Summary Tolerance represents a critical component of addiction. The large conductance calcium-and voltage-activated potassium channel (BK) is a well-established alcohol target, and an important element in behavioral and molecular alcohol tolerance. We tested whether microRNA, a newly-discovered class of gene expression regulators, plays a role in the development of tolerance. We show that in adult mammalian brain alcohol upregulates microRNA (miR-9) and mediates post-transcriptional reorganization in BK mRNA splice variants by miR-9-dependent destabilization of BK mRNAs containing 3’UTRs with a miR-9 Recognition Element (MRE). Different splice variants encode BK isoforms with different alcohol sensitivities. Computational modeling indicates that this miR-9 dependent mechanism contributes to alcohol tolerance. Moreover, this mechanism can be extended to regulation of additional miR-9 targets relevant to alcohol abuse. Our results describe a novel mechanism of multiplex regulation of stability of alternatively spliced mRNA by miRNA in drug adaptation and neuronal plasticity.

The activity of large conductance, Ca 2ϩ -sensitive K ϩ (BK Ca ) channels, known to control neuronal excitability, is increased by ethanol (EtOH) exposure. Moreover, brain cholesterol (CHS) is elevated after chronic exposure to EtOH, suggesting that membrane CHS may play a role in drug tolerance. Here, we use BK Ca channels from human brain (hslo subunits), reconstituted into 1-palmitoyl-2-oleoyl phosphatidylethanolamine/1-palmitoyl-2-oleoyl phosphatidylserine (POPS) bilayers, to examine CHS modulation of EtOH sensitivity. Acute exposure to clinically relevant EtOH levels increases channel activity without modifying conductance. In this minimal system, increases in CHS content within the range found in neuronal membranes lead to progressive antagonism of EtOH action. Furthermore, CHS inhibits basal channel activity with an affinity similar to that of CHS blunting of the alcohol effect. Modification of channel gating by either EtOH or CHS is reduced dramatically by removal of POPS from the bilayer, suggesting a common mechanism(s) of action. Indeed, channel dwell-time analysis indicates that CHS and EtOH exert opposite actions on the stability of channel closed states. However, each agent also acts on distinct dwell states not mirrored by the other, which contribute to the opposite effects of CHS and EtOH on channel gating.

Alcohol is an addictive drug that targets a variety of ion channels and receptors. To address whether the effects of alcohol are compartment specific (soma vs dendrite), we examined the effects of ethanol (EtOH) on large-conductance calcium-activated potassium channels (BK) in cell bodies and dendrites of freshly isolated neurons from the rat nucleus accumbens (NAcc), a region known to be critical for the development of addiction. Compartment-specific drug action was indeed observed. Clinically relevant concentrations of EtOH increased somatic but not dendritic BK channel open probability. Electrophysiological single-channel recordings and pharmacological analysis of the BK channel in excised patches from each region indicated a number of differences, suggestive of a compartment-specific expression of the ␤4 subunit of the BK channel, that might explain the differential alcohol sensitivity. These parameters included activation kinetics, calcium dependency, and toxin blockade. Reverse transcription-PCR showed that both BK channel ␤1 and ␤4 subunit mRNAs are found in the NAcc, although the signal for ␤1 is significantly weaker. Immunohistochemistry revealed that ␤1 subunits were found in both soma and dendrites, whereas ␤4 appeared restricted to the soma. These findings suggest that the ␤4 subunit may confer EtOH sensitivity to somatic BK channels, whereas the absence of ␤4 in the dendrite results in insensitivity to the drug. Consistent with this idea, acute EtOH potentiated ␣␤4 BK currents in transfected human embryonic kidney cells, whereas it failed to alter ␣␤1 BK channel-mediated currents. Finally, an EtOH concentration (50 mM) that increased BK channel open probability strongly decreased the duration of somatic-generated action potential in NAcc neurons.

Rat magnocellular neurones with cell bodies in the supraoptic and paraventricular nuclei of the hypothalamus send their axonal nerve endings into the neurohypophysis, where they release oxytocin or vasopressin into the bloodstream during highly specific firing patterns. Peptide release from the neurohypophysis is closely controlled by an interplay between the duration and frequency of the action potential burst, and the silence that separates the bursts generated by these neurones (Cazalis et al. 1985). Thus, understanding of peptide release from the hypothalamicneurohypophysial system requires characterization of the conductances that shape action potentials in both the cell bodies and their axonal endings. The physical separation of cell bodies and nerve endings in this system allows a unique opportunity to elucidate the differences in neuronal excitability, and underlying conductances in different cellular domains. Large conductance Ca¥-activated K¤ (BK) channels are ubiquitous in neurones. They typically exhibit high permeability and selectivity for K¤, and activation upon 1. Large conductance, Ca¥-activated K¤ (BK) channels were identified in freshly dissociated rat supraoptic neurones using patch clamp techniques. 2. The single channel conductance of cell body BK channels, recorded from inside-out patches in symmetric 145 mÒ K¤, was 246·1 pS, compared with 213 pS in nerve ending BK channels (P < 0·01). 3. At low open probability (Pï), the reciprocal of the slope in the ln(NPï)-voltage relationship (N, number of available channels in the patch) for cell body and nerve ending channels were similar: 11 vs. 14 mVper e-fold change in NPï, respectively. 4. At 40 mV, the [Ca¥]é producing half-maximal activation was 273 nÒ, as opposed to > 1·53 ìÒ for the neurohypophysial channel, indicating the higher Ca¥ sensitivity of the cell body isochannel. 5. Cell body BK channels showed fast kinetics (open time constant, 8·5 ms; fast closed time constant, 1·6 and slow closed time constant, 12·7 ms), identifying them as 'type I' isochannels, as opposed to the slow gating (type II) of neurohypophysial BK channels. 6. Cell body BK activity was reduced by 10 nÒ charybdotoxin (NPï, 37% of control), or 10 nÒ iberiotoxin (NPï, 5% of control), whereas neurohypophysial BK channels are insensitive to charybdotoxin at concentrations as high as 360 nÒ. 7. Whilst blockade of nerve ending BK channels markedly slowed the repolarization of evoked single spikes, blockade of cell body channels was without effect on repolarization of evoked single spikes. 8. Ethanol reversibly increased neurohypophysial BK channel activity (EC50, 22 mÒ; maximal effect, 100 mÒ). In contrast, ethanol (up to 100 mÒ) failed to increase cell body BK channel activity. 9. In conclusion, we have characterized BK channels in supraoptic neuronal cell bodies, and demonstrated that they display different electrophysiological and pharmacological properties from their counterparts in the nerve endings. Keywords: