Distinct Sensitivity of Slo1 Channel Proteins to Ethanol

Abstract

Ethanol levels reached in circulation during moderate-to-heavy alcohol intoxication (50-100 mM) modify Ca 21 -and voltagegated K 1 (BK) channel steady-state activity, eventually altering both physiology and behavior. Ethanol action on BK steadystate activity solely requires the channel-forming subunit slo1 within a bare lipid environment. To identify the protein regions that confer ethanol sensitivity to slo1, we tested the ethanol sensitivity of heterologously expressed slo1 and structurally related channels. Ethanol (50 mM) increased the steady-state activities of mslo1 and Ca 21 -gated MthK, the latter after channel reconstitution into phospholipid bilayers. In contrast, 50-100 mM ethanol failed to alter the steady-state activities of Na 1 /Cl 2 -gated rslo2, H 1 -gated mslo3, and an mslo1/3 chimera engineered by joining the mslo1 region encompassing the N terminus to S6 with the mslo3 cytosolic tail domain (CTD). Collectively, data indicate that the slo family canonical design, which combines a transmembrane 6 (TM6) voltage-gated K 1 channel (K V ) core with CTDs that empower the channel with ion-sensing, does not necessarily render ethanol sensitivity. In addition, the region encompassing the N terminus to the S0-S1 cytosolic loop (missing in MthK) is not necessary for ethanol action. Moreover, incorporation of both this region and an ionsensing CTD to TM6 K V cores (a design common to mslo1, mslo3, and the mslo1/mslo3 chimera) is not sufficient for ethanol sensitivity. Rather, a CTD containing Ca 21 -sensing regulator of conductance for K 1 domains seems to be critical to bestow K V structures, whether of TM2 (MthK) or TM6 (slo1), with sensitivity to intoxicating ethanol levels.

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

“…The ethanol site resides in the CTD of the channel-forming subunit of the BK (slo1) channel. This location is in agreement with our earlier electrophysiological data suggesting that the slo1 CTD was the region that should contain ethanol-sensing site(s) (23). The intracellular topology of the BK channel alcohol-sensing site, however, is rather unusual for a transmembrane channel protein as ethanol-sensing motifs have been mapped to TM regions in many ligand-gated ion channels, such as mammalian GABA, glycine, nACh, and prokaryotic Gloeobacter violaceus (GLIC) channels (5,40).…”

“…After excision, each patch was exposed to control bath solution followed by application of 100 mM ethanol, which is ethanol E max for modulating native BK and recombinant slo1 channel activity (21,34). As consistently reported by us and others (11,23,35) when recording BK currents at sub-μM to low μM Ca 2+ i , 100 mM ethanol caused a ≥twofold reversible increase in mslo1 steady-state activity (NPo) (Fig. 2 A and C).…”

“…The newly identified ethanol-sensing site on BK channel may explain the absolute dependence of ethanol modulation of BK channel function on Ca 2+ i levels (19,23). After computational superposition of predicted ethanol-sensing site in presence vs. nominal zero Ca 2+ , we demonstrate that K361 is not accessible for hydrogen bonding, and ethanol-sensing pocket is protruded by M909 (Fig.…”

“…We have recently established that a CTD containing two Ca 2+ i -sensing, regulator of conductance for K + (RCK) domain(s) seems to be necessary to bestow voltage-gated K + (K V ) structures, whether having two (MthK) or six (mslo1, mouse brain slo1) TM segments, with sensitivity to intoxicating levels of ethanol (23). Primary alignment of CTDs from MthK (residues 117-336) and mslo1 (residues 344-1209) revealed eight regions with sequence similarity (Fig.…”

“…Ethanol sensitivity of slo and related proteins seems restricted to ion channels that are Ca 2+ i -gated (23). Therefore, we next evaluated whether the ethanol-resistance of the double mutant was selective for ethanol or, rather, accompanied by perturbation of gating by physiological changes in Ca 2+ i that activate WT slo1 channels (36)(37)(38).…”