SUMMARY
Large conductance, voltage- and calcium-activated (BK) channels are a molecular target of ethanol. Our data
indicate that ethanol-induced activation of BK channels facilitates the escalation of voluntary ethanol
consumption in mice made dependent to ethanol. We therefore hypothesize that molecular adaptations resulting
from chronic activation of BK channels by ethanol facilitate the progression to dependence, possibly by lowering
ethanol sensitivity. Accordingly, our project aims to elucidate the molecular identity of BK-dependent adaptations
(Aim 1, R21 phase) and to test their functional implication in the transition to dependence (Aim 2, R33 phase)
and in the control of ethanol sensitivity (Aim 3, R33 phase). We will take advantage of a knockin mouse
expressing BK channels that are insensitive to ethanol but function normally otherwise to identify molecular
adaptations to chronic ethanol that selectively result from the action of ethanol on BK channels. Molecular
adaptations that emerge in brain regions relevant to the motivational and affective effects of ethanol (ventral
tegmental area, amygdala, prelimbic prefrontal cortex, and habenula) will be examined in a well-validated mouse
model of ethanol dependence. We will leverage the unprecedented sensitivity and accuracy of data-independent
acquisition mass spectrometry to quantify changes in protein abundance across the entire proteome.
Furthermore, we will implement weighted correlation network analysis to identify proteins that are the most likely
to drive concerted changes in abundance across modules of co-expressed proteins. Nine of these proteins will
be selected at the end of the R21 phase for functional analysis during the R33 phase. We will use virally mediated
RNA interference to knock down candidate proteins in targeted brain regions and evaluate the influence of these
proteins on the time-course, amplitude and persistence of drinking escalation in ethanol-dependent mice. We
predict that some of the proteins controlling drinking escalation will also control acute sensitivity to ethanol, such
that their up- or down-regulation in ethanol-dependent mice would progressively decrease sensitivity to ethanol.
Accordingly, we will also examine the impact of local protein knockdown on the reinforcing and anxiolytic effects
of ethanol. Altogether, the proposed experiments are designed to identify novel molecular determinants of
vulnerability to alcohol use disorders. Our proposal is relevant to the focus of FOA PAR-18-659 because the
proteins identified in this project may pinpoint molecular mechanisms underlying differential sensitivity to alcohol
in the human population.
