An alcohol-sensing site in the calcium- and voltage-gated, large conductance potassium (BK) channel

Abstract

Ethanol alters BK (slo1) channel function leading to perturbation of physiology and behavior. Site(s) and mechanism(s) of ethanol-BK channel interaction are unknown. We demonstrate that ethanol docks onto a water-accessible site that is strategically positioned between the slo1 calcium-sensors and gate. Ethanol only accesses this site in presence of calcium, the BK channel's physiological agonist. Within the site, ethanol hydrogen-bonds with K361. Moreover, substitutions that hamper hydrogen bond formation or prevent ethanol from accessing K361 abolish alcohol action without altering basal channel function. Alcohol interacting site dimensions are approximately 10.7 × 8.6 × 7.1 Å, accommodating effective (ethanol-heptanol) but not ineffective (octanol, nonanol) channel activators. This study presents: (i) to our knowledge, the first identification and characterization of an n-alkanol recognition site in a member of the voltage-gated TM6 channel superfamily; (ii) structural insights on ethanol allosteric interactions with ligand-gated ion channels; and (iii) a first step for designing agents that antagonize BK channel-mediated alcohol actions without perturbing basal channel function.is a psychoactive agent that has been overwhelmingly consumed by mankind across cultures and civilizations. Alcohol actions on central nervous system (CNS) physiology and behavior are largely independent of beverage type but due to ethanol itself (1). Ethanol alters cell excitability by modifying function of transmembrane (TM) ion channel proteins, including K + channels. These channels constitute the most heterogeneous and extensive group of ion channels, its members belonging to TM2, TM4, and TM6 protein superfamilies. Within this myriad of proteins, several K + channels have been shown to modify behavior in response to acute exposure to ethanol concentrations that reach the CNS and other excitable tissues during alcohol drinking (2-5). However, with the sole exception of the TM2, G protein-regulated inward rectifier K + (GIRK) channel (6), there is no structural information on ethanol-K + channel protein interacting sites currently available. Voltage/Ca 2+ -gated, large conductance K + channels (BK), which are members of the TM6 voltage-gated ion channel superfamily, constitute major mediators of alcohol actions in excitable tissues. Acute exposure to ethanol levels reached in CNS during alcohol intoxication alters BK-mediated currents and thus, elicits widespread and profound modifications in physiology and behavior. In rodent models, acute ethanol exposure leads to reduced vasopressin, oxytocin and growth hormone release with consequent perturbation in physiology and behavior (7), altered firing rates in nucleus accumbens (8) and dorsal root ganglia neurons (9), and alcohol-induced cerebral artery constriction (10, 11). Moreover, studies in both mammals and invertebrate models demonstrate that ethanol targeting of neuronal BK is involved in development of alcohol tolerance and dependence (12-16). Although the physiological and be...

Modulation of BK Channel Function by Auxiliary Beta and Gamma Subunits

“…In contrast to the nervous system, ethanol caused inhibition of BK currents in myocytes that led to cerebrovascular constriction, in which the β1 subunit played a major role (Bukiya, Liu, et al, 2009). This group recently identified K361 on BKα as a putative binding site for ethanol (Bukiya, Kuntamallappanavar, et al, 2014). Further studies will be needed to clarify the different effects of different β subunits on the BK channel’s responses to ethanol.…”

Differential potassium channel gene regulation in BXD mice reveals novel targets for pharmacogenetic therapies to reduce heavy alcohol drinking

“…Additionally, preclinical studies have demonstrated that chronic ethanol reduces the function and trafficking of K Ca 2 ( Kcnn ), K V 4.2 ( Kcnd2 ), and K V 7.2 ( Kcnq2 ) channels in the nucleus accumbens (NAc) and hippocampus (Hopf et al, 2010; McGuier et al, 2015; Mulholland, Spencer, Hu, Kroener, & Chandler, 2015; Padula et al, 2015; Spencer, Mulholland, & Chandler, In Press), and pharmacologically enhancing K Ca 2 and K V 7 channel function attenuated voluntary drinking in rodents (Hopf et al, 2011; Knapp, O’Malley, Datta, & Ciraulo, 2014; McGuier et al, 2015; Padula et al, 2013). Ethanol actions on voltage- and calcium-dependent K Ca 1.1 ( Kcnma1 ) channels are involved in acute ethanol tolerance, dependence, and heavy ethanol consumption (Bukiya et al, 2014; Ghezzi, Pohl, Wang, & Atkinson, 2010; Kreifeldt, Le, Treistman, Koob, & Contet, 2013; Treistman & Martin, 2009), and Kcnma1 and Kcnq5 were identified as major hub genes for the acute actions of ethanol (Wolen et al, 2012). Deletion of the gene that encodes K ir 3.3 channels ( Kcnj9 ) enhanced ethanol conditioned place preference (Tipps, Raybuck, Kozell, Lattal, & Buck, 2016) and blunted ethanol-induced excitation of dopamine neurons and increased binge-like ethanol consumption in mice in both a limited-access 2-bottle choice (LA-2BC) model (15% v/v ethanol vs. water) and with limited-access to a single bottle of 20% v/v ethanol (Herman et al, 2015).…”

Modulation of BK Channels by Ethanol

“…Yuan, Leonetti, Pico, Hsiung, & MacKinnon, 2010) and “closed” (Ca 2+ i -free; Wu, Yang, Ye, & Jiang, 2010) states of the BK channel. The model of ethanol docking onto a site of discrete dimensions in the mslo1 channel (cloned from mouse brain) (Bukiya et al, 2014) explains previously described features of modulation of BK current by ethanol and related n-alkanols: 1) ethanol action’s dependence on Ca 2+ i presence (Liu, Bukiya, Kuntamallappanavar, Singh, & Dopico, 2013; Liu, Vaithianathan, Manivannan, Parrill, & Dopico, 2008) (Fig. 1) and 2) the refractoriness of slo1 channels to n-alkanols longer than heptanol (Chu & Treistman, 1997) (Fig.…”

“…More recent studies using engineered BK channel-forming slo1 proteins (Bukiya et al, 2014) and genetic screening or mutation of the BK channel-coding gene in Caenorhabditis elegans (Slo1) (Davis, Scott, Hu, & Pierce-Shimomura, 2014; Davis et al, 2015) have buttressed the idea of an ethanol-recognition site in the BK channel-forming protein (also termed BK α subunit or slo1 channel protein) by satisfying criteria (2) and (3). While criterion (4) has yet to be met, it should be noted that the Molecular Modeling (MM) and Molecular Dynamics (MD) data from Bukiya et al (2014) were based on crystallographic data of the BK channel cytosolic tail domain (CTD) (wherein the ethanol binding site has been mapped) in both “open” (Ca 2+ i -bound; P. Yuan, Leonetti, Pico, Hsiung, & MacKinnon, 2010) and “closed” (Ca 2+ i -free; Wu, Yang, Ye, & Jiang, 2010) states of the BK channel.…”