Allosteric modulation of a neuronal K⁺ channel by 1-alkanols is linked to a key residue in the activation gate

Abstract

Harris, Thanawath, Andrew R. Graber, and Manuel Covarrubias. Allosteric modulation of a neuronal K ϩ channel by 1-alkanols is linked to a key residue in the activation gate. Am J Physiol Cell Physiol 285: C788-C796, 2003; 10.1152/ajpcell.00113.2003.-The selective inhibition of neuronal Shaw2 K ϩ channels by 1-alkanols is conferred by the internal S4-S5 loop, a region that also contributes to the gating of voltage-gated K ϩ channels. Here, we applied alanine scanning mutagenesis to examine the contribution of the S5 and S6 segments to the allosteric modulation of Shaw2 K ϩ channels by 1-alkanols. The internal section of S6 is the main activation gate of K ϩ channels. While several mutations in S5 and S6 modulated the inhibition of the channels by 1-butanol and others had no effect, a single mutation at a key site in S6 (P410A) converted this inhibition into a dramatic dose-dependent potentiation (ϳ2-fold at 15 mM and ϳ6-fold at 50 mM). P410 is the second proline in the highly conserved PVP motif that may cause a significant ␣-helix kink. The P410A currents in the presence of 1-butanol also exhibited novel kinetics (faster activation and slow inactivation). Internal application of 15 mM 1-butanol to inside-out patches expressing P410A did not significantly affect the mean unitary currents (ϳ2 pA at 0 mV) or the mean open time (5-6 ms) but clearly increased the opening frequency and open probability (ϳ2-to 4-fold). All effects displayed a fast onset and were fully reversible upon washout. The results suggest that the allosteric modulation of the Shaw2 K ϩ channel by 1-alkanols depends on a critical link between the PVP motif and activation gating. This study establishes the Shaw2 K ϩ channel as a robust model to investigate the mechanisms of alcohol intoxication and general anesthesia. alcohol; anesthesia; gating; scanning mutagenesis; Shaw channels ALIPHATIC ALCOHOLS (1-alkanols) and general anesthetics directly interact with critical sites in neuronal ion channels and, thereby, alter their function (7,28,29,34,36). Whereas in some instances ion channel function is inhibited, in others it is potentiated. Such effects could result from pore blockade or altered gating and are thought to constitute the physiological basis of acute alcohol intoxication and general anesthesia. However, little is known about the structural basis of these interactions and how they may change gating of ion channels. A current hypothesis proposes that 1-alkanols and general anesthetics bind to amphiphilic protein-protein interfaces involved in the conformational changes that underlie channel gating (2, 10, 14, 28). We have tested this hypothesis by examining the role of regions that contribute to the putative gating machinery of voltage-gated K ϩ channels (Kv channels). The neuronal Shaw2 K ϩ channel from Drosophila melanogaster is suitable for these studies because it is selectively inhibited by pharmacologically relevant concentrations of alcohol (3, 4). Shaw2 K ϩ channels are also special members of the superfamily of Kv channels becau...

Alcohol and anesthetic action at the gate of a voltage-dependent K+ channel

“…To test whether other putative components of the Shaw2 activation machinery may also contribute to 1-alkanol binding, we applied alanine scanning mutagenesis to portions of S5 and S6 [27]. The S5 mutants exhibited normal or modestly altered function and modulation of the inhibition by 1-butanol.…”

“…More significantly, however, as the alanine mutations approached the highly conserved Pro-Val-Pro-Val motif (PVPV), the inhibition by 1-butanol was progressively suppressed; and surprisingly, when the second proline of the PVPV motif was mutated to alanine (PVAV), 1-butanol induced a rapid reversible potentiation of the Shaw2 current. Further biophysical analyses indicated that destabilization of the closed state underlies this response [27]. Prolines in the PVPV motif may introduce a kink in the distal S6 helix, a segment that contributes to the main activation gate [36,38].…”

“…To date, no ion channel has provided 3D structural data on putative alcohol sites. Here, we review our work concerning the interactions between 1-alkanols and the Shaw2 K + channel from Drosophila melanogaster [22,[25][26][27][28]. This work has established this channel as a robust model system to study the molecular basis of the modulation of ion channels by general anesthetic agents.…”

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

“…Currents were recorded 3-7 days postinjection. The Shaw2-F335A mutant was preferred for the experiments reported here because it exhibits higher expression levels and no significantly affected biophysical properties and inhibition by 1-alkanols (24). Patch-clamp recording was conducted as described previously (25) using an Axopatch 200B apparatus (Axon Instruments, Foster City, CA).…”

“…In these experiments, we used the strongly expressing mutant Shaw2-F335A whose electrophysiological properties and 1-alkanol sensitivity are similar to those of the wild-type channel (24). Throughout this report, we refer to Shaw2-F335A as Shaw2.…”