Molecular Features of an Alcohol Binding Site in a Neuronal Potassium Channel

Abstract

Aliphatic alcohols (1-alkanols) selectively inhibit the neuronal Shaw2 K + channel at an internal binding site. This inhibition is conferred by a sequence of 13 residues that constitutes the S4-S5 loop in the pore-forming subunit. Here, we combined functional and structural approaches to gain insights into the molecular basis of this interaction. To infer the forces that are involved, we employed a fast concentration-clamp method (10-90% exchange time = 800 μs) to examine the kinetics of the interaction of three members of the homologous series of 1-alkanols (ethanol, 1-butanol, and 1-hexanol) with Shaw2 K + channels in Xenopus oocyte inside-out patches. As expected for a secondorder mechanism involving a receptor site, only the observed association rate constants were linearly dependent on the 1-alkanol concentration. While the alkyl chain length modestly influenced the dissociation rate constants (decreasing only ∼2-fold between ethanol and 1-hexanol), the secondorder association rate constants increased e-fold per carbon atom. Thus, hydrophobic interactions govern the probability of productive collisions at the 1-alkanol binding site, and short-range polar interactions help to stabilize the complex. We also examined the relationship between the energetics of 1-alkanol binding and the structural properties of the S4-S5 loop. Circular dichroism spectroscopy applied to peptides corresponding to the S4-S5 loop of various K + channels revealed a correlation between the apparent binding affinity of the 1-alkanol binding site and the α-helical propensity of the S4-S5 loop. The data suggest that amphiphilic interactions at the Shaw2 1-alkanol binding site depend on specific structural constraints in the pore-forming subunit of the channel.Alcohol and general anesthetic agents interact with related and relatively specific binding sites in multiple protein targets (1-6). Aliphatic alcohols (e.g., 1-alkanols) have been used to probe the physicochemical properties of these binding sites (7-11). The results of these studies are consistent with the presence of physically circumscribed hydrophobic protein cavities that constitute the alcohol and general anesthetic sites. Also, studies with soluble model proteins have examined the relationship between anesthetic solubility and anesthetic action along with structure-function analysis and thermodynamic arguments and suggest that polar interactions also contribute to the binding of alcohol and inhaled anesthetics (5,9,(12)(13)(14)(15). Polar interactions are also likely to contribute to the binding of general anesthetic agents to ion channels, which are critical physiological targets of these agents (including 1-alkanols). Our earlier work has shown that the Drosophila Shaw2 K + channel is a robust model for investigating the proteinbased theories of alcohol intoxication and general anesthetic action (16)(17)(18) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript channels are members of the superfamily of voltage-gated K + channels (Kv channels)....

Immunosuppressive Effect of Isopropanol: Down-Regulation of Cytokine Production Results from the Alteration of Discrete Transcriptional Pathways in Activated Lymphocytes

“…26). At more physiologically relevant concentrations, alcohols have been shown to induce loss of function of specific proteins, such as ion channels, neurotransmitter receptors, enzymes, and adhesion molecules (27)(28)(29). Structural and biophysical data suggest that binding to the target proteins occurs at discrete sites that are constituted by hydrophobic pockets lined by nonpolar amino acids (26 -29).…”

The Concerted Contribution of the S4-S5 Linker and the S6 Segment to the Modulation of a Kv Channel by 1-Alkanols

“…The side chains of Thr321 and Ser325 are expected to face the hydrophobic S6-b segment. The features of this interface suggest the presence of an amphipathic site that may accommodate 1-alkanols (Shahidullah et al, 2003;Covarrubias et al, 2005). 2000,2003).…”

“…Peptides corresponding to the S4-S5 linkers of Shaw2 and K v 3.4 were synthesized as described previously (Shahidullah et al, 2003). The peptides were dissolved in 5 mM phosphate buffer, pH 6.0, at a final concentration of 50 M. To promote the ␣-helical structure of the peptides, TFE was added to the solutions.…”

“…Curve fitting and data display and analysis were carried out in Origin 7.5 (OriginLab Corp., Northampton, MA) or Sigmaplot 9.0 (Systat Software Inc., Point Richmond, CA). Doseresponse curves were analyzed as described previously (Covarrubias et al, 1995;Harris et al, 2000;Shahidullah et al, 2003). In brief, the normalized equilibrium dose-inhibition curves were empirically described by assuming the Hill equation:…”

“…The voltage-gated Shaw2 K ϩ channel present in the nervous system of Drosophila melanogaster is inhibited by clinically relevant doses of 1-alkanols and inhaled anesthetics in a saturable fashion (Covarrubias et al, 1995;Hodge et al, 2005) (A. Bhattacharji and M. Covarrubias, unpublished data); this modulation involves the 13-amino acid amphipathic S4 -S5 linker ( Fig. 1), which may contribute to the 1-alkanol binding site in the channel (Harris et al, 2000;Shahidullah et al, 2003). In addition, substitution of alanine for a proline at the third position of the highly conserved PxPx motif (PVAV) in the S6 segment of the Shaw2 subunit converts the modulation by 1-alkanols from inhibition to potentiation .…”