The V410M mutation associated with pyrethroid resistance in Heliothis virescens reduces the pyrethroid sensitivity of house fly sodium channels expressed in Xenopus oocytes

Some strains of Heliothis virescens carry a novel sodium channel mutation, corresponding to the replacement of Val410 by Met (designated V410M) in the house fly Vssc1 sodium channel, that is genetically and physiologically associated with pyrethroid resistance. To test the functional significance of this mutation, we created a house fly Vssc1 sodium channel containing the V410M mutation by site-directed mutagenesis, expressed wildtype and specifically mutated sodium channels in Xenopus laevis oocytes, and evaluated the effects of the V410M mutation on the functional and pharmacological properties of the expressed channels by two-electrode voltage clamp. The V410M mutation caused depolarizing shifts of approximately 9mV and approximately 5mV in the voltage dependence of activation and steady-state inactivation, respectively, of Vssc1 sodium channels. The V410M mutation also reduced the sensitivity of Vssc1 sodium channels to the pyrethroid cismethrin at least 10-fold and accelerated the decay of cismethrin-induced sodium tail currents. The degree of resistance conferred by the V410M mutation in the present study is sufficient to account for the degree of pyrethroid resistance in H. virescens that is associated with this mutation. Although Val410 is located in a sodium channel segment identified as part of the binding site for batrachotoxin, the V410M mutation did not alter the sensitivity of house fly sodium channels to batrachotoxin. The effects of the V410M mutation on the voltage dependence and cismethrin sensitivity of Vssc1 sodium channels were indistinguishable from those caused by another sodium channel point mutation, replacement of Leu1014 by Phe (L1014F), that is the cause of knockdown resistance to pyrethroids in the house fly. The positions of the V410M and L1014F mutations in models of the tertiary structure of sodium channels suggest that the pyrethroid binding site on the sodium channel alpha subunit is located at the interface between sodium channel domains I and II.     

Mutations in the house fly Vssc1 sodium channel gene associated with super-kdr resistance abolish the pyrethroid sensitivity of Vssc1/tipE sodium channels expressed in Xenopus oocytes

The super-kdr insecticide resistance trait of the house fly confers resistance to pyrethroids and DDT by reducing the sensitivity of the fly nervous system. The super-kdr genetic locus is tightly linked to the Vssc1 gene, which encodes a voltage-sensitive sodium channel alpha subunit that is the principal site of pyrethroid action. DNA sequence analysis of Vssc1 alleles from several independent super-kdr fly strains identified two amino acid substitutions associated with the super-kdr trait: replacement of leucine at position 1014 with phenylalanine (L1014F), which has been shown to cause the kdr resistance trait in this species, and replacement of methionine at position 918 with threonine (M918T). We examined the functional significance of these mutations by expressing house fly sodium channels containing them in Xenopus laevis oocytes and by characterizing the biophysical properties and pyrethroid sensitivities of the expressed channels using two-electrode voltage clamp. House fly sodium channels that were specifically modified by site-directed mutagenesis to contain the M918T/L1014F double mutation gave reduced levels of sodium current expression in oocytes but otherwise exhibited functional properties similar to those of wildtype channels and channels containing the L1014F substitution. However, M918T/L1014F channels were completely insensitive to high concentrations of the pyrethroids cismethrin and cypermethrin. House fly sodium channels specifically modified to contain the M918T single mutation, which is not known to exist in nature except in association with the L1014F mutation, gave very small sodium currents in oocytes. Assays of these currents in the presence of high concentrations of cismethrin suggest that this mutation alone is sufficient to abolish the pyrethroid sensitivity of house fly sodium channels. These results define the functional significance of the Vssc1 mutations associated with the super-kdr trait of the house fly and are consistent with the hypothesis that the super-kdr trait arose by selection of a second-site mutation (M918T) that confers to flies possessing it even greater resistance than the kdr allele containing the L1014F mutation.     

Cloning and functional characterization of a putative sodium channel auxiliary subunit gene from the house fly (Musca domestica)

The functional expression of cloned Drosophila melanogaster and house fly (Musca domestica) voltage-sensitive sodium channels in Xenopus oocytes is enhanced, and the inactivation kinetics of the expressed channels are accelerated, by coexpression with the tipE protein, a putative sodium channel auxiliary subunit encoded by the tipE gene of D. melanogaster. These results predict the existence of a tipE ortholog in the house fly. Using a PCR-based homology probing approach, we isolated cDNA clones encoding an ortholog of tipE (designated Vssc beta) from adult house fly heads. Clones comprising 3444 bp of cDNA sequence contained a 1317 bp open-reading frame encoding a 438 amino acid protein. The predicted Vssc beta protein exhibited 72% amino acid sequence identity to the entire D. melanogaster tipE protein sequence and 97% identity within the two hydrophobic segments identified as probable transmembrane domains. Coexpression of Vssc beta with the house fly sodium channel alpha subunit (Vssc1) in oocytes enhanced the level of sodium current expression five-fold and accelerated the rate of sodium current inactivation 2.2-fold. Both of these effects were significantly larger in magnitude than the corresponding effects of the D. melanogaster tipE protein on the expression and kinetics of Vssc1 sodium channels. These results identify a second example of a putative sodium channel auxiliary subunit from an insect having functional but not structural homology to vertebrate sodium channel beta subunits.     

Functional Expression of Drosophila para Sodium Channels : Modulation by the Membrane Protein TipE and Toxin Pharmacology

The Drosophila para sodium channel α subunit was expressed in Xenopus oocytes alone and in combination with tipE, a putative Drosophila sodium channel accessory subunit. Coexpression of tipE with para results in elevated levels of sodium currents and accelerated current decay. Para/TipE sodium channels have biophysical and pharmacological properties similar to those of native channels. However, the pharmacology of these channels differs from that of vertebrate sodium channels: (a) toxin II from Anemonia sulcata, which slows inactivation, binds to Para and some mammalian sodium channels with similar affinity (K _d ≅ 10 nM), but this toxin causes a 100-fold greater decrease in the rate of inactivation of Para/TipE than of mammalian channels; (b) Para sodium channels are >10-fold more sensitive to block by tetrodotoxin; and (c) modification by the pyrethroid insecticide permethrin is >100-fold more potent for Para than for rat brain type IIA sodium channels. Our results suggest that the selective toxicity of pyrethroid insecticides is due at least in part to the greater affinity of pyrethroids for insect sodium channels than for mammalian sodium channels.     

Three mutations identified in the voltage-sensitive sodium channel alpha-subunit gene of permethrin-resistant human head lice reduce the permethrin sensitivity of house fly Vssc1 sodium channels expressed in Xenopus oocytes

Point mutations in the para-orthologous sodium channel alpha-subunit of the head louse (M815I, T917I, and L920F) are associated with permethrin resistance and DDT resistance. These mutations were inserted in all combinations using site-directed mutagenesis at the corresponding amino acid sequence positions (M827I, T929I, and L932F) of the house fly para-orthologous voltage-sensitive sodium channel alpha-subunit (Vssc1(WT)) gene and heterologously co-expressed with the sodium channel auxiliary subunit of house fly (Vsscbeta) in Xenopus oocytes. The double mutant possessing M827I and T929I (Vssc1(MITI)/Vsscbeta) caused a approximately 4.0mV hyperpolarizing shift and the triple mutant, Vssc1(MITILF)/Vsscbeta, caused a approximately 3.2mV depolarizing shift in the voltage dependence of activation curves. Vssc1(MITI)/Vsscbeta, Vssc1(TILF)/Vsscbeta, and Vssc1(MITILF)/Vsscbeta caused depolarizing shifts ( approximately 6.6, approximately 7.6, and approximately 8.8mV, respectively) in the voltage dependence of steady-state inactivation curves. The M827I and L932F mutations reduced permethrin sensitivity when expressed alone but the T929I mutation, either alone or in combination, virtually abolished permethrin sensitivity. Thus, the T929I mutation is the principal cause of permethrin resistance in head lice. Comparison of the expression rates of channels containing single, double and triple mutations with that of Vssc1(WT)/Vsscbeta channels indicates that the M827I mutation may play a role in rescuing the decreased expression of channels containing T929I.     

Pyrethroids Differentially Alter Voltage-Gated Sodium Channels from the Honeybee Central Olfactory Neurons

The sensitivity of neurons from the honey bee olfactory system to pyrethroid insecticides was studied using the patch-clamp technique on central ‘antennal lobe neurons’ (ALNs) in cell culture. In these neurons, the voltage-dependent sodium currents are characterized by negative potential for activation, fast kinetics of activation and inactivation, and the presence of cumulative inactivation during train of depolarizations. Perfusion of pyrethroids on these ALN neurons submitted to repetitive stimulations induced (1) an acceleration of cumulative inactivation, and (2) a marked slowing of the tail current recorded upon repolarization. Cypermethrin and permethrin accelerated cumulative inactivation of the sodium current peak in a similar manner and tetramethrin was even more effective. The slow-down of channel deactivation was markedly dependent on the type of pyrethroid. With cypermethrin, a progressive increase of the tail current amplitude along with successive stimulations reveals a traditionally described use-dependent recruitment of modified sodium channels. However, an unexpected decrease in this tail current was revealed with tetramethrin. If one considers the calculated percentage of modified channels as an index of pyrethroids effects, ALNs are significantly more susceptible to tetramethrin than to permethrin or cypermethrin for a single depolarization, but this difference attenuates with repetitive activity. Further comparison with peripheral neurons from antennae suggest that these modifications are neuron type specific. Modeling the sodium channel as a multi-state channel with fast and slow inactivation allows to underline the effects of pyrethroids on a set of rate constants connecting open and inactivated conformations, and give some insights to their specificity. Altogether, our results revealed a differential sensitivity of central olfactory neurons to pyrethroids that emphasize the ability for these compounds to impair detection and processing of information at several levels of the bees olfactory pathway.     

Tetrodotoxin-sensitive sodium current in native Xenopus oocytes

Depolarization of oocytes of Xenopus laevis usually elicits mainly passive currents, and a calcium-dependent chloride current. However, oocytes obtained from some donors show, in addition, a transient inward current on depolarization to potentials beyond ca. -40 mV. This current is abolished by tetrodotoxin at submicromolar concentrations, and is prolonged by veratrine; thus, it probably arises through sodium channels of a type similar to those found in nerve and muscle cells. However, the kinetics of the sodium currents varied between oocytes from different donors; this result suggests that genes encoding different sodium channels may be expressed in oocytes from different donors. The presence of these native channels may complicate experiments to study the expression of exogenous sodium channels encoded by foreign messenger RNAs injected into the oocyte.     

Independent and joint modulation of rat Nav1.6 voltage-gated sodium channels by coexpression with the auxiliary β1 and β2 subunits

The Na_v1.6 voltage-gated sodium channel α subunit isoform is the most abundant isoform in the brain and is implicated in the transmission of high frequency action potentials. Purification and immunocytochemical studies imply that Na_v1.6 exist predominantly as Na_v1.6 + β1 + β2 heterotrimeric complexes. We assessed the independent and joint effects of the rat β1 and β2 subunits on the gating and kinetic properties of rat Na_v1.6 channels by recording whole-cell currents in the two-electrode voltage clamp configuration following transient expression in Xenopus oocytes. The β1 subunit accelerated fast inactivation of sodium currents but had no effect on the voltage dependence of their activation and steady-state inactivation and also prevented the decline of currents following trains of high-frequency depolarizing prepulses. The β2 subunit selectively retarded the fast phase of fast inactivation and shifted the voltage dependence of activation towards depolarization without affecting other gating properties and had no effect on the decline of currents following repeated depolarization. The β1 and β2 subunits expressed together accelerated both kinetic phases of fast inactivation, shifted the voltage dependence of activation towards hyperpolarization, and gave currents with a persistent component typical of those recorded from neurons expressing Na_v1.6 sodium channels. These results identify unique effects of the β1 and β2 subunits and demonstrate that joint modulation by both auxiliary subunits gives channel properties that are not predicted by the effects of individual subunits.     

An important role of a pyrethroid-sensing residue F1519 in the action of the N-alkylamide insecticide BTG 502 on the cockroach sodium channel

Deltamethrin, a pyrethroid insecticide, and BTG 502, an alkylamide insecticide, target voltage-gated sodium channels. Deltamethrin binds to a unique receptor site and causes prolonged opening of sodium channels by inhibiting deactivation and inactivation. Previous ²²Na⁺ influx and receptor binding assays using mouse brain synaptoneurosomes showed that BTG 502 antagonized the binding and action of batrachotoxin (BTX), a site 2 sodium channel neurotoxin. However, the effect of BTG 502 has not been examined directly on sodium channels expressed in Xenopus oocytes. In this study, we examined the effect of BTG 502 on wild-type and mutant cockroach sodium channels expressed in Xenopus oocytes. Toxin competition experiments confirmed that BTG 502 antagonizes the action of BTX and possibly shares a common receptor site with BTX. However, unlike BTX which causes persistent activation of sodium channels, BTG 502 reduces the amplitude of peak sodium current. A previous study showed that BTG 502 was more toxic to pyrethroid-resistant house flies possessing a super-kdr (knockdown resistance) mechanism than to pyrethroid-susceptible house flies. However, we found that the cockroach sodium channels carrying the equivalent super-kdr mutations (M918T and L1014F) were not more sensitive to BTG 502 than the wild-type channel. Instead, a kdr mutation, F1519I, which reduces pyrethroid binding, abolished the action of BTG 502. These results provide evidence the actions of alkylamide and pyrethroid insecticides require a common sodium channel residue.     

A Single Crossing-Over Event in Voltage-Sensitive Na+ Channel Genes May Cause Critical Failure of Dengue Mosquito Control by Insecticides

The voltage-sensitive sodium (Na⁺) channel (Vssc) is the target site of pyrethroid insecticides. Pest insects develop resistance to this class of insecticide by acquisition of one or multiple amino acid substitution(s) in this channel. In Southeast Asia, two major Vssc types confer pyrethroid resistance in the dengue mosquito vector Aedes aegypti, namely, S989P+V1016G and F1534C. We expressed several types of Vssc in Xenopus oocytes and examined the effect of amino acid substitutions in Vssc on pyrethroid susceptibilities. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to permethrin by 100- and 25-fold, respectively, while S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to permethrin by 1100-fold. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to deltamethrin by 10- and 1-fold (no reduction), respectively, but S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to deltamethrin by 90-fold. These results imply that pyrethroid insecticides are highly likely to lose their effectiveness against A. aegypti if such a Vssc haplotype emerges as the result of a single crossing-over event; thus, this may cause failure to control this key mosquito vector. Here, we strongly emphasize the importance of monitoring the occurrence of triple mutations in Vssc in the field population of A. aegypti.     

Ionic currents of the nodal membrane underlying the fastest saltory conduction in myelinated giant nerve fibers of the shrimp Penaeus japonicus

The myelinated giant nerve fiber of the shrimp, Penaeus japonicus, is known to have the fastest velocity of saltatory impulse conduction among all nerve fibers so far studied, owing to its long distances between nodal regions and large diameter. For a better understanding of the basis of this fast conduction, a medial giant fiber of the ventral nerve cord of the shrimp was isolated, and ionic currents of its presynaptic membrane (a functional node) were examined using the sucrose-gap voltage-clamp method. Inward currents induced by depolarizing voltage pulses had a maximum value of 0.5 μA and a reversal potential of 120 mV. These currents were completely suppressed by tetrodotoxin and greatly prolonged by scorpion toxin, suggesting that they are the Na current. Both activation and inactivation kinetics of the Na current were unusually rapid in comparison with those of vertebrate nodes. According to a rough estimation of the excitable area, the density of Na current reached 500 mA/cm². In many cases, the late outward currents were induced only by depolarizing pulses larger than 50 mV in amplitude. The slope conductance measured from late currents were mostly smaller than that measured from the Na current, suggesting a low density of K channels in the synaptic membrane. These characteristics are in good harmony with the fact that the presynaptic membrane plays a role as functional node in the fastest impulse conduction of this nerve fiber.     

Potential-dependent changes in ionic selectivity of batrachotoxin-modified sodium channels of frog nerve fiber

Ionic currents through sodium channels modified by batrachotoxin were measured by the voltage clamp method on a myelinated frog nerve fiber membrane. The reversal potential (E_rev) of steady-state currents was shown to be on the average 5 mV less positive than E_rev corresponding to the initial (peak) values of the currents. The results of control experiments using procaine and tetrodotoxin showed that the change in E_rev observed during a depolarizing pulse is not connected with the presence of unmodified sodium channels or unblocked potassium channels, with nonlinearity of leakage, or with a change in transmembrane gradients of current-carrving cations. In experiments with measurement of "instant" currents it was shown that E_rev becomes less positive as the amplitude and duration of preliminary depolarization increase. The results support the view that sodium-potassium selectivity of batrachotoxin-modified sodium channels depends on potential.     

The sodium current underlying action potentials in guinea pig hippocampal CA1 neurons

Neurons were acutely dissociated from the CA1 region of hippocampal slices from guinea pigs. Whole-cell recording techniques were used to record and control membrane potential. When the electrode contained KF, the average resting potential was about -40 mV and action potentials in cells at -80 mV (current-clamped) had an amplitude greater than 100 mV. Cells were voltage-clamped at 22-24 degrees C with electrodes containing CsF. Inward currents generated with depolarizing voltage pulses reversed close to the sodium equilibrium potential and could be completely blocked with tetrodotoxin (1 microM). The amplitude of these sodium currents was maximal at about -20 mV and the amplitude of the tail currents was linear with potential, which indicates that the channels were ohmic. The sodium conductance increased with depolarization in a range from -60 to 0 mV with an average half-maximum at about -40 mV. The decay of the currents was not exponential at potentials more positive than -20 mV. The time to peak and half-decay time of the currents varied with potential and temperature. Half of the channels were inactivated at a potential of -75 mV and inactivation was essentially complete at -40 to -30 mV. Recovery from inactivation was not exponential and the rate varied with potential. At lower temperatures, the amplitude of sodium currents decreased, their time course became longer, and half-maximal inactivation shifted to more negative potentials. In a small fraction of cells studied, sodium currents were much more rapid but the voltage dependence of activation and inactivation was very similar.     

A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Numerous point mutations in sodium channel genes have been identified in pyrethroid-resistant insect species, and many have been confirmed to reduce or abolish sensitivity of channels expressed in Xenopus oocytes to pyrethroids. Recently, several novel mutations were reported in sodium channel genes of pyrethroid-resistant Aedes mosquito populations. One of the mutations is a phenylalanine (F) to cysteine (C) change in segment 6 of domain III (IIIS6) of the Aedes mosquito sodium channel. Curiously, a previous study showed that alanine substitution of this F did not alter the action of deltamethrin, a type II pyrethroid, on a cockroach sodium channel. In this study, we changed this F to C in a pyrethroid-sensitive cockroach sodium channel and examined mutant channel sensitivity to permethrin as well as five other type I or type II pyrethroids in Xenopus oocytes. Interestingly, the F to C mutation drastically reduced channel sensitivity to three type I pyrethroids, permethrin, NRDC 157 (a deltamethrin analogue lacking the ??-cyano group) and bioresemthrin, but not to three type II pyrethroids, cypermethrin, deltamethrin and cyhalothrin. These results confirm the involvement of the F to C mutation in permethrin resistance, and raise the possibility that rotation of type I and type II pyrethroids might be considered in the control of insect pest populations where this particular mutation is present.     

Expression and Functional Characterization of the Human Ether-à-go-go-Related Gene (<Emphasis Type="Italic">HERG</Emphasis>) K<Superscript>+</Superscript> Channel Cardiac Splice Variant in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Oocytes

HERG C_Cardiac, a C-terminal splice variant of the human ether-à-go-go-related gene (HERG A), was identified and found to be 100% homologous to HERG_USO. Real-time polymerase chain reaction data indicated that in the human heart HERG C_Cardiac mRNA was expressed eight times more than HERG A, whereas in human ventricular tissue it was expressed six times more than HERG A. A HERG C_Cardiac-green fluorescence protein (GFP) construct was heterologously expressed in Xenopus oocytes. Confocal micrographs revealed that HERG C_Cardiac was mainly expressed in the plasma membrane. HERG C_Cardiac channel expressed in oocytes produced slower inactivating outward currents and faster deactivating tail currents than those of HERG A channel. Equal amounts of HERG A and HERG C_Cardiac cRNA coinjected into oocytes formed intermediate HERG A + HERG C_Cardiac heteromultimers, which was reconfirmed by immunoprecipitation experiments with a HERG A N-terminal antibody. These heteromultimers had different inactivation, deactivation and activation kinetics from those of HERG A and HERG C_Cardiac channels. HERG A + HERG C_Cardiac heteromultimers significantly reduced the model action potential mean amplitude and increased the fast and slow inactivation τ values of the action potential repolarization phase, suggesting involvement of HERG A and HERG C_Cardiac heteromultimers in modulation of the refractory interval.     

A voltage-dependent gating transition induces use-dependent block by tetrodotoxin of rat IIA sodium channels expressed in Xenopus oocytes.

We have utilized molecular biological techniques to demonstrate that rat IIA sodium channels expressed in Xenopus oocytes were blocked by tetrodotoxin (TTX) in a use-dependent manner. This use dependence was the result of an increased affinity of the channels for TTX upon depolarization, most likely due to a conformational change in the channel. Using a mutant with a slower macroscopic rate of inactivation, we have demonstrated that this conformational change is not the transition into the fast-inactivated state. The transition is probably one occurring during activation of the channel, as suggested by the fact that one sodium channel mutant demonstrated comparable depolarizing shifts in the voltage dependence of both activation and use-dependent block by TTX. The transition occurred at potentials more negative than those resulting in channel conductance, suggesting that the conformational change that causes use-dependent block by TTX is a closed-state voltage-dependent gating transition.     

Point mutations in domain III of a Drosophila neuronal Na channel confer resistance to allethrin

Voltage-gated sodium channels are the presumed site of action of pyrethroid insecticides and DDT. We screened several mutant sodium channel Drosophila lines for resistance to type I pyrethroids. In insecticidal bioassays the para(74) and para(DN7) fly lines showed greater than 4-fold resistance to allethrin relative to the allethrin sensitive Canton-S control line. The amino acid substitutions of both mutants are in domain III. The point mutation associated with para(74) lies within the S6 transmembrane region and the amino acid substitution associated with para(DN7) lies within the S4-S5 linker region. These sites are analogous to the mutations in domain II underlying knockdown resistance (kdr) and super-kdr, naturally occurring forms of pyrethroid resistance found in houseflies and other insects. Electrophysiological studies were performed on isolated Drosophila neurons from wild type and para(74) embryos placed in primary culture for three days to two weeks. The mutant para(74) sodium currents were kinetically similar to wild type currents, in activation, inactivation and time to peak. The only observed difference between para(74) and wild-type neurons was in the affinity of the type I pyrethroid, allethrin. Application of 500 nM allethrin caused removal of inactivation and prolonged tail currents in wild type sodium channels but had little or no effect on para(74) mutant sodium channels.     

AtALMT12 represents an R‐type anion channel required for stomatal movement in Arabidopsis guard cells

Stomatal pores formed by a pair of guard cells in the leaf epidermis control gas exchange and transpirational water loss. Stomatal closure is mediated by the release of potassium and anions from guard cells. Anion efflux from guard cells involves slow (S‐type) and rapid (R‐type) anion channels. Recently the SLAC1 gene has been shown to encode the slow, voltage‐independent anion channel component in guard cells. In contrast, the R‐type channel still awaits identification. Here, we show that AtALMT12, a member of the aluminum activated malate transporter family in Arabidopsis, represents a guard cell R‐type anion channel. AtALMT12 is highly expressed in guard cells and is targeted to the plasma membrane. Plants lacking AtALMT12 are impaired in dark‐ and CO₂‐induced stomatal closure, as well as in response to the drought‐stress hormone abscisic acid. Patch‐clamp studies on guard cell protoplasts isolated from atalmt12 mutants revealed reduced R‐type currents compared with wild‐type plants when malate is present in the bath media. Following expression of AtALMT12 in Xenopus oocytes, voltage‐dependent anion currents reminiscent to R‐type channels could be activated. In line with the features of the R‐type channel, the activity of heterologously expressed AtALMT12 depends on extracellular malate. Thereby this key metabolite and osmolite of guard cells shifts the threshold for voltage activation of AtALMT12 towards more hyperpolarized potentials. R‐Type channels, like voltage‐dependent cation channels in nerve cells, are capable of transiently depolarizing guard cells, and thus could trigger membrane potential oscillations, action potentials and initiate long‐term anion and K⁺ efflux via SLAC1 and GORK, respectively.     

Quantal charge redistributions accompanying the structural transitions of sodium channels

Asymmetric displacement currents, I _g , associated with the gating of nerve sodium channels have been recorded in cell-attached macropatches of Xenopus laevis oocytes injected with exogenous mRNA coding for rat-brain-II sodium channels. The I _g properties were found to be similar to those of gating currents previously observed in native nerve preparations. I _g fluctuations were measured in order to ascertain the discreteness of the conformational changes which precede the channel opening. The autocorrelation of the fluctuations is consistent with a shot-like character of the elementary I _g contributions. The variance of the fluctuations indicates that most of the gating-charge movement that accompanies the activation of a single sodium channel occurs in 2 to 3 brief packets, each carrying an equivalent of about 2.3 electron charges.