In Utero Antiretroviral Exposure and Risk of Neurodevelopmental Problems in HIV-Exposed Uninfected 5-Year-Old Children.

Studies have observed neurodevelopmental (ND) challenges among young children perinatally HIV-exposed yet uninfected (CHEU) with in utero antiretroviral (ARV) exposure, without clear linkage to specific ARVs. Atazanavir (ATV) boosted with ritonavir has been a preferred protease inhibitor recommended for pregnant women, yet associations of ATV with ND problems in CHEU have been reported. Studies among early school-age children are lacking. The pediatric HIV/AIDS cohort study (PHACS) surveillance monitoring for antiretroviral therapy (ART) toxicities (SMARTT) study evaluated 5-year-old monolingual English-speaking CHEU using the behavior assessment system for children, Wechsler preschool and primary scales of intelligence, and test of language development-primary. A score ≥1.5 standard deviations worse than population norms defined a signal within each domain. Analyses of risk for signals were stratified by timing of any ARV initiation. Associations between ARV exposure and risk of ND signals were assessed using proportional odds models, adjusting for confounders. Among 230 children exposed to ARVs at conception, 15% had single and 8% had multiple ND problems; ATV exposure was not associated with higher risk of signals [adjusted cumulative odds ratio (cOR) = 0.66, confidence interval (CI): 0.28-1.56]. However, among 461 children whose mothers initiated ARVs during pregnancy, 21% had single and 12% had multiple ND problems; ATV exposure was associated with higher risk of signals (cOR = 1.70, CI: 0.82-3.54). The specific regimen tenofovir/emtricitabine/ATV was associated with higher risk (cOR = 2.31, CI: 1.08-4.97) relative to regimens using a zidovudine/lamivudine backbone combined with non-ATV ARVs. It remains important to monitor neurodevelopment of CHEU during early childhood and investigate the impact and the role of timing of in utero exposure to specific ARVs.

Mapping the interaction site for a ß-scorpion toxin in the pore module of domain III of voltage-gated Na(+) channels.

Activation of voltage-gated sodium (Na(v)) channels initiates and propagates action potentials in electrically excitable cells. ß-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of Na(V) channels. CssIV stabilizes the voltage sensor in domain II in its activated state via a voltage-sensor trapping mechanism. Amino acid residues required for the action of CssIV have been identified in the S1-S2 and S3-S4 extracellular loops of domain II. The extracellular loops of domain III are also involved in toxin action, but individual amino acid residues have not been identified. We used site-directed mutagenesis and voltage clamp recording to investigate amino acid residues of domain III that are involved in CssIV action. In the IIISS2-S6 loop, five substitutions at four positions altered voltage-sensor trapping by CssIV(E15A). Three substitutions (E1438A, D1445A, and D1445Y) markedly decreased voltage-sensor trapping, whereas the other two substitutions (N1436G and L1439A) increased voltage-sensor trapping. These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with CssIV. N1436G enhanced voltage-sensor trapping via increased binding affinity to the resting state, whereas L1439A increased voltage-sensor trapping efficacy. Based on these results, a three-dimensional model of the toxin-channel interaction was developed using the Rosetta modeling method. These data provide additional molecular insight into the voltage-sensor trapping mechanism of toxin action and define a three-point interaction site for ß-scorpion toxins on Na(V) channels. Binding of α- and ß-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on Na(V) channels acts synergistically to modify channel gating and paralyze prey.

Structure-function map of the receptor site for ß-scorpion toxins in domain II of voltage-gated sodium channels.

Voltage-gated sodium (Na(v)) channels are the molecular targets of ß-scorpion toxins, which shift the voltage dependence of activation to more negative membrane potentials by a voltage sensor-trapping mechanism. Molecular determinants of ß-scorpion toxin (CssIV) binding and action on rat brain sodium channels are located in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular linkers of the voltage-sensing module in domain II. In IIS1-S2, mutations of two amino acid residues (Glu(779) and Pro(782)) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly(845), define a hot spot of high-impact residues. Two of these substitutions (A841N and L846A) reduced voltage sensor trapping. The other three substitutions (N842R, V843A, and E844N) increased voltage sensor trapping. These bidirectional effects suggest that the IIS3-S4 loop plays a primary role in determining both toxin affinity and efficacy. A high resolution molecular model constructed with the Rosetta-Membrane modeling system reveals interactions of amino acid residues in sodium channels that are crucial for toxin action with residues in CssIV that are required for its effects. In this model, the wedge-shaped CssIV inserts between the IIS1-S2 and IIS3-S4 loops of the voltage sensor, placing key amino acid residues in position to interact with binding partners in these extracellular loops. These results provide new molecular insights into the voltage sensor-trapping model of toxin action and further define the molecular requirements for the development of antagonists that can prevent or reverse toxicity of scorpion toxins.

Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

Scorpion ß-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin-based therapeutics to prevent lethality from scorpion envenomation.