gramicidin-a and 1-chloro-1-2-2-trifluorocyclobutane

Volatile anesthetic agent, 1-chloro-1,2,2-trifluorocyclobutane (F3), was found to alter gramicidin A channel function by enhancing Na(+) transport (. Biophys. J. 77:739-746). Whether this functional change is associated with structural alternation is evaluated by circular dichroism and nuclear magnetic resonance spectroscopy. The circular dichroism and nuclear magnetic resonance results indicate that at low millimolar concentrations, 1-chloro-1,2,2-trifluorocyclobutane causes minimal changes in gramicidin A channel structure in sodium dodecyl sulfate micelles. All hydrogen bonds between channel backbones are well maintained in the presence of 1-chloro-1,2,2-trifluorocyclobutane, and the channel structure is stable. The finding supports the notion that low affinity drugs such as volatile anesthetics and alcohols can cause significant changes in protein function without necessarily producing associated changes in protein structure. To understand the molecular mechanism of general anesthesia, it is important to recognize that in addition to structural changes, other protein properties, including dynamic characteristics of channel motions, may also be of functional significance.

Topics: Anesthetics; Chlorofluorocarbons; Circular Dichroism; Cyclobutanes; Dose-Response Relationship, Drug; Gramicidin; Hydrogen Bonding; Ion Channels; Magnetic Resonance Spectroscopy; Micelles; Models, Molecular; Protons; Sodium; Sodium Dodecyl Sulfate; Tryptophan

Although interactions of general anesthetics with soluble proteins have been studied, the specific interactions with membrane bound-proteins that characterize general anesthesia are largely unknown. The structural modulations of anesthetic interactions with synaptic ion channels have not been elucidated. Using gramicidin A as a simplified model for transmembrane ion channels, we have recently demonstrated that a pair of structurally similar volatile anesthetic and nonimmobilizer, 1-chloro-1,2,2-trifluorocyclobutane (F3) and 1,2-dichlorohexafluorocyclobutane (F6), respectively, have distinctly different effects on the channel function. Using high-resolution NMR structural analysis, we show here that neither F3 nor F6 at pharmacologically relevant concentrations can significantly affect the secondary structure of the gramicidin A channel. Although both the anesthetic F3 and the nonimmobilizer F6 can perturb residues at the middle section of the channel deep inside the hydrophobic region in the sodium dodecyl sulfate micelles, only F3, but not F6, can significantly alter the chemical shifts of the tryptophan indole N-H protons near the channel entrances. The results are consistent with the notion that anesthetics cause functional change of the channel by interacting with the amphipathic domains at the peptide-lipid-water interface.

Topics: Anesthetics, General; Biophysical Phenomena; Biophysics; Chlorofluorocarbons; Cyclobutanes; Gramicidin; In Vitro Techniques; Ion Channels; Lipids; Magnetic Resonance Spectroscopy; Micelles; Protein Structure, Secondary; Tryptophan; Water

Although it plays no clinical role in general anesthesia, gramicidin A, a transmembrane channel peptide, provides an excellent model for studying the specific interaction between volatile anesthetics and membrane proteins at the molecular level. We show here that a pair of structurally similar volatile anesthetic and nonimmobilizer (nonanesthetic), 1-chloro-1,2,2-trifluorocyclobutane (F3) and 1, 2-dichlorohexafluorocyclobutane (F6), respectively, interacts differently with the transmembrane peptide. With 400 microM gramicidin A in a vesicle suspension of 60 mM phosphatidylcholine-phosphatidylglycerol (PC/PG), the intermolecular cross-relaxation rate constants between (19)F of F3 and (1)H in the chemical shift regions for the indole and backbone amide protons were 0.0106 +/- 0.0007 (n = 12) and 0.0105 +/- 0.0014 (n = 8) s(-1), respectively. No cross-relaxation was measurable between (19)F of F6 and protons in these regions. Sodium transport study showed that with 75 microM gramicidin A in a vesicle suspension of 66 mM PC/PG, F3 increased the (23)Na apparent efflux rate constant from 149.7 +/- 7.2 of control (n = 3) to 191.7 +/- 12.2 s(-1) (n = 3), and the apparent influx rate constant from 182.1 +/- 15.4 to 222.8 +/- 21.7 s(-1) (n = 3). In contrast, F6 had no effects on either influx or efflux rate. It is concluded that the ability of general anesthetics to interact with amphipathic residues near the peptide-lipid-water interface and the inability of nonimmobilizer to do the same may represent some characteristics of anesthetic-protein interaction that are of importance to general anesthesia.

Topics: Amino Acid Sequence; Anesthetics, General; Biophysical Phenomena; Biophysics; Chlorofluorocarbons; Cyclobutanes; Gramicidin; In Vitro Techniques; Ion Transport; Membrane Proteins; Molecular Sequence Data; Sodium; Structure-Activity Relationship