d-Alanine: Distribution, origin, physiological relevance, and implications in disease.

d-Alanine (d-Ala) is an unusual endogenous amino acid present in invertebrates and vertebrates. Compared to its l-isomer, the characterization of d-Ala is challenging because of the need for chiral resolution and the low amounts of the d-enantiomer present. With recent improvements in measurement capabilities, research on d-Ala, along with other d-amino acids, has been growing, especially as the functional significance of d-Ala in the mammalian nervous and endocrine systems is becoming known. Here we provide an overview of the distribution, origin, function, and disease implications of d-Ala.

Role of alanine:glyoxylate aminotransferase 2 in metabolism of asymmetric dimethylarginine in the settings of asymmetric dimethylarginine overload and bilateral nephrectomy.

BACKGROUND: Asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict complications and mortality in cardiovascular and renal diseases. Alanine:glyoxylate aminotransferase 2 (AGXT2) can metabolize both ADMA and SDMA; however, this metabolic pathway is still poorly understood. The goal of our study was to test the hypothesis that AGXT2 is compensatory upregulated in the settings of ADMA overload and bilateral nephrectomy. METHODS: ADMA was infused for 3 days using osmotic minipumps in mice. Half of the mice underwent bilateral nephrectomy 24 h before the end of the infusion. RESULTS: Infusion of ADMA caused a 3- to 4-fold increase in plasma and urine ADMA levels and a 2- to 3-fold increase in plasma and urine levels of the ADMA-specific metabolite of AGXT2 α-keto-δ-(N,N-dimethylguanidino)valeric acid (DMGV). Bilateral nephrectomy led to an ∼4-fold increase of plasma SDMA levels, but did not change plasma ADMA levels. Interestingly, plasma levels of DMGV were elevated 32-fold in the mice, which underwent bilateral nephrectomy. Neither bilateral nephrectomy nor ADMA infusion caused upregulation of AGXT2 expression or activity. CONCLUSIONS: Our data demonstrate that short-term elevation of systemic levels of ADMA leads to a dramatic increase of DMGV formation without upregulation of AGXT2 expression or activity, which suggests that AGXT2-mediated pathway of ADMA metabolism is not saturated under normal conditions and may play a major role in the maintenance of ADMA homeostasis in the setting of local or systemic elevation of ADMA levels.

Structure-activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation.

BACKGROUND AND PURPOSE: The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. EXPERIMENTAL APPROACH: CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and ß-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. KEY RESULTS: Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at ß-arrestin translocation was reduced by ninefold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and ß-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY(1(a)) receptor. CONCLUSIONS AND IMPLICATIONS: Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity.

Flight muscle-specific Pro-Ala-rich extension of troponin is important for maintaining the insect-type myofilament lattice integrity.

Insect flight muscle (IFM) can oscillate at frequencies up to 1000Hz, owing to its capability of stretch activation (SA). It is a highly specialized form of cross striated muscles, and its peculiar features include the IFM-specific isoform of troponin-I (troponin-H or TnH) with an unusually long Pro-Ala-rich extension at the C-terminus. Although we have shown that this extension does not directly take part in SA, questions remain as to what its real role is and why it is expressed only in IFM. Here we explored the structural role of the extension, be comparing X-ray diffraction patterns and electron micrographs of bumblebee IFM fibers before and after enzymatic removal of the extension. The removal had a dramatic effect on diffraction patterns: In IFMs in general, the equatorial 2,0 reflection is much stronger than the 1,1 reflection, but after removal, their intensities became almost equal (stronger 1,1 is a feature of vertebrate skeletal muscle). Electron micrographs revealed that a substantial fraction of the thin filaments showed a tendency to move towards the vertebrate position (the trigonal position between three thick filaments), while the rest of the thin filaments remained in their original insect position (midway between two neighboring thick filaments). Therefore, one of the roles of the extension is suggested to keep the filament lattice in the correct configuration for IFM. This insect-type lattice structure is preserved among IFMs from varied insect orders but not in body muscles, suggesting that the maintenance of this lattice structure is important for flight functions.

Role of a conserved glycine triplet in the NSS amino acid transporter KAAT1.

K+-coupled amino acid transporter 1 (KAAT1) belongs to the NSS family of solute transporters and it is expressed in the midgut and in salivary glands of Manduca sexta larvae. As more than 80% of family members, KAATI shows a stretch of three glycines (G85-G87) that according to the structure of the prototype transporter LeuT, is located close to the access of the permeation pathway. In this work the role of the triplet has been investigated by alanine and cysteine scanning methods in protein heterologously expressed in Xenopus laevis oocytes. All the mutants were functional but the surface expression level was reduced for G85A and G87A mutants and unaffected for G86A mutant. All presented altered amino acid uptake and transport associated currents in the presence of each of the cations (Na+, K+, Li+) that can be exploited by the wt. G87A mutant induced increased uncoupled fluxes in the presence of all the cations. Cross-linking studies, performed by the treatment of cysteine mutants with the oxidative complex Cu(Il)(l,10-phenanthroline)3, showed that limiting the flexibility of the region by covalent blockage of position 87, causes a significant reduction of amino acid uptake. Na+ protected G87C mutant from oxidation, both directly and indirectly. The conserved glycine triplet in KAAT1 plays therefore a complex role that allows initial steps of cation interaction with the transporter.

Insights into the mechanism of activation of the phosphorylation-independent response regulator NblR. Role of residues Cys69 and Cys96.

Cyanobacteria respond to environmental stress conditions by adjusting their photosynthesis machinery. In Synechococcus sp. PCC 7942, phycobilisome degradation and other acclimation responses after nutrient or high light stress require activation by the phosphorylation-independent response regulator NblR. Structural modelling of its receiver domain suggested a role for Cys69 and Cys96 on activation of NblR. Here, we investigate this hypothesis by engineering Cys to Ala substitutions. In vivo and in vitro analyses indicated that mutations Cys69Ala and/or Cys96Ala have a minor impact on NblR function, structure, size, or oligomerization state of the protein, and that Cys69 and Cys96 do not seem to form disulphide bridges. Our results argue against the predicted involvement of Cys69 and Cys96 on NblR activation by redox sensing.

A single amino acid controls the functional switch of human constitutive androstane receptor (CAR) 1 to the xenobiotic-sensitive splicing variant CAR3.

The constitutive androstane receptor (CAR) is constitutively activated in immortalized cell lines independent of xenobiotic stimuli. This feature of CAR has limited its use as a sensor for xenobiotic-induced expression of drug-metabolizing enzymes. Recent reports, however, reveal that a splicing variant of human CAR (hCAR3), which contains an insertion of five amino acids (APYLT), exhibits low basal but xenobiotic-inducible activities in cell-based reporter assays. Nonetheless, the underlying mechanisms of this functional shift are not well understood. We have now generated chimeric constructs containing various residues of the five amino acids of hCAR3 and examined their response to typical hCAR activators. Our results showed that the retention of alanine (hCAR1+A) alone is sufficient to confer the constitutively activated hCAR1 to the xenobiotic-sensitive hCAR3. It is noteworthy that hCAR1+A was significantly activated by a series of known hCAR activators, and displayed activation superior to that of hCAR3. Moreover, intracellular localization assays revealed that hCAR1+A exhibits nuclear accumulation upon 6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime (CITCO) treatment in COS1 cells, which differs from the spontaneous nuclear distribution of hCAR1 and the nontranslocatable hCAR3. Mammalian two-hybrid and glutathione S-transferase pull-down assays further demonstrated that hCAR1+A interacts with the coactivator SRC-1 and GRIP-1 at low level before activation, while at significantly enhanced level in the presence of CITCO. Thus, the alanine residue in the insertion of hCAR3 seems in charge of the xenobiotic response of hCAR3 through direct and indirect mechanisms. Activation of hCAR1+A may represent a sensitive avenue for the identification of hCAR activators.

Effect of blood lactate-guided conditioning of horses with exercises of differing durations and intensities on heart rate and biochemical blood variables.

The velocity at which blood lactate concentration ([LA]) of 4 mmol/L is reached (v(4)) is widely used to determine fitness, but there are few published data on using [LA] as a guide for the exercise speed for training in horses. In this study, the effect of 3 conditioning programs with [LA] guided exercise speeds on v(4), v(200) (speed at a heart rate of 200 beats/min), blood [LA], plasma FFA ([FFA]), and alanine concentrations ([alanine]), before and after exercise, as well as heart rate during exercise, of horses was examined. Six 2-yr-old Haflinger stallions underwent an initial treadmill-based standard exercise test (SET). A regression analysis [LA]-speed relationship was used to calculate v(1.5), v(2.5), and v(4). Horses were then randomly assigned to 1 of 3 conditioning programs according to a 3 x 6 Latin square design. During 6 wk, horses exercised on the treadmill every other day for 45 min at their calculated v(1.5) or v(2.5) or 25 min at their v(4). Each conditioning period (CDP) was followed by 5 wk without conditioning. At 2 and 9 d, and 5 wk, after the end of the CDP, the horses performed another SET to evaluate again the v(4) and v(200). Blood [LA], plasma [FFA], and [alanine] were measured before and after heart rate during exercise sessions 1, 11, and 21 in each CDP. None of the exercise programs had an effect on v(4) and v(200) (P > 0.05). The increase of the mean [LA] after exercise decreased during CDP (P < 0.05), and the increase of mean heart rate during exercise tended to decrease as well (P = 0.07). There was no difference among the conditioning programs. Plasma [FFA] before exercise was not influenced by the CDP (P > 0.05). The plasma [FFA] was always greater after exercise (P < 0.05), but there was no difference among conditioning programs. Overall, the increase was greatest after the 21st exercise session compared with the 1st and 11th exercise sessions (P < 0.05). The mean plasma [alanine] before exercise remained similar during all CDP (P > 0.05). Mean plasma [alanine] of the horses was increased after all exercise sessions measured (P < 0.05). There was no difference among conditioning programs (P > 0.05). It is concluded that conditioning with the exercise types used had small effects. This could have been because the exercise stress was too small, but also because the workload was not increased during the CDP.

Oxidation of cysteine 645 of cobalamin-independent methionine synthase causes a methionine limitation in Escherichia coli.

Cobalamin-independent methionine synthase (MetE) catalyzes the final step in Escherichia coli methionine biosynthesis but is inactivated under oxidative conditions, triggering a methionine deficiency. This study demonstrates that the mutation of MetE cysteine 645 to alanine completely eliminates the methionine auxotrophy imposed by diamide treatment, suggesting that modulation of MetE activity via cysteine 645 oxidation has significant physiological consequences for oxidatively stressed cells.

Structure-function relationships of the non-lanthionine-containing peptide (class II) bacteriocins produced by gram-positive bacteria.

This review focuses on the structure and mode-of-action of non-lanthionine-containing peptide bacteriocins produced by Gram-positive bacteria. These bacteriocins may be divided into four groups: (i) the anti-listerial one-peptide pediocin-like bacteriocins that have very similar amino acid sequences, (ii) the two-peptide bacteriocins that consist of two different peptides, (iii) the cyclic bacteriocins, and (iv) the linear non-pediocin-like one-peptide bacteriocins. These bacteriocins are largely cationic, contain 20 to 70 residues, and kill cells through membrane-permeabilization. The pediocin-like bacteriocins are the ones that are best characterized. Upon contact with target membranes, their cationic N-terminal half forms a beta-sheet-like structure that binds to the target cell surface, while their more hydrophobic helical-containing C-terminal half penetrates into the hydrophobic core of target-cell membranes and apparently binds to the mannose phosphotransferase permease in a manner that results in membrane leakage. Immunity proteins that protect cells from being killed by pediocin-like bacteriocins bind to the bacteriocin-permease complex and prevent bacteriocin-induced membrane-leakage. Recent structural analyses of two-peptide bacteriocins indicate that they form a helix-helix structure that penetrates into cell membranes. Also these bacteriocins may act by binding to integrated membrane proteins. It is proposed that many membrane-active peptide bacteriocins kill target-cells through basically the same mechanism; the common theme being that a membrane-penetrating part of bacteriocins bind to a membrane embedded region of an integrated membrane protein, thereby causing conformational alterations in the protein that in turn lead to membrane-leakage and cell death.

An intact connexin N-terminus is required for function but not gap junction formation.

The cytoplasmic N-termini of connexins have been implicated in protein trafficking, oligomerization and channel gating. To elucidate the role of the N-terminus in connexin37 (CX37), we studied mutant constructs containing partial deletions of its 23 N-terminal amino acids and a construct with a complete N-terminus in which residues 2-8 were replaced with alanines. All mutants containing nine or more N-terminal amino acids form gap junction plaques in transiently transfected HeLa cells, whereas most of the longer deletions do not. Although wild-type CX37 allowed intercellular transfer of microinjected neurobiotin in HeLa cells and formed conducting hemichannels in Xenopus oocytes, none of the mutant constructs tested show evidence of channel function. However, in coexpression experiments, N-terminal mutants that formed gap junction plaques potently inhibit hemichannel conductance of wild-type CX37 suggesting their co-oligomerization. We conclude that as much as half the length of the connexin N-terminus can be deleted without affecting formation of gap junction plaques, but an intact N-terminus is required for hemichannel gating and intercellular communication.

Mutational analysis of conserved glycine residues 142, 143 and 146 reveals Gly(142) is critical for tetramerization of CTP synthase from Escherichia coli.

CTPS (cytidine 5'-triphosphate synthase) catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source. Binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers. In the present study, site-directed mutagenesis was used to replace the fully conserved glycine residues 142 and 143 within the UTP-binding site and 146 within the CTP-binding site of Escherchia coli CTPS. CD spectral analyses of wild-type CTPS and the glycine mutants showed a slight reduction of approximately 15% in alpha-helical content for G142A and G143A relative to G146A and wild-type CTPS, suggesting some local alterations in structure. Relative to wild-type CTPS, the values of k(cat)/K(m) for ammonia-dependent and glutamine-dependent CTP formation catalysed by G143A were reduced 22- and 16-fold respectively, whereas the corresponding values for G146A were reduced only 1.4- and 1.8-fold respectively. The glutaminase activity (k(cat)) of G146A was similar to that exhibited by the wild-type enzyme, whereas that of G143A was reduced 7.5-fold. G146A exhibited substrate inhibition at high concentrations of ammonia and a partial uncoupling of glutamine hydrolysis from CTP production. Although the apparent affinity (1/[S](0.5)) of G143A and G146A for UTP was reduced approximately 4-fold, G146A exhibited increased co-operativity with respect to UTP. Thus mutations in the CTP-binding site can affect UTP-dependent activity. Surprisingly, G142A was inactive with both ammonia and glutamine as substrates. Gel-filtration HPLC experiments revealed that both G143A and G146A were able to form active tetramers in the presence of ATP and UTP; however, nucleotide-dependent tetramerization of G142A was significantly impaired. Our observations highlight the sensitivity of the structure of CTPS to mutations in the UTP- and CTP-binding sites, with Gly(142) being critical for nucleotide-dependent oligomerization of CTPS to active tetramers. This 'structural sensitivity' may limit the number and/or types of mutations that could be selected for during the development of resistance to cytotoxic pyrimidine nucleotide analogues.

Functional and structural roles of conserved cysteine residues in the carboxyl-terminal domain of the follicle-stimulating hormone receptor in human embryonic kidney 293 cells.

The carboxyl-terminal segment of G protein-coupled receptors has one or more conserved cysteine residues that are potential sites for palmitoylation. This posttranslational modification contributes to membrane association, internalization, and membrane targeting of proteins. In contrast to other members of the glycoprotein hormone receptor family (the LH and thyroid-stimulating hormone receptors), it is not known whether the follicle-stimulating hormone receptor (FSHR) is palmitoylated and what are the effects of abolishing its potential palmitoylation sites. In the present study, a functional analysis of the FSHR carboxyl-terminal segment cysteine residues was carried out. We constructed a series of mutant FSHRs by substituting cysteine residues with alanine, serine, or threonine individually and together at positions 629 and 655 (conserved cysteines) and 627 (nonconserved). The results showed that all three cysteine residues are palmitoylated but that only modification at Cys629 is functionally relevant. The lack of palmitoylation does not appear to greatly impair coupling to G(s) but, when absent at position 629, does significantly impair cell surface membrane expression of the partially palmitoylated receptor. All FSHR Cys mutants were capable of binding agonist with the same affinity as the wild-type receptor and internalizing on agonist stimulation. Molecular dynamics simulations at a time scale of approximately 100 nsec revealed that replacement of Cys629 resulted in structures that differed significantly from that of the wild-type receptor. Thus, deviations from wild-type conformation may potentially contribute to the severe impairment in plasma membrane expression and the modest effects on signaling exhibited by the receptors modified in this particular position.

Use of NMR metabolomics to analyze the targets of D-cycloserine in mycobacteria: role of D-alanine racemase.

D-Cycloserine (DCS) is only used with multidrug-resistant strains of tuberculosis because of serious side effects. DCS is known to inhibit cell wall biosynthesis, but the in vivo lethal target is still unknown. We have applied NMR-based metabolomics combined with principal component analysis to monitor the in vivo effect of DCS on Mycobacterium smegmatis. Our analysis suggests DCS functions by inhibiting multiple protein targets.

Factors affecting guanine nucleotide binding to rat AMPA receptors.

Glutamate receptors are competitively inhibited by guanine nucleotides. Insight into the physiological function of this inhibition would be greatly advanced if nucleotide binding could be eliminated through mutations without altering other aspects of receptor function, or if compounds were discovered that selectively prevent nucleotide binding. It was previously reported that a lysine in the chick kainate binding protein (cKBP) is specifically involved in guanine nucleotide binding. In the present study we mutated the equivalent lysine in the rat AMPA receptor subunit GluR1 flip to alanine (K445A) and assessed changes in nucleotide affinity from the displacement of [(3)H]fluorowillardiine. As in the cKBP, the affinity for nucleotides was greatly reduced while the binding affinity for agonists remained unchanged. The reduction in affinity was largest for GTP (factor of 5.8) and GDP (4.4) and minor for GMP and guanosine. This suggests that K445 is involved in stabilizing the second phosphate of the nucleotide. Given that bulkier analogs like GDP-fucose are also accommodated at this site, it seems likely that nucleotides bind in such a way that their phosphates project out of the cleft. In excised-patch recordings using short pulses of glutamate, the K445A mutation increased the EC(50) for the peak response 1.8-fold and accelerated desensitization and deactivation. This indicates that the effects of this mutation are not as specific as previously suggested. Efforts to selectively eliminate inhibition by nucleotides may therefore depend on mapping out further the docking site. In a first attempt using point mutations we ruled out several amino acids around the cleft as being involved in nucleotide binding. Also, the AMPA receptor modulator PPNDS which competitively inhibits nucleotide binding to purinergic receptors did not affect nucleotide inhibition, suggesting that there are major differences in the topography between purinergic and glutamate receptors. Thus new approaches, including crystallography, may be called for to identify residues uniquely involved in nucleotide binding.

Agonist-dependent consequences of proline to alanine substitution in the transmembrane helices of the calcitonin receptor.

BACKGROUND AND PURPOSE: Transmembrane proline (P) residues in family A G protein-coupled receptors (GPCRs) form functionally important kinks in their helices. These residues are little studied in family B GPCRs but experiments with the VPAC1 receptor and calcitonin receptor-like receptor (CL) show parallels with family A receptors. We sought to determine the function of these residues in the insert negative form of the human calcitonin receptor, a close relative of CL. EXPERIMENTAL APPROACH: Proline residues within the transmembrane domains of the calcitonin receptor (P246, P249, P280, P326, P336) were individually mutated to alanine (A) using site-directed mutagenesis. Receptors were transiently transfected into Cos-7 cells using polyethylenimine and salmon and human calcitonin-induced cAMP responses measured. Salmon and human calcitonin competition binding experiments were also performed and receptor cell-surface expression assessed by whole cell ELISA. KEY RESULTS: P246A, P249A and P280A were wild-type in terms of human calcitonin-induced cAMP activation. P326A and P336A had reduced function (165 and 12-fold, respectively). In membranes, human calcitonin binding was not detectable for any mutant receptor but in whole cells, binding was detected for all mutants apart from P326A. Salmon calcitonin activated mutant and wild-type receptors equally, although B(max) values were reduced for all mutants apart from P326A. CONCLUSIONS AND IMPLICATIONS: P326 and P336 are important for the function of human calcitonin receptors and are likely to be involved in generating receptor conformations appropriate for agonist binding and receptor activation. However, agonist-specific effects were observed , implying distinct conformations of the human calcitonin receptor.

Unveiling hidden catalytic contributions of the conserved His/Trp-III in tyrosine recombinases: assembly of a novel active site in Flp recombinase harboring alanine at this position.

The catalytic pentad of tyrosine recombinases, that assists the tyrosine nucleophile, includes a conserved histidine/tryptophan (His/Trp-III). Flp and Cre harbor tryptophan at this position; most of their kin recombinases display histidine. Contrary to the conservation rule, Flp(W330F) is a much stronger recombinase than Flp(W330H). The hydrophobicity of Trp330 or Phe330 is utilized in correctly positioning Tyr343 during the strand cleavage step of recombination. Why then is phenylalanine almost never encountered in the recombinase family at this conserved position? Using exogenous nucleophiles and synthetic methylphosphonate or 5'-thiolate substrates, we decipher that Trp330 also assists in the activation of the scissile phosphate and the departure of the 5'-hydroxyl leaving group. These two functions are consistent with the hydrogen bonding property of Trp330 as well as its location in structures of the Flp recombination complexes. However, van der Waals contact between Trp330 and Arg308 may also be important for the phosphate activation step. A structure based suppression strategy permits the inactive variant Flp(W330A) to be rescued by a second site mutation A339M. Modeling alanine and methionine at positions 330 and 339, respectively, in the Flp crystal structure suggests a plausible mechanism for active site restoration. Successful suppression suggests the possibility of evolving, by design, new active site configurations for tyrosine recombination.

The cold denatured state is compact but expands at low temperatures: hydrodynamic properties of the cold denatured state of the C-terminal domain of L9.

A point mutation of a small globular protein, the C-terminal domain of L9 destabilizes the protein and leads to observable cold-denaturation at temperatures above zero. The cold denatured state is in slow exchange with the native state on the NMR time scale, and this allows the hydrodynamic properties of the cold unfolded state and the native state to be measured under identical conditions using pulsed-field gradient NMR diffusion measurements. This provides the first experimental measurement of the hydrodynamic properties of a cold unfolded protein and its folded form under identical conditions. Hydrodynamic radii of the cold-induced unfolded states were measured for a set of temperatures ranging from 2 degrees C to 25 degrees C at pD 6.6 in the absence of denaturant. The cold unfolded state is compact compared to the urea or acid unfolded state and a trend of increasing radii of hydration is observed as the temperature is lowered. These observations are confirmed by experiments on the same protein at pD 8.0, where it is more stable, in the presence of a modest concentration of urea. The expansion of the cold-denatured state at lower temperatures is consistent with the temperature dependence of hydrophobic interactions.

An alanine in segment 3 of domain III (IIIS3) of the cockroach sodium channel contributes to the low pyrethroid sensitivity of an alternative splice variant.

In a previous study, we showed that two alternative exons (G1 and G2 encoding IIIS3-S4) were involved in the differential sensitivity of two cockroach sodium channel splice variants, BgNa(v)1-1 and BgNa(v)2-1 (previously called KD1 and KD2), to deltamethrin, a pyrethroid insecticide (Tan, et al., 2002b. Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels. J. Neurosci. 22, 5300-5309.). Here, we report the identification of an amino acid residue in exon G2 that contributes to the low deltamethrin sensitivity of BgNa(v)2-1. Replacement of A1356 in BgNa(v)2-1 with the corresponding V1356 in BgNa(v)1-1 enhanced the sensitivity of the BgNa(v)2-1 channel to deltamethrin by six-fold. Conversely, substitution of V1356 with A1356 in BgNa(v)1-1 produced a recombinant BgNa(v)1-1 channel that was 5-fold more resistant to deltamethrin. These results demonstrate that A1356 contributes to the low sensitivity of BgNa(v)2-1 to deltamethrin. A1356V substitution also shifted the voltage-dependence of activation by 10 mV in the hyperpolarizing direction. Possible mechanisms by which this amino acid change affects the action of pyrethroids on the sodium channel are discussed.