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  • A considerable body of evidence suggests that alcohols volat

    2021-11-29

    A considerable body of evidence suggests that alcohols, volatile anesthetics and inhaled drugs of abuse act at discrete sites on glycine and GABAA receptors, specifically within circumscribed protein pockets (Mascia et al., 2000, Mihic et al., 1997, Beckstead et al., 2000). An amino NAD+ residue (serine-267) in the second transmembrane segment of the α1 GlyR is thought to play an important role in the enhancing effects of ethanol and volatile anesthetics and, upon mutation, this enhancement is diminished or abolished. Mutation of the S267 residue to glutamine (S267Q), which results in the loss of ethanol enhancement of the α1 GlyR (Findlay et al., 2002), led to a decreased TFA effect (Fig. 1B), suggesting that TFA may act on this receptor in a manner similar to that of ethanol and volatile anesthetics. However, this conclusion is at odds with the finding that TFA modulation of the GlyR does not extend to other members of the cys-loop receptor family. Considering that a similar anesthetic binding pocket has been characterized in the closely-related GABAA receptors (Jung and Harris, 2006), we were surprised to find that the α1β2γ2S GABAA receptor was completely resistant to the effects of TFA. TFA thus displays rather remarkable selectivity as an allosteric modulator at glycine but not GABAA or 5-HT3 receptors. This of particular interest given that other GlyR modulators, such as inhalants, anesthetics, zinc, picrotoxin and tropisetron (Lynch, 2004), are far less specific, acting on multiple members of the cys-loop receptor family. Thus, TFA is one of the only known compounds to show GlyR-specific modulation, suggesting that this compound, or derivatives of this compound, may be useful for characterizing GlyR-specific effects in complex systems that contain other cys-loop receptors. However, the GlyR is not the only ion channel that displays sensitivity to TFA. A study by Han et al. (2001) showed that TFA reversibly activated ATP-sensitive potassium (KATP) channels in ventricular myocytes. Analysis of single channel recordings suggested that TFA acts on this channel by increasing the durations of bursts of channel openings and decreasing the sensitivity of KATP channels to ATP. The authors hypothesized that this action of TFA may be responsible for the cardioprotective effects of isofluorane in vivo (Han et al., 2001). However, our report is the first to identify a target within the central nervous system for TFA, expanding the possible secondary effects of this compound. TFA may also act at other biochemical sites in vivo. For example, it alters the proliferation rates of certain cell types, increasing the growth rates of some, while reducing the growth of others (Cornish et al., 1999). The exact mechanisms underlying these effects on cell division are unknown; however, as a chaotropic anion, TFA and other compounds of this class could conceivably affect membrane function, enzymatic catalysis, secondary protein structure, and protein stability. It is important to note that in our study, no such non-specific membrane effects were seen. The most common source of TFA in vivo is the metabolism of volatile anesthetics. Post-surgical blood TFA levels were monitored by Gauntlett et al. (1989) in two patients pretreated with the enzyme-inducing agent isoniazid and then anesthetized with isoflurane. TFA concentrations as high as 40μM were observed two days after surgery and remained elevated for at least a week. TFA is thought to bind to peptides and proteins in the body, thus remaining in the system long after the anesthetic has been cleared, leading to unwanted side effects such as halothane-induced hepatitis following a second administration of volatile anesthetics (Holaday, 1977, Gut et al., 1995). This concern becomes more prevalent with the increased use of synthesized peptides as potential therapeutic drugs. Following HPLC purification, peptides carry an unknown, and often variable, amount of TFA. This can lead to wide variability in effects across different batches of the same synthesized peptide. Reports comparing the effects of peptides with and without TFA contamination have been mixed. While some groups find that the presence of TFA alters the behavior or conformation of the tested peptide (Roux et al., 2007), others have found that the in vitro efficacy and toxicity are the same for TFA and other counter ions (Pini et al., 2011). However, these peptides often have very different toxicity profiles in vivo, with the TFA-bound peptides showing higher toxicity (Cornish et al., 1999, Pini et al., 2011). Even for peptides that show no differences in effect in the presence or absence of TFA, the secondary effects of TFA, such as the GlyR modulation reported here, and the extended lifetime of TFA in the body could drastically alter the effect of the peptide in vivo. Thus, our results have implications for the growing field of peptide-based drug design.