Acute alcohol tolerance is intrinsic to the BKCa protein, but is modulated by the lipid environment

Abstract

Ethanol tolerance, in which exposure leads to reduced sensitivity, is an important component of alcohol abuse and addiction. The molecular mechanisms underlying this process remain poorly understood. The BKCa channel plays a central role in the behavioral response to ethanol in Caenorhabditis elegans (Davies, A. G., Pierce-Shimomura, J. T., Kim, H., VanHoven, M. K., Thiele, T. R., Bonci, A., Bargmann, C. I., and McIntire, S. L. (2003) Cell 115, 655-666) and Drosophila (Cowmeadow, R. B., Krishnan, H. R., and Atkinson, N. S. (2005) Alcohol. Clin. Exp. Res. 29, 1777-1786) . In neurons, ethanol tolerance in BKCa channels has two components: a reduced number of membrane channels and decreased potentiation of the remaining channels (Pietrzykowski, A. Z., Martin, G. E., Puig, S. I., Knott, T. K., Lemos, J. R., and Treistman, S. N. (2004) J. Neurosci. 24, 8322-8332) . Here, heterologous expression coupled with planar bilayer techniques examines two additional aspects of tolerance in human BKCa channels. 1) Is acute tolerance observed in a single channel protein complex within a lipid environment reduced to only two lipids? 2) Does lipid bilayer composition affect the appearance of acute tolerance? We found that tolerance was observable in BKCa channels in membrane patches pulled from HEK cells and when they are placed into reconstituted 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine membranes. Furthermore, altering bilayer thickness by incorporating the channel into lipid mixtures of 1,2-dioleoyl-3-phosphatidylethanolamine with phosphatidylcholines of increasing chain length, or with sphingomyelin, strongly affected the sensitivity of the channel, as well as the time course of the acute response. Ethanol sensitivity changed from a strong potentiation in thin bilayers to inhibition in thick sphingomyelin/1,2-dioleoyl-3-phosphatidylethanolamine bilayers. Thus, tolerance can be an intrinsic property of the channel protein-lipid complex, and bilayer thickness plays an important role in shaping the pattern of response to ethanol. As a consequence of these findings the protein-lipid complex should be treated as a unit when studying ethanol action.

Figure 1. A schematic of the experimental strategy employed to examine the BK_Ca channel in a natural lipid environment and in an artificial membrane

Stably transfected HEK-293 cells were grown in artificial medium (42) and then half of the cells were used directly in the patch recording experiments. The other half were used to isolate membrane fragments for the bilayer recordings.

Figure 2. Voltage dependence of BK_Ca channels expressed in HEK-293 cells

A, currents through a single channel recorded in inside-out patch clamp mode in the presence of 1 μm free Ca²⁺, shows an increase in activity with depolarization from a holding potential of −80 to +20 mV. B, an I-V plot (channel current versus membrane potential) averaged over 10 recordings yielded a BK_Ca channel unitary conductance of 267 pS. O and C represent the open and closed states of the channel, respectively.

Figure 3. BK_Ca channels expressed in HEK-293 cells develop rapid acute tolerance to ethanol exposures

A, continuous recording of an inside-out patch before and after application of 50 mm ethanol. The holding potential is −50 mV. The traces shown were taken during the first 10 s of 1-min segments of the recording at 1,2,4,8, and 15 min after the application of 50 mm ethanol, as indicated. B, normalized plot of nPo ratio (with ethanol/control) over the time course of recording from 8 patches. Each data point is the nPo calculated during 30 s of the recording.

Figure 4. BK_Ca channels extracted from transfected HEK-293 cells and reconstituted into POPE/POPS bilayers show acute tolerance

A, continuous recording of a BK_Ca channel in a POPE/POPS bilayer before and 1 min after the application of 50 mm ethanol (indicated by the arrow; buffer conditions: 300/150 KCl (cis/trans), [Ca²⁺]free about 8 μm). The holding potential is 20 mV. The traces shown were taken during the first 10 s of each 1-min recording, as marked in the figure. B, normalized plot of nPo ratio (with ethanol/control) from 7 trials in POPE/POPS bilayers (filled symbols). Three control experiments (open symbols) showed that BK_Ca channel activities are stable over the time course of recording in the absence of alcohol. The inset in B shows the measured ethanol concentration in the cis/trans recording chambers during a recording time of 10 min.

Figure 5. Initial ethanol sensitivity of the BK_Ca channel changes with an increase in bilayer thickness from robust activation in bilayers of PC (14:1)/DOPE to inhibition in bilayers of SPM/DOPE

A, recordings of BK_Ca current in each bilayer before and after the application of 50 mm ethanol (2 min after the exposure to ethanol). The buffer condition is 300/150 KCl (cis/trans), and 20 μm [Ca²⁺]_free for bilayers of DOPE with PC series, and 10 μm for SPM/DOPE bilayers. The holding potential is 20 mV for all bilayers. B, scatter plots of BK_Ca channel response at 2 min after exposure to 50 mm ethanol in each bilayer. Each point represents a single experiment in a given bilayer. The average nPo (with ethanol/control) are shown as mean ± S.E. at the top of each column, along with the number of experiments in the average.

Figure 6. The sustained response of BK_Ca channels to ethanol is altered in lipid bilayers of different thickness

A, traces of BK_Ca current from a continuous recording in a PC (20:1)/DOPE bilayer before and after the indicated times since exposures to 50 mm ethanol, showing the development of in session acute ethanol tolerance. The buffer condition is 300/150 KCl (cis/trans), and 20 μm [Ca²⁺]_free, holding potential is 20 mV. Normalized plots of nPo ratio (with ethanol/control, closed symbols) over the time course of recording in PC (20:1)/DOPE bilayer (B), PC (24:1)/DOPE bilayer (C), and SPM/DOPE bilayer (D). In each case the open symbols plot the activity in the absence of alcohol and the arrows indicate the first response 1 min after alcohol addition.