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

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.Alcohol (ethyl alcohol, ethanol) 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/Ca²⁺-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 behavioral consequences of ethanol disruption of BK function have been well documented, it remains unknown whether alcohol modification of BK function results from drug interaction with a defined recognition site(s) in a protein target vs. physical perturbation of the proteolipid environment where the BK protein resides. Thus, location and structural characteristics of the ethanol-recognition site(s), as well as nature of chemical bonds between ethanol and functional target that lead to modification of BK function, remain unknown.Ethanol-induced regulation of BK channels is fine-tuned by many factors, including the BK channel-forming slo1 protein (α subunit) isoform (17) and its modification by phosphorylation (18), BK channel accessory (β) subunits (11), the channel-activating ionic ligand (Cai2+) (19) and the lipid microenvironment around the BK protein complex (20). However, ethanol perturbation of BK function is sustained when the slo1 protein is probed with the alcohol in cell-free membrane patches (19–21) or after protein reconstitution into artificial lipid bilayers (22). We recently demonstrated that perturbation of slo1 function by ethanol concentrations reached in blood during alcohol intoxication does not extend to Na⁺-gated slo2 and pH-gated slo3 channels, which are phylogenetically and structurally related to slo1. However, ethanol sensitivity does extend to a prokaryotic K⁺ channel from Methanobacterium thermoautotrophicum (MthK) (23), a TM2 ion channel that shares basic Cai2+-driven gating mechanisms with slo1 (24). Collectively, these studies lead us to hypothesize that ethanol-recognition site(s) involved in alcohol modification of BK current exists in the slo1 cytosolic Cai2+-sensing tail domain (CTD).Based on crystallographic data of the slo1 CTD and primary alignment of slo1-related ion channels that share ethanol sensitivity, we first identified eight putative ethanol recognition regions in the slo1 CTD. Using computational modeling, point amino acid substitutions and electrophysiology, we identified a distinct pocket as the ethanol-recognition site that leads to alcohol modification of BK current. This site has a few common characteristics of alcohol-binding protein sequences (25), yet presents features that differ from those of the alcohol site described in GIRK (6). In opposition to GIRK currents, which can be potentiated by alcohol in absence of G proteins (6), ethanol modulation of BK currents is dependent on the presence of Cai2+ (19). Our data strongly suggest that ethanol access to the newly identified BK ethanol-recognition site depends on the Cai2+ levels associated with the slo1 CTD. Thus, current data not only provide a structural basis for understanding Cai2+–alcohol allosterism on BK channels but could render structural insights on other ligand-gated channels that are activated by ethanol in presence of their natural ligand (26–30). Finally, present data document that the newly identified site plays a critical role in BK channel sensitivity to long-chain alkanols and explain the reported chain length differential sensitivity (“cutoff”) of linear n-alkanols to modify BK current (31).Identification of a distinct alcohol-sensing site in BK channels opens the door for rational design of pharmaceuticals to counteract widespread effects of alcohol intoxication in the body without altering basal BK channel function. Because this site is present in human BK protein ({"type":"entrez-protein","attrs":{"text":"AAA92290.1","term_id":"758791","term_text":"AAA92290.1"}}AAA92290.1), it is possible that genetic, epigenetic or other modifications of the alcohol-sensing site in BK channels could contribute to differential sensitivity to alcohol intoxication in humans. In addition, considering that individuals with low alcohol sensitivity are prone to developing heavy drinking (32), an altered profile of alcohol-sensing site on BK channels might be included as a potential predictor, along with other targets, for developing alcohol preference.