg protein/kg) caused dose-dependent hypotension, followed by gradual recovery (2-20 min) to basal levels, and induced dose-dependent (1-20 μg/mL) vasodilation in pre-contracted arteries, what was more pronounced when the endothelium remained intact. These effects were partially counteracted by pre-treatment with indomethacin (cyclooxygenase inhibitor). Prior incubation of Bl-V with commercial pentavalent Bothrops antivenom also attenuated the cardiovascular effects induced by the venom, in spite of it not being among the venoms used for the development of the bothropic antivenom. Through an approach based on two chromatographic steps and mass spectrometry (MALDI-ToF and MALDI-ISD), a component with acute cardiovascular effects was isolated and identified as the basic phospholipase blD-PLA

Voltage sensor domains (VSDs) regulate ion channels and enzymes by undergoing conformational changes depending on membrane electrical signals. The molecular mechanisms underlying the VSD transitions are not fully understood. Here, we show that some mutations of I241 in the S1 segment of the Shaker Kv channel positively shift the voltage dependence of the VSD movement and alter the functional coupling between VSD and pore domains. Among the I241 mutants, I241W immobilized the VSD movement during activation and deactivation, approximately halfway between the resting and active states, and drastically shifted the voltage activation of the ionic conductance. This phenotype, which is consistent with a stabilization of an intermediate VSD conformation by the I241W mutation, was diminished by the charge-conserving R2K mutation but not by the charge-neutralizing R2Q mutation. Interestingly, most of these effects were reproduced by the F244W mutation located one helical turn above I241. Electrophysiology recordings using nonnatural indole derivatives ruled out the involvement of cation-Π interactions for the effects of the Trp inserted at positions I241 and F244 on the channel’s conductance, but showed that the indole nitrogen was important for the I241W phenotype. Insight into the molecular mechanisms responsible for the stabilization of the intermediate state were investigated by creating in silico the mutations I241W, I241W/R2K, and F244W in intermediate conformations obtained from a computational VSD transition pathway determined using the string method. The experimental results and computational analysis suggest that the phenotype of I241W may originate in the formation of a hydrogen bond between the indole nitrogen atom and the backbone carbonyl of R2. This work provides new information on intermediate states in voltage-gated ion channels with an approach that produces minimum chemical perturbation.

Several naturally occurring polypeptide neurotoxins target specific sites on the voltage-gated sodium channels. Of these, the gating modifier toxins alter the behavior of the sodium channels by stabilizing transient intermediate states in the channel gating pathway. Here we have used an integrated approach that combines electrophysiological and spectroscopic measurements to determine the structural rearrangements modified by the β-scorpion toxin Ts1. Our data indicate that toxin binding to the channel is restricted to a single binding site on domain II voltage sensor. Analysis of Cole-Moore shifts suggests that the number of closed states in the activation sequence prior to channel opening is reduced in the presence of toxin. Measurements of charge–voltage relationships show that a fraction of the gating charge is immobilized in Ts1-modified channels. Interestingly, the charge–voltage relationship also shows an additional component at hyperpolarized potentials. Site-specific fluorescence measurements indicate that in presence of the toxin the voltage sensor of domain II remains trapped in the activated state. Furthermore, the binding of the toxin potentiates the activation of the other three voltage sensors of the sodium channel to more hyperpolarized potentials. These findings reveal how the binding of β-scorpion toxin modifies channel function and provides insight into early gating transitions of sodium channels.

A potent insecticidal toxin, β/δ-PrIT1, molecular mass of 5598.86 [M+H](+), was characterized from Phoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidal spider toxins from the genus Phoneutria. β/δ-PrIT1 was very toxic (LD50 = 4 nmol/g) to flies (Musca domestica), but not to mice (Mus musculus). Kinetic studies showed that (125)I-β/δ-PrIT1 binds to two distinct sites in insect sodium channels, with close affinity (Kd1 = 34.7 pM and Kd2 = 35.1 pM). Its association is rather fast (t1/2(1) = 1.4 min, t1/2(2) = 8.5 min) and its dissociation is a slower process (t1/2(1) = 5.4 min, t1/2(2) = 32.8 min). On rat brain synaptosomes β/δ-PrIT1 partially competed (∼30%) with the beta-toxin (125)I-CssIV, but did not compete with the alpha-toxin of reference (125)I-AaII, nor with the beta-toxin (125)I-TsVII. On cockroach nerve cord synaptosomes, β/δ-PrIT1 did not compete with the anti-insect toxin (125)I-LqqIT1, but it competed (IC50 = 80 pM) with the "alpha-like" toxin (125)I-BomIV. In cockroach neurons, β/δ-PrIT1 inhibited the inactivation of Nav-channels and it shifted the sodium channel activation to hyperpolarizing potentials. These results indicate two different binding sites for β/δ-PrIT1, leading to two different pharmacological responses. β/δ-PrIT1 is one of the most toxic spider toxins to insects without apparent toxicity to mammals, and provide new model for the development of insecticides.

The voltage-dependent displacement of the scorpion Tityus serrulatus α-toxin Ts3 was investigated in native sodium channels of GH3 cells by examining the removal of its effects in toxin-free solution. Toxin at saturating concentration was pulsed (∼1 s) directly onto the cell, thus causing an eight-fold increase of the slow component (τs=6 ms) of fast inactivation, and a three-fold increase of the time constant of its fast component. At 0 mV, maximal conductance was achieved in cells before and after treatment with Ts3, and no displacement of the toxin could be detected. Toxin displacement occurred if stronger depolarising pulses (>100 mV) were applied. The rate of displacement depended on the amplitude and duration of the pulses, and was not related with outward Na+ flux. We propose a model in which activation does not require complete movement of segment S4 of domain IV (IVS4) and that a more extensive movement of this segment is needed for normal fast inactivation. A kinetic model is presented that can account for the typical effects of site 3 toxins. British Journal of Pharmacology (2004) 142, 1115–1122. doi:10.1038/sj.bjp.0705793

A number of neurotoxins from venoms of invertebrates and plants are ligands for voltage-gated Na + channels and are useful tools for studying Na + channel function and structure. Using whole-cell recordings from vagal afferent nodose neurons, we studied neurotoxins that target Na + channels. We asked whether Ts3 (an α-scorpion toxin) and/or veratridine (a lipid-soluble toxin), could modify the TTX-resistant Na + current generated by vagal afferent nodose neurons. Nodose TTX-resistant current was not affected by Ts3, whereas Ts3 slowed inactivation of the current generated by TTX-sensitive current component. We found that veratridine inhibited the TTX-resistant Na + currents on rat nodose neurons. Interestingly, veratridine-modified Na + channels developed a persistent current that accounted for the large tail current observed. We propose that veratridine modifies TTX-resistant Na + channels through a mechanism distinct from its actions on other voltage-gated Na + channels.

Voltage sensor domains (VSDs) regulate ion channels and enzymes by transporting electrically charged residues across a hydrophobic VSD constriction called the gating pore or hydrophobic plug. How the gating pore controls the gating charge movement presently remains debated. Here, using saturation mutagenesis and detailed analysis of gating currents from gating pore mutations in the Shaker Kv channel, we identified statistically highly significant correlations between VSD function and physicochemical properties of gating pore residues. A necessary small residue at position S240 in S1 creates a "steric gap" that enables an intracellular access pathway for the transport of the S4 Arg residues. In addition, the stabilization of the depolarized VSD conformation, a hallmark for most Kv channels, requires large side chains at positions F290 in S2 and F244 in S1 acting as "molecular clamps," and a hydrophobic side chain at position I237 in S1 acting as a local intracellular hydrophobic barrier. Finally, both size and hydrophobicity of I287 are important to control the main VSD energy barrier underlying transitions between resting and active states. Taken together, our study emphasizes the contribution of several gating pore residues to catalyze the gating charge transfer. This work paves the way toward understanding physicochemical principles underlying conformational dynamics in voltage sensors.

In nerves and muscles, action potentials are initiated by the rapid activation of voltage-gated sodium (Nav) channels and terminated by the delayed activation of voltage-gated potassium (Kv) channels. This sequential activation, which is the prerequisite for the genesis of the action potential, requires faster activation kinetics of the voltage-sensor domains (VSD) in Nav as compared to Kv channels. Despite on decades of investigations, the molecular determinants and mechanisms underlying this phenomenon remain elusive. Here, we show that this differential gating is mostly imparted by six conserved hydrophilic residues Thr or Ser located in the S2 and S4 segments of the VSD in domains I-III of Nav channels while these positions are commonly occupied by hydrophobic residues in the VSD of Kv channels. Hydrophilic substitutions at the S2 position in the Shaker Kv channel accelerate the gating charge transfer by decreasing its energy barrier while hydrophilic substitutions at the S4 position speed up VSD activation by destabilizing its resting conformation. Interestingly, these hydrophilic residues are present in a Nav-related gene expressed in an evolutionary-distant unicellular choanoflagellate, suggesting that rapidly-gated Nav channels evolved before the emergence of metazoans and their nervous systems. We also show that the physiological co-expression of the ubiquitous regulatory β1 subunit further accelerates VSD movement in both a neuronal and a muscular Nav, providing a molecular basis for the β1-dependent fast gating mode previously detected from the ionic conductance. Our study uncovers the fundamental molecular determinants and possible mechanisms that enabled the differential gating in sodium and potassium channels and the emergence of the action potential. This work was supported by NIH grant GM030376.

Voltage-sensing in ion channels and some phosphatases relies on the movement of positively-charged residues (gating charges) carried by the fourth (S4) helix of the voltage-sensor domain (VSD). Structures of Kv channels revealed the position of the gating charges in the open/inactivated conformation of the VSD yet the pathway they follow during gating remains unclear. To this aim, we previously showed that at hyperpolarized potentials, the first gating charge of the Shaker K+ channel (R362) resides within atomic proximity of I241, located in the middle of the S1 segment. Here, we show that I241 mutations shift the voltage dependence of channels activation toward positive potentials and this effect is most pronounced for the I241W mutation. This mutation isolates two equal components of the charge movement during activation, thereby stabilizing the S4 in an intermediate position between resting and active states. Using non-natural amino acid incorporation, electrophysiology recordings and molecular dynamic simulations, we show that this S4-immobilization involves a specific interaction between I241W and R365, the second gating charge. We found that this interaction requires the presence of the nitrogen atom of the indole ring and is not affected by a dispersion of its Pi electrons. This interaction is present regardless of whether the S4 moves from its resting, active or relaxed state but the charge component that is being isolated during deactivation only represents 30% compared to 50% during activation. Taken together, our results indicate that each of the gating charges move in slightly different pathways, the overall S4 motion is similar during activation and deactivation, and the electric field is shaped differently between activation and deactivation.

In voltage-dependent ion channels, the movement of the voltage-sensing S4 helices produces gating currents. The charge displaced as a function of the membrane potential (Q-V) is well described by a sequential two-state Boltzmann relation, indicating that there are at least two steps of gating charge movement from their Resting state to the Active state. In addition, it has been shown that at a maintained positive potential, the S4 helices of voltage-gated Na, Ca and K channels and the voltage sensitive phosphatase Ci-VSP, undergo a slower secondary conformational transition stabilizing the sensor in a Relaxed (inactivated) state. From the Relaxed state, the Q-V relation exhibits a strong shift towards negative potentials when compared to the Q-V relation measured from the resting state. We engineered gating perturbations in the Shaker potassium channel, by substituting specific aromatic residues in positions spatially close to the S4. One of these mutants, in position I241 of S1, part of the hydrophobic plug of the voltage sensor, when mutated to tryptophan (I241W), produces a strong split in the Q-V when measured from the resting state. By labeling M356C with TMRM we also find the same split in the fluorescence-voltage curve. We propose that the presence of the tryptophan in the 241 position favors an interaction with one of the positively-charged arginines along the S4, thus stabilizing a fleeting intermediate state in the gating pathway. However, in the I241W mutant, the split in the Q-V almost disappears when the gating currents are measured from the relaxed state and the same result is seen with the fluorescence-voltage curve. This result and the effect of other tryptophan perturbations near the S4 segment strongly support the existence of two structurally distinct gating pathways for the movement of the S4 helices. Supported: NIHGM030376.

A family of 4kDa neurotoxic peptides was purified from venoms of Phoneutria spiders. All have six cysteine residues, and low similarity with other neurotoxins. Three toxins caused moderate inhibition of L-type Ca(2+) channels. The structure of toxin PRTx27C3 was modeled and compared with toxin ADO1. The importance of four residues is suggested.

α-Scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an α-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence–voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

Recently, we proposed a quantitative model to explain the molecular mechanism of action of the Tityus serrulatus Ts3 alpha-toxin on sodium channels. In this model, the toxin acts as a stop that prevents the segment S4 of domain IV from reaching its outermost position, thus impairing the normal fast inactivation without affecting activation. In the present work, we analyze the predictions of the proposed model with regard to the voltage-dependent transitions to and from inactivation. Our results show that the recovery from inactivation was significantly faster in Ts3-bound channels and that there was no significant voltage dependence. The transition to inactivated state from open state in Ts3-modified channels presented a small but significant voltage dependence, which may derive from an intrinsic voltage dependence of inactivation or by a short movement of IVS4 in the presence of bound Ts3. We also studied the thermodynamic parameters of the voltage-dependent displacement of Ts3 from its binding site. We have observed that the activation energy to remove the toxin is 27 kJ/mol, part of which derives from the imposed depolarizing potential and the movement of an equivalent electrical charge of 0.54 e(0). These results support the proposed model.

Voltage-sensors (VS) are protein domains which transduce changes in membrane potential into conformational changes thereby controlling the opening of a pore or the activity of a phosphatase domain. This operation creates measurable gating currents (Ig) by a local rearrangement of charged groups in the VS. Although there is high conservation of these gating charges among VS, the kinetics of Ig varies greatly, spreading over several orders of magnitudes. These kinetic differences explain the temporal separation of Na+ and K+ conductances in excitable cells, critical for the generation and propagation of the action potential. Despite its fundamental importance, the molecular mechanism underlying this phenomenon is not well understood. Here, we identified in the Shaker K+ channel two hydrophobic residues Ile241 and Ile287 located in S1 and S2 respectively which directly interact with S4 arginines during gating charge movement. We show that Shaker gating kinetics can be speeded up ∼ 3 fold by mutating Ile287 to more hydrophilic residues threonine or serine. Interestingly, voltage-dependent sodium channel (Nav) possesses threonines in domains I-III at positions homologous to Ile287 and exhibit Ig ∼ 10 times faster than that of Shaker. Mutating these threonines to isoleucines in Nav1.4 slows down Ig 2-3 times. Moreover, the sensing currents of the voltage-sensitive phosphatase Ci-VSP, which are ∼ 8 times slower than that of Shaker, can be speeded 3-4 fold by substituting Ile126, (homologous to Ile241 in Shaker) to threonine. Taken together, our data indicate that the side chains of these two key positions form a hydrophobic plug separating the external and internal media. This plug constitutes the main energy barrier for charge crossing and its amplitude is modulated by its degree of hydrophobicity, thus controlling the kinetics of the sensor. Support: NIH-GM030376.

ProTxII, a peptide extracted from the venom of the tarantula Thrixopelma pruriens, binds to multiple voltage-gated sodium channel isoforms. In NaV1.2 ProTxII reduces the sodium conductance and decreases total gating charge (Sokolov et al., Mol. Pharm. 73:1020). In this isoform the toxin's effect could be partially reversed with strong depolarization indicating interactions with one or more of the voltage sensors. Mutagenesis data further implicated the domain II voltage sensor in the toxin-channel interaction in NaV1.2. Here we have shown that, as was seen for NaV1.2, ProTxII (2-5μM) produced a decrease in maximum conductance (∼60%) and a decrease in total gating charge (∼20%) in the NaV1.4 isoform expressed in Xenopus oocytes. Unlike NaV1.2, however, these effects on NaV1.4 could not be reversed with strong depolarization. We also discovered a single residue, S660, located at the S3-S4 linker/S4 boundary of DII, which when mutated, renders this channel insensitive to toxin. We used site-specific fluorescent measurements to determine the effect of ProTxII on the movement of individual voltage sensors. In the presence of ProTxII the voltage dependence of the fluorescent signal of DIIS4 was modified, but not eliminated, indicating that this voltage sensor is not completely immobilized upon toxin binding. The fluorescent signals measured from domains I and III were not significantly affected by ProTxII implicating a specific interaction with DII in producing the effect of ProTxII on NaV1.4. Supported by GM30376 (FB) and NS061535-01(GBE).

SummaryMost action potentials are produced by the sequential activation of voltage-gated sodium (Nav) and potassium (Kv) channels. This is mainly achieved by the rapid conformational rearrangement of voltage-sensor (VS) modules in Nav channels, with activation kinetics up to 6-fold faster than Shaker-type Kv channels. Here, using mutagenesis and gating current measurements, we show that a 3-fold acceleration of the VS kinetics in Nav versus Shaker Kv channels is produced by the hydrophilicity of two “speed-control” residues located in the S2 and S4 segments in Nav domains I–III. An additional 2-fold acceleration of the Nav VS kinetics is provided by the coexpression of the β1 subunit, ubiquitously found in mammal tissues. This study uncovers the molecular bases responsible for the differential activation of Nav versus Kv channels, a fundamental prerequisite for the genesis of action potentials.

The Kv7 family of voltage-gated K+ channels consists of five members (Kv7.1- Kv7.5), each showing specific tissue distribution and physiological role. Mutations in Kv7.1 cause the long QT syndrome, whereas Kv7.4 mutations underlie a rare form of slowly progressive deafness (DFNA2); finally, mutations in Kv7.2 and Kv7.3 genes have been identified in families affected by Benign Familial Neonatal Seizures. Disease-causing mutations often affect residues in the voltage-sensing domain of Kv7 subunits. In the present study, we have characterized the ionic and gating current of homomeric neuronally-expressed Kv7 channels (Kv7.2-Kv7.5) using the cut-open oocyte voltage clamp. The ionic and gating current were recorded at 18°C and also at 28°C to speed up gating charge movement. Increasing the recording temperature from 18°C to 28°C caused an accelerated activation/deactivation kinetics of the ionic currents in all homomeric Kv7 channels (the Q10 for the activation kinetics at 0 mV was 3.8, 4.1, 8.3, and 2.8 for Kv7.2, Kv7.3, Kv7.4 and Kv7.5). Moreover, currents carried by Kv7.4 channels (and less so Kv7.2) also showed a significant increase in their maximal value. Gating currents were only resolved in Kv7.4 and Kv7.5 channels, possibly because of an higher membrane channel density; ON gating charges at saturated potential (+40mV) amounted to 1.34±0.34 (Kv7.4) and 0.79±0.20 nC (Kv7.5). At 28°C, Kv7.4 gating currents had the following salient properties: 1) similar time integral of ON and OFF, indicating charge conservation; 2) a left-shift in the V1/2 of the ON gating charge when compared to ionic currents; 3) a rising phase in the OFF gating charge after depolarizations to values >0 mV. These results represent the first description of Kv7.4 and Kv7.5 gating currents and may help to clarify the molecular consequence of disease-causing mutations affecting channel gating. Supported by NIH GM30376 and Telethon GGP07125.