Effect of a fluvalinate-resistance-associated sodium channel mutation from varroa mites on cockroach sodium channel sensitivity to fluvalinate, a pyrethroid insecticide

Fluvalinate is a pyrethroid insecticide that is widely used in the control of the varroa mite (Varroa destructor), an ecto-parasite of the honeybee. Previously we identified four fluvalinate-resistance-associated mutations in the sodium channel gene of the varroa mite. One of the mutations caused a leucine (L) to proline (P) change at 1770 in the linker connecting domains III and IV of the sodium channel. Interestingly, at the position corresponding to the L to P mutation, all known insect (including honeybee) sodium channel proteins already naturally contain a P residue (e.g., P1577 in the cockroach sodium channel BgNa(v)). To determine whether insect sodium channels are less sensitive to fluvalinate than arachnid sodium channels, we replaced P1577 with an L in a BgNa(v) variant (BgNa(v)1-1) and examined the sensitivity of the recombinant channel to fluvalinate. The P1577L substitution did not alter the gating properties of the BgNa(v)1-1 channel expressed in Xenopus oocytes. However, the BgNa(v)1-1(P1577L) channel was five-fold more sensitive to fluvalinate compared with the BgNa(v)1-1 channel. These results not only implicate the L to P mutation in fluvalinate resistance in varroa mites, but also suggest a possible contribution of L1770 to the higher sensitivity of varroa mites to fluvalinate than their insect hosts.     

Substitutions in the domain III voltage-sensing module enhance the sensitivity of an insect sodium channel to a scorpion beta-toxin

Scorpion β-toxins bind to the extracellular regions of the voltage-sensing module of domain II and to the pore module of domain III in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain II in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant β-toxin, Lqh-dprIT(3), from Leiurus quinquestriatus hebraeus with insect sodium channels from Blattella germanica (BgNa(v)). Like other scorpion β-toxins, Lqh-dprIT(3) shifts the voltage dependence of activation of BgNa(v) channels expressed in Xenopus oocytes to more negative membrane potentials but only after strong depolarizing prepulses. Notably, among 10 BgNa(v) splice variants tested for their sensitivity to the toxin, only BgNa(v)1-1 was hypersensitive due to an L1285P substitution in IIIS1 resulting from a U-to-C RNA-editing event. Furthermore, charge reversal of a negatively charged residue (E1290K) at the extracellular end of IIIS1 and the two innermost positively charged residues (R4E and R5E) in IIIS4 also increased the channel sensitivity to Lqh-dprIT(3). Besides enhancement of toxin sensitivity, the R4E substitution caused an additional 20-mV negative shift in the voltage dependence of activation of toxin-modified channels, inducing a unique toxin-modified state. Our findings provide the first direct evidence for the involvement of the domain III voltage-sensing module in the action of scorpion β-toxins. This hypersensitivity most likely reflects an increase in IIS4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation.     

Analysis of the action of lidocaine on insect sodium channels

A new class of sodium channel blocker insecticides (SCBIs), which include indoxacarb, its active metabolite, DCJW, and metaflumizone, preferably block inactivated states of both insect and mammalian sodium channels in a manner similar to that by which local anesthetic (LA) drugs block mammalian sodium channels. A recent study showed that two residues in the cockroach sodium channel, F1817 and Y1824, corresponding to two key LA-interacting residues identified in mammalian sodium channels are not important for the action of SCBIs on insect sodium channels, suggesting unique interactions of SCBIs with insect sodium channels. However, the mechanism of action of LAs on insect sodium channels has not been investigated. In this study, we examined the effects of lidocaine on a cockroach sodium channel variant, BgNa(v)1-1a, and determined whether F1817 and Y1824 are also critical for the action of LAs on insect sodium channels. Lidocaine blocked BgNa(v)1-1a channels in the resting state with potency similar to that observed in mammalian sodium channels. Lidocaine also stabilized both fast-inactivated and slow-inactivated states of BgNa(v)1-1a channels, and caused a limited degree of use- and frequency-dependent block, major characteristics of LA action on mammalian sodium channels. Alanine substitutions of F1817 and Y1824 reduced the sensitivity of the BgNa(v)1-1a channel to the use-dependent block by lidocaine, but not to tonic blocking and inactivation stabilizing effects of lidocaine. Thus, similar to those on mammalian sodium channels, F1817 and Y1824 are important for the action of lidocaine on cockroach sodium channels. Our results suggest that the receptor sites for lidocaine and SCBIs are different on insect sodium channels.     

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.     

Identification of new batrachotoxin-sensing residues in segment IIIS6 of the sodium channel

Ion permeation through voltage-gated sodium channels is modulated by various drugs and toxins. The atomistic mechanisms of action of many toxins are poorly understood. A steroidal alkaloid batrachotoxin (BTX) causes persistent channel activation by inhibiting inactivation and shifting the voltage dependence of activation to more negative potentials. Traditionally, BTX is considered to bind at the channel-lipid interface and allosterically modulate the ion permeation. However, amino acid residues critical for BTX action are found in the inner helices of all four repeats, suggesting that BTX binds in the pore. In the octapeptide segment IFGSFFTL in IIIS6 of a cockroach sodium channel BgNa(V), besides Ser_3i15 and Leu_3i19, which correspond to known BTX-sensing residues of mammalian sodium channels, we found that Gly_3i14 and Phe_3i16 are critical for BTX action. Using these data along with published data as distance constraints, we docked BTX in the Kv1.2-based homology model of the open BgNa(V) channel. We arrived at a model in which BTX adopts a horseshoe conformation with the horseshoe plane normal to the pore axis. The BTX ammonium group is engaged in cation-π interactions with Phe_3i16 and BTX moieties interact with known BTX-sensing residues in all four repeats. Oxygen atoms at the horseshoe inner surface constitute a transient binding site for permeating cations, whereas the bulky BTX molecule would resist the pore closure, thus causing persistent channel activation. Our study reinforces the concept that steroidal sodium channel agonists bind in the inner pore of sodium channels and elaborates the atomistic mechanism of BTX action.     

Synergistic interaction between two cockroach sodium channel mutations and a tobacco budworm sodium channel mutation in reducing channel sensitivity to a pyrethroid insecticide

Pyrethroid insecticide resistance due to reduced nerve sensitivity, known as knockdown resistance (kdr or kdr-type), is linked to multiple point mutations in the para-homologous sodium channel genes. Previously we demonstrated that two mutations (E434K and C764R) in the German cockroach sodium channel greatly enhanced the ability of the L993F mutation (a known kdr -type mutation) to reduce sodium channel sensitivity to deltamethrin, a pyrethroid insecticide. Neither E434K nor C764R alone, however, altered sodium channel sensitivity. To examine whether E434K and C764R also enhance the effect of pyrethroid resistance-associated sodium channel mutations identified in other insects, we introduced a V to M mutation (V409M) into the cockroach sodium channel protein at the position that corresponds to the V421M mutation in the Heliothis virescens sodium channel protein. We found that the V409M mutation alone modified the gating properties of the sodium channel and reduced channel sensitivity to deltamethrin by 10-fold. Combining the V409M mutation with either the E434K or C764K alone did not reduce the V409M channel sensitivity to deltamethrin further. However, the triple mutation combination (V409M, E434K and C764R) dramatically reduced channel sensitivity by 100-fold compared with the wild-type channel. These results suggest that the E434K and C764R mutations are important modifiers of sodium channel sensitivity to pyrethroid insecticides.     

Modulation of the cardiac sodium channel Nav1.5 by fibroblast growth factor homologous factor 1B

We have previously shown that fibroblast growth factor homologous factor 1B (FHF1B), a cytosolic member of the fibroblast growth factor family, associates with the sensory neuron-specific channel Na(v)1.9 but not with the other sodium channels present in adult rat dorsal root ganglia neurons. We show in this study that FHF1B binds to the C terminus of the cardiac voltage-gated sodium channel Na(v)1.5 and modulates the properties of the channel. The N-terminal 41 amino acid residues of FHF1B are essential for binding to Na(v)1.5, and the conserved acidic rich domain (amino acids 1773-1832) in the C terminus of Na(v)1.5 is sufficient for association with this factor. Binding of the growth factor to recombinant wild type human Na(v)1.5 in human embryonic kidney 293 cells produces a significant hyperpolarizing shift in the voltage dependence of channel inactivation. An aspartic acid to glycine substitution at position 1790 of the channel, which underlies one of the LQT-3 phenotypes of cardiac arrythmias, abolishes the interaction of the Na(v)1.5 channel with FHF1B. This is the first report showing that interaction with a growth factor can modulate properties of a voltage-gated sodium channel.     

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.     

A naturally occurring amino acid substitution in the voltage-dependent sodium channel selectivity filter affects channel gating

The pore of sodium channels contains a selectivity filter made of 4 amino acids, D/E/K/A. In voltage sensitive sodium channel (Nav) channels from jellyfish to human the fourth amino acid is Ala. This Ala, when mutated to Asp, promotes slow inactivation. In some Nav channels of pufferfishes, the Ala is replaced with Gly. We studied the biophysical properties of an Ala-to-Gly substitution (A1529G) in rat Nav1.4 channel expressed in Xenopus oocytes alone or with a β1 subunit. The Ala-to-Gly substitution does not affect monovalent cation selectivity and positively shifts the voltage-dependent inactivation curve, although co-expression with a β1 subunit eliminates the difference between A1529G and WT. There is almost no difference in channel fast inactivation, but the β1 subunit accelerates WT current inactivation significantly more than it does the A1529G channels. The Ala-to-Gly substitution mainly influences the rate of recovery from slow inactivation. Again, the β1 subunit is less effective on speeding recovery of A1529G than the WT. We searched Nav channels in numerous databases and noted at least four other independent Ala-to-Gly substitutions in Nav channels in teleost fishes. Thus, the Ala-to-Gly substitution occurs more frequently than previously realized, possibly under selection for alterations of channel gating.     

Molecular mechanism of synthetic pyrethroid and organophosphate resistance in field isolates of <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> tick collected from a northern state of India

The frequently used chemical control method to manage Rhipicephalus microplus is limited by the emergence of resistance populations. Understanding of resistance mechanisms is essential to develop strategy for sustainable management. The present study was focused on working out the molecular mechanisms of resistance against synthetic pyrethroids (SPs) and organophosphates (OPs) in field isolates of R. microplus collected from six districts of Uttar Pradesh, India. Adult immersion test with discriminating concentrations (AIT-DC) was used to determine resistance status of isolates to SPs (deltamethrin, cypermethrin) and OPs (diazinon, coumaphos). All the six isolates were found resistant to SPs with resistance factor (RF) of 2.9–58.6 and to one of the OP compounds, diazinon having RF of 3.5–13.7 but susceptible to coumaphos (RF?<?1.4). Three R. microplus genes, viz. para-sodium channel domain II S4-5 linker, carboxylesterase (372 bp) and acetylcholinesterase 2 (1692 bp) were sequenced and compared with respective sequences of reference susceptible IVRI-I, reference OP resistant population (IVRI-III), IVRI-IV and multi-acaricide resistant population (IVRI-V) of R. microplus. A C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to L64I amino acid substitution was detected in all six isolates. The G1120A mutation in the carboxylesterase gene could not be detected in any isolate. Five nucleotide substitutions viz., G138A, G889A, T1090A, C1234T and G1403A were identified in the acetylcholinesterase 2 gene leading to four amino acid substitutions. The findings of the study corroborate the role of mutation in sodium channel and acetylcholinesterase 2 genes in SP and OP resistance in this part of India.     

Chimeric analysis of Na(+)/Ca(2+) exchangers NCX1 and NCX3 reveals structural domains important for differential sensitivity to external Ni(2+) or Li(+).

Externally applied Ni(2+), which apparently competes with Ca(2+) in all three isoforms of Na(+)/Ca(2+) exchanger, inhibits exchange activity of NCX1 or NCX2 with a 10-fold higher affinity than that of NCX3, whereas stimulation of exchange by external Li(+) is significantly greater in NCX2 and NCX3 than in NCX1 (Iwamoto, T., and Shigekawa, M. (1998) Am. J. Physiol. 275, C423-C430). Here we identified structural domains in the exchanger that confer differential sensitivity to Ni(2+) or Li(+) by measuring intracellular Na(+)-dependent (45)Ca(2+) uptake in CCL39 cells stably expressing NCX1/NCX3 chimeras or mutants. We found that two segments in the exchanger corresponding mostly to the internal alpha-1 and alpha-2 repeats are individually responsible for the alteration of Ni(2+) sensitivity, both together accounting for approximately 80% of the difference between NCX1 and NCX3. In contrast, the segment corresponding to the alpha-2 repeat fully accounts for the differential Li(+) sensitivity between the isoforms. The Ni(2+) sensitivity was mimicked, respectively, by simultaneous substitution of two amino acids in the alpha-1 repeat (N125G/T127I in NCX1 and G159N/I161T in NCX3) and substitution of one amino acid in the alpha-2 repeat (V820A in NCX1 and A809V in NCX3). On the other hand, the Li(+) sensitivity was mimicked by double substitution mutation in the alpha-2 repeat (V820A/Q826V in NCX1 and A809V/V815Q in NCX3). Single substitution mutations at Asn(125) and Val(820) of NCX1 caused significant alterations in the interactions of the exchanger with Ca(2+) and Ni(2+), and Ni(2+) and Li(+), respectively, although the extent of alteration varied depending on the nature of side chains of substituted residues. Since the above four important residues are mostly in the putative loops of the alpha repeats, these regions might form an ion interaction domain in the exchanger.     

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.     

Interaction between tetraethylammonium and amino acid residues in the pore of cloned voltage-dependent potassium channels

Extracellular tetraethylammonium (TEA) inhibits currents in Xenopus oocytes that have been injected with mRNAs encoding voltage-dependent potassium channels. Concentration-response curves were used to measure the affinity of TEA; this differed up to 700-fold among channels RBK1 (KD 0.3 mM), RGK5 (KD 11 mM), and RBK2 (KD greater than 200 mM). Studies in which chimeric channels were expressed localized TEA binding to the putative extracellular loop between trans-membrane domains S5 and S6. Site-directed mutagenesis of residues in this region identified the residue Tyr379 of RBK1 as a crucial determinant of TEA sensitivity; substitution of Tyr in the equivalent positions of RBK2 (Val381) and RGK5 (His401) made these channels as sensitive to TEA as RBK1. Nonionic forces are involved in TEA binding because (i) substitution of the Phe for Tyr379 in RBK1 increased its affinity, (ii) protonation of His401 in RGK5 selectively reduced its affinity, and (iii) the affinity of TEA was unaffected by changes in ionic strength. The results suggest an explanation for the marked differences in TEA sensitivity that have been observed among naturally occurring and cloned potassium channels and indicate that the amino acid corresponding to residue 379 in RBK1 lies within the external mouth of the ion channel.     

Mutations on M3 helix of Plutella xylostella glutamate-gated chloride channel confer unequal resistance to abamectin by two different mechanisms

Abamectin is one of the most widely used avermectins for agricultural pests control, but the emergence of resistance around the world is proving a major threat to its sustained application. Abamectin acts by directly activating glutamate-gated chloride channels (GluCls) and modulating other Cys-loop ion channels. To date, three mutations occurring in the transmembrane domain of arthropod GluCls are associated with target-site resistance to abamectin: A309V in Plutella xylostella GluCl (PxGluCl), G323D in Tetranychus urticae GluCl1 (TuGluCl1) and G326E in TuGluCl3. To compare the effects of these mutations in a single system, A309V/I/G and G315E (corresponding to G323 in TuGluCl1 and G326 in TuGluCl3) substitutions were introduced individually into the PxGluCl channel. Functional analysis using Xenopus oocytes showed that the A309V and G315E mutations reduced the sensitivity to abamectin by 4.8- and 493-fold, respectively. In contrast, the substitutions A309I/G show no significant effects on the response to abamectin. Interestingly, the A309I substitution increased the channel sensitivity to glutamate by one order of magnitude (∼12-fold). Analysis of PxGluCl homology models indicates that the G315E mutation interferes with abamectin binding through a steric hindrance mechanism. In contrast, the structural consequences of the A309 mutations are not so clear and an allosteric modification of the binding site is the most likely mechanism. Overall the results show that both A309V and G315E mutations may contribute to target-site resistance to abamectin and may be important for the future prediction and monitoring of abamectin resistance in P. xylostella and other arthropod pests.     

Deltamethrin-induced changes in synaptosomal transport of 3H-epinephrine in the squid optic lobes

In fresh synaptosomal preparation from the squid optic lobe 3H-epinephrine transport was significantly affected by deltamethrin at greater than 10(-9) M. Under the experimental conditions such an effect of deltamethrin manifested as either a reduction (at nondepolarized state) or an increase (at depolarized state) in the final level of 3H-epinephrine accumulated in the synaptosomes. Only a part of such a deltamethrin effect was due to its effect on the sodium channel. The remainder of the deltamethrin effect was strongly influenced by agents or treatments which are known to influence internal Ca2+ concentration, suggesting a possible involvement of Ca2+ regulatory mechanisms in the process of stimulation of transmitter release by deltamethrin.     

A conserved determinant in the V1 loop of HIV-1 modulates the V3 loop to prime low CD4 use and macrophage infection

The CD4 binding site (CD4bs) on the HIV-1 envelope plays a major role in determining the capacity of R5 viruses to infect primary macrophages. Thus, envelope determinants within or proximal to the CD4bs have been shown to control the use of low CD4 levels on macrophages for infection. These residues affect the affinity for CD4 either directly or indirectly by altering the exposure of CD4 contact residues. Here, we describe a single amino acid determinant in the V1 loop that also modulates macrophage tropism. Thus, we identified an E153G substitution that conferred high levels of macrophage infectivity for several heterologous R5 envelopes, while the reciprocal G153E substitution abrogated infection. Shifts in macrophage tropism were associated with dramatic shifts in sensitivity to the V3 loop monoclonal antibody (MAb), 447-52D and soluble CD4, as well as more modest changes in sensitivity to the CD4bs MAb, b12. These observations are consistent with an altered conformation or exposure of the V3 loop that enables the envelope to use low CD4 levels for infection. The modest shifts in b12 sensitivity suggest that residue 153 impacts on the exposure of the CD4bs. However, the more intense shifts in sCD4 sensitivity suggest additional mechanisms that likely include an increased ability of the envelope to undergo conformational changes following binding to suboptimal levels of cell surface CD4. In summary, we show that a conserved determinant in the V1 loop modulates the V3 loop to prime low CD4 use and macrophage infection.     

Characterization of mutants of human immunodeficiency virus type 1 that have escaped neutralization by a monoclonal antibody to the gp120 V2 loop.

The biologically cloned human immunodeficiency virus type 1 (HIV-1) RF isolate is sensitive to neutralization by the murine monoclonal antibody (MAb) G3-4 to a conformationally sensitive epitope in the V2 loop of HIV-1 gp120. To assess how variation in the V2 amino acid sequence affects neutralization by this MAb, we cultured RF in the presence of G3-4 to select neutralization escape mutants. Three such mutants resistant to G3-4 neutralization were generated from three independent experiments. Solubilized gp120 from each of these escape mutants had a reduced affinity for G3-4 and also for two other V2 MAbs that were able to bind the wild-type RF gp120. PCR sequencing of the entire gp120 of the wild-type RF virus and the escape mutants showed that amino acid substitutions had occurred only at two positions, Y177H and L179P, both in V2. Experimental introduction of the Y177H substitution into the RF V2 loop in the context of the NL4-3 molecular clone re-created the G3-4-resistant phenotype. The L179P mutant was not viable. Thus, our findings confirm that the HIV-1 V2 loop contains the conformationally sensitive neutralization epitope recognized by G3-4 and that a single amino acid substitution within this region can result in escape variants that arise from immune selection pressure.     

Molecular localization of the inhibitory arachidonic acid binding site to the pore of hIK1

We previously demonstrated that the endogenously expressed human intermediate conductance, Ca(2+)-activated K(+) channel (hIK1) was inhibited by arachidonic acid (AA) (Devor, D. C., and Frizzell, R. A. (1998) Am. J. Physiol. 274, C138-C148). Here we demonstrate, using the excised, inside-out patch-clamp technique, that hIK1, heterologously expressed in HEK293 cells, is inhibited 82 +/- 2% (n = 16) with 3 microm AA, being half-maximally inhibited (IC(50)) at 1.4 +/- 0.7 microm. In contrast, AA does not inhibit the Ca(2+)-dependent, small conductance K(+) channel, rSK2, another member of the KCNN gene family. Therefore, we utilized chimeric hIK1/rSK2 channels to define the AA binding domain on hIK1 to the S5-Pore-S6 region of the channel. Subsequent site-directed mutagenesis revealed that mutation of Thr(250) to Ser (T250S) resulted in a channel with limited sensitivity to block by AA (8 +/- 2%, n = 8), demonstrating that Thr(250) is a key molecular determinant for the inhibition of hIK1 by AA. Likewise, when Val(275) in S6 was mutated to Ala (V275A) AA inhibited only 43 +/- 11% (n = 9) of current flow. The double mutation T250S/V275A eliminated the AA sensitivity of hIK1. Introducing the complimentary single amino acid substitutions into rSK2 (S359T and A384V) conferred partial AA sensitivity to rSK2, 21 +/- 3% and 31 +/- 3%, respectively. Further, introducing the double mutation S359T/A384V into rSK2 resulted in a 63 +/- 8% (n = 9) inhibition by AA, thereby demonstrating the ability to introduce this inhibitory AA binding site into another member of the KCNN gene family. These results demonstrate that AA interacts with the pore-lining amino acids, Thr(250) and Val(275) in hIK1, conferring inhibition of hIK1 by AA and that AA and clotrimazole share similar, if not identical, molecular sites of interaction.     

A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K(+) channels by G-protein betagamma-subunits

To determine the interaction site(s) of ATP-sensitive K(+) (K(ATP)) channels for G-proteins, sulfonylurea receptor (SUR2A or SUR1) and pore-forming (Kir6.2) subunits were reconstituted in the mammalian cell line, COS-7. Intracellular application of the G-protein betagamma2-subunits (G(betagamma)(2)) caused a reduction of ATP-induced inhibition of Kir6.2/SUR channel activities by lessening the ATP sensitivity of the channels. G(betagamma)(2) bound in vitro to both intracellular (loop-NBD) and C-terminal segments of SUR2A, each containing a nucleotide-binding domain (NBD). Furthermore, a single amino acid substitution in the loop-NBD of SUR (Arg656Ala in SUR2A or Arg665Ala in SUR1) abolished the G(betagamma)(2)-dependent alteration of the channel activities. These findings provide evidence that G(betagamma) modulates K(ATP) channels through a direct interaction with the loop-NBD of SUR.