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.     

Actions of the pyrethroid insecticides cismethrin and cypermethrin on house fly Vssc1 sodium channels expressed in Xenopus oocytes

Voltage-sensitive sodium channels encoded by the Vssc1 gene of the house fly (Musca domestica) were expressed in Xenopus laevis oocytes in combination with the tipE gene product of Drosophila melanogaster and were characterized by two-electrode voltage clamp. Vssc1/tipE sodium channels expressed in oocytes were highly sensitive to tetrodotoxin; half-maximal inhibition of sodium currents by tetrodotoxin was obtained at a concentration of 2.4 nM. Cismethrin, a pyrethroid that produces Type I effects on intact nerve, slowed the inactivation of sodium currents carried by Vssc1/tipE channels during a depolarizing pulse and induced a tail current after repolarization that decayed with a first-order time constant of approximately 650 ms. The voltage dependence of activation and steady-state inactivation of cismethrin-modified channels were shifted to more negative potentials. Cypermethrin, a pyrethroid with Type II effects on intact nerve, also prolonged the inactivation of Vssc1/tipE sodium channels and induced a tail current. However, the cypermethrin-induced tail current was extremely persistent, decaying with a first-order time constant of approximately 42 s. Unlike cismethrin, the effect of cypermethrin was use dependent, requiring repeated depolarizing pulses for the full development of modified sodium currents. The divergent effects of cismethrin and cypermethrin on Vssc1/tipE sodium channels expressed in oocytes are consistent with the actions of these and related compounds on sodium channels in invertebrate and vertebrate nerve preparations and provide insight into the mechanisms underlying the production of Type I and II effects on neuronal excitability. Arch. Insect Biochem. Physiol. 38:126–136, 1998. © 1998 Wiley-Liss, Inc.     

Novel sodium channel gene mutations in Blattella germanica reduce the sensitivity of expressed channels to deltamethrin

Pyrethroid insecticides alter the normal gating of voltage-gated sodium channels in the nervous system. Three sodium channel mutations (E434K, C764R, L993F) were recently identified in pyrethroid resistant German cockroach populations. In this report, we show that the L993F mutation decreased sodium channel sensitivity to the pyrethroid, deltamethrin, by five-fold in Xenopus oocytes. In contrast, neither E434K nor C764R alone decreased channel sensitivity to deltamethrin. However, E434K or C764R combined with L993F reduced deltamethrin sensitivity by 100-fold. Furthermore, concomitant presence of all three mutations (KRF) reduced channel sensitivity to deltamethrin by 500-fold. None of the mutations significantly affected channel gating. However, sodium current amplitudes from the mutant sodium channel carrying either E434K or C764R alone were much reduced compared to those of the wild-type channel or the channel carrying the double or triple mutations (KF, RF and KRF). These results indicated that evolution of sodium channel insensitivity in the German cockroach is achieved by sequential selection of a primary mutation L993F and two secondary mutations E434K and C764R, and concomitant presence of all three mutations dramatically reduced sodium channel sensitivity to deltamethrin.     

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.     

Voltage-dependent regulation of modal gating in the rat SkM1 sodium channel expressed in Xenopus oocytes

The TTX-sensitive rat skeletal muscle sodium channel (rSkM1) exhibits two modes of inactivation (fast vs slow) when the alpha subunit is expressed alone in Xenopus oocytes. In this study, two components are found in the voltage dependence of normalized current inactivation, one having a V1/2 in the expected voltage range (approximately -50 mV, I(N)) and the other with a more hyperpolarized V1/2 (approximately -130 mV, IH) at a holding potential of -90 mV. The I(N) component is associated with the gating mode having rapid inactivation and recovery from inactivation of the macroscopic current (N-mode), while IH corresponds to the slow inactivation and recovery mode (H-mode). These two components are interconvertible and their relative contribution to the total current varies with the holding potential: I(N) is favored by hyperpolarization. The interconversion between the two modes is voltage dependent and is well fit to a first-order two-state model with a voltage dependence of e-fold/8.6 mV and a V1/2 of -62 mV. When the rat sodium channel beta 1-subunit is coinjected with rSkM1, IH is essentially eliminated and the inactivation kinetics of macroscopic current becomes rapid. These two current components and their associated gating modes may represent two conformations of the alpha subunit, one of which can be stabilized either by hyperpolarization or by binding of the beta 1 subunit.     

Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes

Vasopressin stimulates the activity of the epithelial Na channel (ENaC) through the cAMP/PKA pathway in the cortical collecting tubule, or in similar amphibian epithelia, but the mechanism of this regulation is not yet understood. This stimulation by cAMP could not be reproduced with the rat or Xenopus ENaC expressed in Xenopus oocyte. Recently, it was shown that the α-subunit cloned from the guinea-pig colon (αgp) could confer the ability to be activated by the membrane-permeant cAMP analogue 8-chlorophenyl-thio-cAMP (cpt-cAMP) to channels produced by expression of αgp, βrat and γrat ENaC subunits. In this study we investigate the mechanism of this activation. Forskolin treatment, endogenous production of cAMP by activation of coexpressed β adrenergic receptors, or intracellular perfusion with cAMP did not increase the amiloride-sensitive Na current, even though these maneuvers stimulated CFTR (cystic fibrosis transmembrane conductance regulator)-mediated Cl currents. In contrast, extracellular 8-cpt-cAMP increased αgp, βrat and γrat ENaC activity but had no effect on CFTR. Swapping intracellular domains between the cpt-cAMP-sensitive αgp and the cpt-cAMP-resistant αrat-subunit showed that neither the N-terminal nor the C-terminal of α ENaC was responsible for the effect of cpt-cAMP. The mechanisms of activation of ENaC by cpt-cAMP and of CFTR by the cAMP/PKA pathway are clearly different. cpt-cAMP seems to increase the activity of ENaC formed by αgp and βγrat by interacting with the extracellular part of the protein. Received: 19 January 2001/Revised: 27 April 2001     

Three kinds of currents in the canine betaine-GABA transporter BGT-1 expressed in Xenopus laevis oocytes

The cloned canine betaine-GABA cotransporter BGT-1 has been heterologously expressed in Xenopus laevis oocytes in order to characterize its electrophysiological properties. Voltage-clamp experiments on transfected oocytes reveal the presence of three types of membrane current which are absent in non-injected oocytes: (i) an organic substrate-independent current (uncoupled current); (ii) a transport-associated current, seen upon addition of betaine or GABA; (iii) presteady-state currents induced by voltage changes. The three kinds of current are analogous to those reported in structurally similar cotransporters. The transport-associated current is strictly dependent on the presence of Na(+). The good correlation between the amount of charge underlying the presteady-state currents and the transport-associated current indicates that both processes are due to the activity of the transporter.     

Functional association of the beta 1 subunit with human cardiac (hH1) and rat skeletal muscle (mu 1) sodium channel alpha subunits expressed in Xenopus oocytes

Native cardiac and skeletal muscle Na channels are complexes of alpha and beta 1 subunits. While structural correlates for activation, inactivation, and permeation have been identified in the alpha subunit and the expression of alpha alone produces functional channels, beta 1- deficient rat skeletal muscle (mu 1) and brain Na channels expressed in Xenopus oocytes do not gate normally. In contrast, the requirement of a beta 1 subunit for normal function of Na channels cloned from rat heart or human heart (hH1) has been disputed. Coinjection of rat brain beta 1 subunit cRNA with hH1 (or mu 1) alpha subunit cRNA into oocytes increased peak Na currents recorded 2 d after injection by 240% (225%) without altering the voltage dependence of activation. In mu 1 channels, steady state inactivation was shifted to more negative potentials (by 6 mV, p < 0.01), but the shift of 2 mV was not significant for hH1 channels. Nevertheless, coexpression with beta 1 subunit speeded the decay of macroscopic current of both isoforms. Ensemble average hH1 currents from cell-attached patches revealed that coexpression of beta 1 increases the rate of inactivation (quantified by time to 75% decay of current; p < 0.01 at -30, -40, and -50 mV). Use- dependent decay of hH1 Na current during repeated pulsing to -20 mV (1 s, 0.5 Hz) after a long rest was reduced to 16 +/- 2% of the first pulse current in oocytes coexpressing alpha and beta 1 subunits compared to 35 +/- 8% use-dependent decay for oocytes expressing the alpha subunit alone. Recovery from inactivation of mu 1 and hH1 Na currents after 1-s pulses to -20 mV is multiexponential with three time constants; coexpression of beta 1 subunit decreased all three recovery time constants. We conclude that the beta 1 subunit importantly influences the function of Na channels produced by coexpression with either the hH1 or mu 1 alpha subunits.     

A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.     

SPLUNC1 expression reduces surface levels of the epithelial sodium channel (ENaC) in Xenopus laevis oocytes

Throughout the body, the epithelial Na⁺ channel (ENaC) plays a critical role in salt and liquid homeostasis. In cystic fibrosis airways, for instance, improper regulation of ENaC results in hyperabsorption of sodium that causes dehydration of airway surface liquid. This dysregulation then contributes to mucus stasis and chronic lung infections. ENaC is known to undergo proteolytic cleavage, which is required for its ability to conduct Na⁺ ions. We have previously shown that the short, palate lung and nasal epithelial clone (SPLUNC1) binds to and inhibits ENaC in both airway epithelia and in Xenopus laevis oocytes. In this study, we found that SPLUNC1 was more potent at inhibiting ENaC than either SPLUNC2 or long PLUNC1 (LPLUNC1), two other PLUNC family proteins that are also expressed in airway epithelia. Furthermore, we were able to shed light on the potential mechanism of SPLUNC1''s inhibition of ENaC. While SPLUNC1 did not inhibit proteolytic activity of trypsin, it significantly reduced ENaC currents by reducing the number of ENaCs in the plasma membrane. A better understanding of ENaC''s regulation by endogenous inhibitors may aid in the development of novel therapies designed to inhibit hyperactive ENaC in cystic fibrosis epithelia.Key words: mucociliary clearance, chronic airway disease, cystic fibrosis, protease, airway surface liquid, Na⁺ absorption     

Histidine(118) in the S2-S3 linker specifically controls activation of the KAT1 channel expressed in Xenopus oocytes

The guard cell K(+) channel KAT1, cloned from Arabidopsis thaliana, is activated by hyperpolarization and regulated by a variety of physiological factors. Low internal pH accelerated the activation kinetics of the KAT1 channel expressed in Xenopus oocytes with a pK of approximately 6, similar to guard cells in vivo. Mutations of histidine-118 located in the putative cytoplasmic linker between the S2 and S3 segments profoundly affected the gating behavior and pH dependence. At pH 7.2, substitution with a negatively charged amino acid (glutamate, aspartate) specifically slowed the activation time course, whereas that with a positively charged amino acid (lysine, arginine) accelerated. These mutations did not alter the channel's deactivation time course or the gating behavior after the first opening. Introducing an uncharged amino acid (alanine, asparagine) at position 118 did not have any obvious effect on the activation kinetics at pH 7.2. The charged substitutions markedly decreased the sensitivity of the KAT1 channel to internal pH in the physiological range. We propose a linear kinetic scheme to account for the KAT1 activation time course at the voltages where the opening transitions dominate. Changes in one forward rate constant in the model adequately account for the effects of the mutations at position 118 in the S2-S3 linker segment. These results provide a molecular and biophysical basis for the diversity in the activation kinetics of inward rectifiers among different plant species.     

Functional studies of the effect of NO donor on human CLCN1 polymorphism/mutants expressed in Xenopus laevis oocytes

In this study, we investigated the effect of NO donor, diethylamine/nitric oxide (DEA/NO), on the electrophysiological behavior of human skeletal muscle chloride channel (CLCN1). The wild-type and variants of CLCN1, including one polymorphism (P727L) and four mutants (T631I, D644G, G482R, and S471F), were expressed in Xenopus oocytes and the ionic current was measured by two-electrode voltage-clamp method. Our results revealed that there is no significant difference in the current-voltage relationships and half-voltage values of open probability between wild-type and variants of CLCN1 except for G482R. Application of the DEA-NO (0.1 mM) significantly increases the channel conductance of wild-type, T631I, D644G, and S471F, but not P727L. This indicates that P727L polymorphism causes loss of sensitivity of CLCN1 to the DEA/NO treatment, which could be due to a conformational change caused by proline substitution. The data suggest that the polymorphic changes may affect the function of CLCN1 in response to the treatment of chemical compounds.     

Electrogenic uptake of nucleosides and nucleoside-derived drugs by the human nucleoside transporter 1 (hCNT1) expressed in Xenopus laevis oocytes

The concentrative pyrimidine-preferring nucleoside transporter 1 (hCNT1), cloned from human fetal liver, was expressed in Xenopus laevis oocytes. Using the two-electrode voltage-clamp technique, it is shown that translocation of nucleosides by this transporter generates sodium inward currents. Membrane hyperpolarization (from -50 to -150 mV) did not affect the K(0.5) for uridine, although it increased the transport current approximately 3-fold. Gemcitabine (a pyrimidine nucleoside-derived drug) but not fludarabine (a purine nucleoside-derived drug) induced currents in oocytes expressing the hCNT1 transporter. The K(0.5) value for gemcitabine at -50 mV membrane potential was lower than that for natural substrates, although this drug induced a lower current than uridine and cytidine, thus suggesting that the affinity binding of the drug transporter is high but that translocation occurs more slowly. The analysis of the currents generated by the hCNT1-mediated transport of nucleoside-derived drugs used in anticancer and antiviral therapies will be useful in the characterization of the pharmacological profile of this family of drug transporters and will allow rapid screening for uptake of newly developed nucleoside-derived drugs.     

NMDA and glycine regulate the affinity of the Mg2+-block site in NR1-1a/NR2A NMDA receptor channels expressed in Xenopus oocytes

NMDA receptors are glutamate-regulated ion channels of critical importance for many neurophysiological and neuropathological processes. Mg2+ blocks the NMDA receptor by binding to the channel pore with an apparent affinity that depends on the membrane potential. We have investigated the effect of NMDA and the required co-agonist glycine on the affinity of the Mg2+ block site in NR1-1a/NR2A NMDA receptors expressed in Xenopus oocytes. We found that NMDA and glycine increase the IC50 value of the Mg2+-block site at pH 7.4 and in the presence of physiological concentration of Ca2+. The increase the IC50 value may correspond to a decrease in Mg2+-block affinity. This effect may result in an increased influx of Ca2+, and this influx may constitute up to a third of the total Ca2+ influx induced by NMDA. At high pH, or at low concentrations of Ca2+, NMDA and glycine have an opposite effect and instead decreased the IC50 value of the Mg2+-block. These results indicate that glutamate and glycine can regulate the affinity of the Mg2+-block site. This effect may have implications for the understanding the role of NMDA receptors both under physiological and pathophysiological conditions.     

Spectrum of mutations in the major human skeletal muscle chloride channel gene (CLCN1) leading to myotonia.

Autosomal dominant myotonia congenita and autosomal recessive generalized myotonia (GM) are genetic disorders characterized by the symptom of myotonia, which is based on an electrical instability of the muscle fiber membrane. Recently, these two phenotypes have been associated with mutations in the major muscle chloride channel gene CLCN1 on human chromosome 7q35. We have systematically screened the open reading frame of the CLCN1 gene for mutations by SSC analysis (SSCA) in a panel of 24 families and 17 single unrelated patients with human myotonia. By direct sequencing of aberrant SSCA conformers were revealed 15 different mutations in a total of 18 unrelated families and 13 single patients. Of these, 10 were novel (7 missense mutations, 2 mutations leading to frameshift, and 1 mutation predicted to affect normal splicing). In our overall sample of 94 GM chromosomes we were able to detect 48 (51%) mutant GM alleles. Three mutations (F413C), R894X, and a 14-bp deletion in exon 13) account for 32% of the GM chromosomes in the German population. Our finding that A437T is probably a polymorphism is in contrast to a recent report that the recessive phenotype GM is associated with this amino acid change. We also demonstrate that the R894X mutation may act as a recessive or a dominant mutation in the CLCN1 gene, probably depending on the genetic background. Functional expression of the R894X mutant in Xenopus oocytes revealed a large reduction, but not complete abolition, of chloride currents. Further, it had a weak dominant negative effect on wild-type currents in coexpression studies. Reduction of currents predicted for heterozygous carriers are close to the borderline value, which is sufficient to elicit myotonia.     

Effect of linker insertion mutations in the human immunodeficiency virus type 1 gag gene on activation of viral protease expressed in bacteria.

We have expressed the human immunodeficiency virus type 1 (HIV-1) protease (PR) in bacteria as a Gag-PR polyprotein (J. Luban and S.P. Goff, J. Virol. 65:3203-3212, 1991). The protein displays enzymatic activity, cleaving the Gag polyprotein precursor Pr55gag to the expected products. The PR enzyme is only active as a dimer, and we hypothesized that PR activation might be used as an indicator of polyprotein multimerization. We constructed 25 linker insertion mutations throughout gag and assessed the PR activity of mutant Gag-PR polyproteins by the appearance of Gag cleavage products in bacterial lysates. All mutant constructs produced stable protein in bacteria. PR activity of the majority of the Gag-PR mutants was indistinguishable from that of the wild type. Six mutants, one with an insertion in the matrix (MA), four with insertions in the capsid (CA), and one with insertions in the nucleocapsid (NC), globally disrupted polyprotein processing. When PR was provided in trans on a separate plasmid, the Gag proteins were cleaved with wild-type efficiency. These results suggest that the gag mutations identified as disruptive of polyprotein processing did not conceal the scissile bonds of the polyprotein. Rather, the mutations prevented PR activation in the context of a Gag-PR polyprotein, perhaps by preventing polyprotein dimerization.     

Polymorphism of intron-1 in the voltage-gated sodium channel gene of Anopheles gambiae s.s. populations from Cameroon with emphasis on insecticide knockdown resistance mutations

Sequence variation at the intron-1 of the voltage-gated sodium channel gene in Anopheles gambiae M- and S-forms from Cameroon was assessed to explore the number of mutational events originating knockdown resistance ( kdr ) alleles. Mosquitoes were sampled between December 2005 and June 2006 from three geographical areas: (i) Magba in the western region; (ii) Loum, Tiko, Douala, Kribi, and Campo along the Atlantic coast; and (iii) Bertoua, in the eastern continental plateau. Both 1014S and 1014F kdr alleles were found in the S-form with overall frequencies of 14% and 42% respectively. Only the 1014F allele was found in the M-form at lower frequency (11%). Analysis of a 455 bp region of intron-1 upstream the kdr locus revealed four independent mutation events originating kdr alleles, here named MS1 -1014F, S1-1014S and S2-1014S kdr- intron-1 haplotypes in S-form and MS3-1014F kdr- intron-1 haplotype in the M-form. Furthermore, there was evidence for mutual introgression of kdr 1014F allele between the two molecular forms, MS1 and MS3 being widely shared by them. Although no M/S hybrid was observed in analysed samples, this wide distribution of haplotypes MS1 and MS3 suggests inter-form hybridizing at significant level and emphasizes the rapid diffusion of the kdr alleles in Africa. The mosaic of genetic events found in Cameroon is representative of the situation in the West–Central African region and highlights the importance of evaluating the spatial and temporal evolution of kdr alleles for a better management of insecticide resistance.