Molecular Features of an Alcohol Binding Site in a Neuronal Potassium 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, 12-15). 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 protein-based theories of alcohol intoxication and general anesthetic action (16-18). Shaw2 K+ channels are members of the superfamily of voltage-gated K+ channels (Kv channels). Pharmacologically relevant concentrations of 1-alkanols selectively inhibit the Shaw2 K+ channel by stabilizing the closed state at an internal site, which is probably determined by the 13-amino acid sequence that constitutes the internal S4–S5 loop in the pore-forming subunit of the channel (16-20). Between ethanol and 1-hexanol, the apparent equilibrium dissociation constants decrease with the 1-alkanol carbon chain length according to the Meyer–Overton rule (17, 18). The binding free energy change per methylene group (−3 kJ/mol) corresponds to the free energy change necessary to transfer 1-alkanols from water to alkanes (21, 22). With longer chain 1-alkanols (longer than 1-heptanol), the binding free energy change levels off. Thus, as expected for the interaction of general anesthetic agents with their targets, a hydrophobic effect at a circumscribed site plays a significant role in the inhibition of Shaw2 K+ channels by 1-alkanols. However, it is not clear how the hydrophobic effect and the predicted polar interactions control the kinetics of 1-alkanol binding. Also, although the S4–S5 loop in the Shaw2 K+ channel clearly confers the inhibition by 1-alkanols, it is not known what structural features of this segment are responsible for this unique response. Here, we employed a combination of functional and structural approaches to shed some light on the solutions to these problems. Using a fast solution exchange system (concentration-clamp), we investigated the kinetics of the inhibition of Shaw2 K+ channels by ethanol, 1-butanol, and 1-hexanol. All three 1-alkanols exhibited second-order binding kinetics, which allowed the estimation of the second-order association rate constants and the dissociation rate constants. From the analysis of the relationship between alkyl chain length and the binding and unbinding rate constants, we inferred the main forces that control the interaction between the Shaw2 K+ channel and 1-alkanols. Then, the analysis of several peptides corresponding to the S4–S5 loop by CD1 spectroscopy revealed a relationship between the apparent binding affinity and the α-helical propensity. The observations are discussed in terms of a working hypothesis, whereby hydrophobic and weak short-range polar forces help to determine the extent of binding of 1-alkanols to a relatively specific internal site that is constrained by the secondary structure of the S4–S5 loop in the pore-forming subunit of the Shaw2 K+ channel.