Probing the Outer Mouth Structure of the hERG Channel with Peptide Toxin Footprinting and Molecular Modeling

Abstract Previous studies have shown that the unusually long S5-P linker lining human ether a-go-go related gene’s (hERG’s) outer vestibule is critical for its channel function: point mutations at high-impact positions here can interfere with the inactivation process and, in many cases, also reduce the pore’s K+ selectivity. Because no data are available on the equivalent region in the available K channel crystal structures to allow for homology modeling, we used alternative approaches to model its three-dimensional structure. The first part of this article describes mutant cycle analysis used to identify residues on hERG’s outer vestibule that interact with specific residues on the interaction surface of BeKm-1, a peptide toxin with known NMR structure and a high binding affinity to hERG. The second part describes molecular modeling of hERG’s pore domain. The transmembrane region was modeled after the crystal structure of KvAP pore domain. The S5-P linker was docked to the transmembrane region based on data from previous NMR and mutagenesis experiments, as well as a set of modeling criteria. The models were further restrained by contact points between hERG’s outer vestibule and the bound BeKm-1 toxin molecule deduced from the mutant cycle analysis. Based on these analyses, we propose a working model for the open conformation of the outer vestibule of the hERG channel, in which the S5-P linkers interact with the pore loops to influence ion flux through the pore

BeKm-1 Is a HERG-Specific Toxin that Shares the Structure with ChTx but the Mechanism of Action with ErgTx1

Peptide toxins with disulfide-stabilized structures have been used as molecular calipers to probe the outer vestibule structure of K channels. We want to apply this approach to the human ether-a-go-go-related gene (HERG) channel, whose outer vestibule is unique in structure and function among voltage-gated K channels. Our focus here is BeKm-1, a HERG-specific peptide toxin that can suppress HERG in the low nM concentration range. Although BeKm-1 shares the three-dimensional scaffold with the well-studied charybdotoxin, the two use different mechanisms in suppressing currents through their target K channels. BeKm-1 binds near, but not inside, the HERG pore, and it is possible that BeKm-1-bound HERG channels can conduct currents although with markedly altered voltage-dependence and kinetics of gating. BeKm-1 and ErgTx1 differ in three-dimensional scaffold, but the two share mechanism of action and have overlapping binding sites on the HERG channel. For both, residues in the middle of the S5-P linker (the putative 583–597 helix) and residues at the pore entrance are critical for binding, although specific contact points vary between the two. Toxin foot printing using BeKm-1 and ErgTx1 will likely provide complementary information about the unique outer vestibule structure of the HERG channel

Retinoic Acid-Differentiated Neuroblastoma SH-SY5Y Is an Accessible In Vitro Model to Study Native Human Acid-Sensing Ion Channels 1a (ASIC1a)

Human neuroblastoma SH-SY5Y is a prominent neurobiological tool used for studying neuropathophysiological processes. We investigated acid-sensing (ASIC) and transient receptor potential vanilloid-1 (TRPV1) and ankyrin-1 (TRPA1) ion channels present in untreated and differentiated neuroblastoma SH-SY5Y to propose a new means for their study in neuronal-like cells. Using a quantitative real-time PCR and a whole-cell patch-clamp technique, ion channel expression profiles, functionality, and the pharmacological actions of their ligands were characterized. A low-level expression of ASIC1a and ASIC2 was detected in untreated cells. The treatment with 10 μM of retinoic acid (RA) for 6 days resulted in neuronal differentiation that was accompanied by a remarkable increase in ASIC1a expression, while ASIC2 expression remained almost unaltered. In response to acid stimuli, differentiated cells showed prominent ASIC-like currents. Detailed kinetic and pharmacological characterization suggests that homomeric ASIC1a is a dominant isoform among the present ASIC channels. RA-treatment also reduced the expression of TRPV1 and TRPA1, and minor electrophysiological responses to their agonists were found in untreated cells. Neuroblastoma SH-SY5Y treated with RA can serve as a model system to study the effects of different ligands on native human ASIC1a in neuronal-like cells. This approach can improve the characterization of modulators for the development of new neuroprotective and analgesic drugs

Comparative Study of the Aftereffect of CO2 Inhalation or Tiletamine–Zolazepam–Xylazine Anesthesia on Laboratory Outbred Rats and Mice

CO2 inhalation is currently the most common method of euthanasia for laboratory rats and mice, and it is often used for further terminal blood sampling for clinical biochemical assays. Lately, this method has been criticized due to animal welfare issues associated with some processes that develop after CO2 inhalation. The stress reaction and the value of the clinical laboratory parameters significantly depend on the used anesthetics, method, and the site of blood sampling. Especially in small rodents, an acute terminal state followed by a cascade of metabolic reactions that can affect the studied biochemical profile may develop and cause unnecessary suffering of animals. The aim of this study was to compare the stability of biochemical parameters of outbred Sprague Dawley rats and CD-1 mice serum collected after CO2 inhalation or the intramuscular injection of tiletamine–zolazepam–xylazine (TZX). The serum content of total protein and albumin, cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotr ansferase (ALT), alkaline phosphatase (ALP), total bilirubin, and creatinine was decreased by the injection of TZX in comparison with CO2 inhalation. In addition, the levels of calcium, phosphates, chlorides and potassium were lowered by TZX vs. CO2 administration, while the level of sodium increased. Finally, the level of the majority of serum clinical biochemical parameters in rats and mice tend to be overestimated after CO2 inhalation, which may lead to masking the possible effect of anti-inflammatory drugs in animal tests. Injection anesthesia for small rodents with TZX is a more feasible method for terminal blood sampling, which also reduces the suffering of animals