Distinctly Different Interactions of Anesthetic and Nonimmobilizer with Transmembrane Channel Peptides

Abstract

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.

Monitoring changes of paramagnetically-shifted 31P signals in phospholipid vesicles

“…Phospholipid vesicles are used as model systems for a wide range of investigations, including vesicle permeation by amyloidogenic peptides, [10,46] changes in membrane dynamics upon protein binding [22] and the transport of ions and small molecules across a membrane [15,16]. The results presented here indicate that using phospholipid vesicles as a mimetic of membrane interaction care should be taken using paramagnetic shift reagents and freshly prepared vesicles that any changes in 31 P peak position or appearance over the early time points of an experiment is not simply a result of this "maturation" or reequilibration process, but arises from the action under investigation.…”

“…The use of other, often more dilute, NMR-active nuclei can alleviate this problem, depending on their location in or around the vesicle bilayer. For example, the functioning of ion channels, and their inhibition by various compounds such as toxins or anaesthetics has been investigated using 7 Li and 23 Na NMR and the use of paramagnetic shift reagents based on dysprosium (III) [15,16]. The lipids themselves, can be observed directly using the single phosphorus atom located in the head group of each molecule.…”

Large-scale molecular dynamics simulations of general anesthetic effects on the ion channel in the fully hydrated membrane: The implication of molecular mechanisms of general anesthesia

“…It has long been recognized that anesthetic binding, particularly when volatile anesthetics are involved, cannot be viewed as stationary (13)(14)(15)(16). Rapid binding kinetics, along with the fact that a huge number of structurally diverse anesthetics can produce similar effects, seems to suggest that a ''one-size-fits-all'' anesthetic pocket is not plausible.…”

“…The gramicidin A (gA) channel was chosen as a convenient model, because the high-resolution structure of this channel has been solved by NMR (17,18) and thus can be used as the starting structure for simulation (19,20). In addition, we have accumulated an extensive amount of experimental data about anesthetic effects on this channel and found that the channel conductance can be modulated by anesthetics but not by structurally similar molecules devoid of anesthetic effects (15), suggesting the theoretical relevance of the model to anesthetic action. We also found that anesthetics, but not nonanesthetics (or nonimmobilizers), can interact specifically with the amphiphilic-anchoring tryptophan residues of gA channel at the lipid-water interface and that this interaction requires the channel conformation in the membranous environment (14,15,21).…”

“…In addition, we have accumulated an extensive amount of experimental data about anesthetic effects on this channel and found that the channel conductance can be modulated by anesthetics but not by structurally similar molecules devoid of anesthetic effects (15), suggesting the theoretical relevance of the model to anesthetic action. We also found that anesthetics, but not nonanesthetics (or nonimmobilizers), can interact specifically with the amphiphilic-anchoring tryptophan residues of gA channel at the lipid-water interface and that this interaction requires the channel conformation in the membranous environment (14,15,21). These experimental results are the essential premise for the initial setup of the simulation system.…”