16 research outputs found
Establishing an Ion Pair Interaction in the Homomeric {rho}1 {gamma}-Aminobutyric Acid Type A Receptor That Contributes to the Gating Pathway
{gamma}-Aminobutyric acid type A (GABAA) receptors are members of the Cys-loop superfamily of ligand-gated ion channels. Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state, but the mechanism of gating is not well understood. Here we utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study the gating interface of the human homopentameric {rho}1 GABAA receptor. We have identified an ion pair interaction between two conserved charged residues, Glu92 in loop 2 of the extracellular domain and Arg258 in the pre-M1 region. We hypothesize that the salt bridge exists in the closed state by kinetic measurements and free energy analysis. Several other charged residues at the gating interface are not critical to receptor function, supporting previous conclusions that it is the global charge pattern of the gating interface that controls receptor function in the Cys-loop superfamily
Multiple Tyrosine Residues Contribute to GABA Binding in the GABA_C Receptor Binding Pocket
The ligand binding site of Cys-loop receptors is dominated by aromatic amino acids. In GABA_C receptors, these are predominantly tyrosine residues, with a number of other aromatic residues located in or close to the binding pocket. Here we examine the roles of these residues using substitution with both natural and unnatural amino acids followed by functional characterization. Tyr198 (loop B) has previously been shown to form a cation−π interaction with GABA; the current data indicate that none of the other aromatic residues form such an interaction, although the data indicate that both Tyr102 and Phe138 may contribute to stabilization of the positively charged amine of GABA. Tyr247 (loop C) was very sensitive to substitution and, combined with data from a model of the receptor, suggest a π–π interaction with Tyr241 (loop C); here again functional data show aromaticity is important. In addition the hydroxyl group of Tyr241 is important, supporting the presence of a hydrogen bond with Arg104 suggested by the model. At position Tyr102 (loop D) size and aromaticity are important; this residue may play a role in receptor gating and/or ligand binding. The data also suggest that Tyr167, Tyr200, and Tyr208 have a structural role while Tyr106, Trp246, and Tyr251 are not critical. Comparison of the agonist binding site “aromatic box” across the superfamily of Cys-loop receptors reveals some interesting parallels and divergences
A Unified View of the Role of Electrostatic Interactions in Modulating the Gating of Cys Loop Receptors
In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on {gamma}-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily
Salvianolic Acid B Promotes the Survival of Random-Pattern Skin Flaps in Rats by Inducing Autophagy
Random-pattern skin flap transplantation is frequently applied in plastic and reconstructive surgery. However, the distal part of the flap often suffers necrosis due to ischemia. In this study, the effects of salvianolic acid B (Sal B) on flap survival were evaluated, and the underlying mechanisms were investigated. Sal B improved the survival area, reduced tissue edema, and increased the number of microvessels in skin flaps after 7 days, whereas an autophagy inhibitor (3-methyladenine) reversed the Sal B-induced increase in flap viability. In addition, Sal B stimulated angiogenesis, inhibited apoptosis, reduced oxidative stress, and upregulated autophagy in areas of ischemia. Moreover, the effects of Sal B on angiogenesis, apoptosis, and oxidative stress were reversed by autophagy inhibition. Overall, our findings suggest that Sal B has pro-angiogenesis, anti-apoptosis, and anti-oxidative stress effects by stimulating autophagy, which enhances the survival of random-pattern skin flaps
Molecular Studies of Alpha-Scorpion Toxin Interactions with Voltage-gated Sodium Channels
Thesis (Ph.D.)--University of Washington, 2012Voltage-gated sodium channels are responsible for initiation and propagation of the action potential in vertebrate nerve and muscle. They are also the molecular targets for a large number of paralytic neurotoxins. Alpha-scorpion toxins, including LqhII (Leiurus quinquestriatus hebraeus, type II), bind to the extracellular domain on the sodium channel and inhibit channel fast inactivation. Their binding prolongs sodium channel opening, leading to repetitive firing, depolarization and conduction block. As a consequence, these toxins can kill organisms by inducing paralysis and cardiac arrhythmia. Using site-directed mutagenesis, we have identified residues that constitute the functional interaction surfaces of alpha-scorpion toxin and its receptor site on the voltage-gated sodium channel. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for LqhII, and mutant E1613R had ~73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the receptor site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of sodium channels and reveals its mode of interaction with a gating modifier toxin. The bioactive surface of LqhII has recently been shown to be made of a conserved core domain (Phe-15, Arg-18, Trp-38, and Asn-44) and a variable NC domain (Lys-2, Thr-57, Lys-58). In this work, possible interactions on surfaces of alpha-scorpion toxin and its receptor site on the voltage-gated sodium channel were tested by thermodynamic mutant cycle analysis. Single mutations at key amino acid residues important for activity on toxin and sodium channel were constructed by mutagenesis. We have identified an intermolecular interaction between extracellular loop of sodium channel and alpha-scorpion toxins. We demonstrated a specific aromatic-aromatic interaction between amino acid residue Phe1610 and Trp38 of LqhII, a residue that is conserved among many alpha-scorpion toxins. Toxin dissociation was accelerated by depolarization and increased by mutations at both sites, whereas association rates at negative membrane potentials were not changed for mutation at Phe1610, but slightly increased for mutation at Trp38. These results constrain the possible orientation of alpha-scorpion toxin with respect to the gating-module of DIV in sodium channel and suggest that upon interaction, the core-domain of LqhII is in close proximity to the sodium channel. We found that an antianginal and anti-ischemic drug, ranolazine, attenuated sustained Na+ current induced by alpha-scorpion toxin, with a 50% inhibitory concentration (IC50) of 102 ± 10.7 uM. It also attenuated the peak Na+ currents, with an IC50 of 334 ± 2.6 uM. The results demonstrate that ranolazine has antagonist effect against alpha-scorpion toxin. Consistent with this effect on sodium channels, ranolazine reduces the lethal paralytic effects of LqhII in mice
Correction for Wang et al., Mapping the receptor site for α-scorpion toxins on a Na <sup>+</sup> channel voltage sensor
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