Several neurotransmitters, including acetylcholine, regulate neuronal tone by suppressing a non-inactivating low-threshold voltage-gated potassium current generated by the M-channel. Agonist dependent control of the M-channel is mediated by calmodulin, activation of anchored protein kinase C (PKC), and depletion of the phospholipid messenger phosphatidylinositol 4,5-bisphosphate (PIP2). In this report, we show how this trio of second messenger responsive events acts synergistically and in a stepwise manner to suppress activity of the M-current. PKC phosphorylation of the KCNQ2 channel subunit induces dissociation of calmodulin from the M-channel complex. The calmodulin-deficient channel has a reduced affinity towards PIP2. This pathway enhances the effect of concomitant reduction of PIP2, which leads to disruption of the M-channel function. These findings clarify how a common lipid cofactor, such as PIP2, can selectively regulate ion channels.

Alcohol use disorder (AUD) is a debilitating condition marked by cyclic patterns of craving, use, and withdrawal. These pathological behaviors are mediated by multiple neurotransmitter systems utilizing glutamate, GABA, dopamine, ATP, and adenosine. In particular, purines such as ATP and adenosine have been demonstrated to alter the phase and function of the circadian clock and are reciprocally regulated by the clock itself. Importantly, chronic ethanol intake has been demonstrated to disrupt the molecular circadian clock and is associated with altered circadian patterns of activity and sleep. Moreover, ethanol has been demonstrated to disrupt purinergic signaling, while dysfunction of the purinergic system has been implicated in conditions of drug abuse such as AUD. In this review, we summarize our current knowledge regarding circadian disruption by ethanol, focusing on the reciprocal relationship that exists between oscillatory neurotransmission and the molecular circadian clock. In particular, we offer detailed explanations and hypotheses regarding the concerted regulation of purinergic signaling and circadian oscillations by neurons and astrocytes, and review the diverse mechanisms by which purinergic dysfuction may contribute to circadian disruption or alcohol abuse. Finally, we describe the mechanisms by which ethanol may disrupt or hijack endogenous circadian rhythms to induce the maladaptive behavioral patterns associated with AUD.

Depression is a well-known risk factor for developing relapse drinking, but the neuronal mechanisms underlying the interactions between depression and alcohol use disorders remain elusive. Accumulating evidence has associated depression with hyperactivity of the lateral habenula (LHb), an epithalamic structure in the brain that encodes aversive signals. Glutamate receptors contribute substantially to the excitability of LHb neurons. Glutamatergic synapses in LHb neurons largely express GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) that can be modulated by Ca2+/calmodulin-dependent protein II (CaMKII). In the current study, we tested the hypothesis that withdrawal from repeated cycles of ethanol drinking triggers an increase in LHb AMPAR and CaMKII activity concomitant with depression-like symptoms, and their inhibitions bring a reduction in depressive-like behaviors and alcohol consumption. Western blotting revealed a higher level of phosphorylated AMPAR GluA1 subunit at a CaMKII locus (GluA1-Ser831) in the LHb of ethanol-withdrawn rats than that of age-matched naïve counterparts. In ethanol-withdrawn rats, pharmacological inhibition of LHb AMPAR activity significantly mitigated the depressive-like behavior and ethanol drinking and seeking behaviors, but affected neither sucrose intake nor locomotor activity; and inhibition of LHb CaMKII activity, or chemogenetic inhibition of LHb activity produced similar effects. Conversely, activation of LHb AMPARs induced depressive-like behaviors in ethanol-naïve rats. These results demonstrate that CaMKII-AMPAR signaling in the LHb exemplifies a molecular basis for depressive-like symptoms during ethanol withdrawal and that inhibition of this signaling pathway may offer a new therapeutic approach to address the comorbidity of alcohol abuse and depression.

Background: Calmodulin binding to KCNQ subunit is required for maintaining the M-current. Results: Protein kinase CK2-mediated phosphorylation of CaM enhances KCNQ2 current. KCNQ2 subunit tethers protein kinase CK2 and protein phosphatase 1. Conclusion: Phosphorylation status of CaM regulates the M-current. Significance: Phosphorylation status of calmodulin regulates neuronal excitability via M-current modulation.

Alcohol use disorders (AUDs) and anxiety disorders (ADs) are often seen concurrently, but their underlying cellular basis is unclear. For unclear reasons, the lateral habenula (LHb), a key brain region involved in the pathophysiology of ADs, becomes hyperactive after ethanol withdrawal. M-type K channels (M-channels), important regulators of neuronal activity, are abundant in the LHb, yet little is known about their role in AUDs and associated ADs. We report here that in rats at 24 h withdrawal from systemic ethanol administration (either by intraperitoneal injection, 2 g/kg, twice/day, for 7 days; or intermittent drinking 20% ethanol in a two-bottle free choice protocol for 8 weeks), the basal firing rate and the excitability of LHb neurons in brain slices was higher, whereas the amplitude of medium afterhyperpolarization and M-type K currents were smaller, when compared to ethanol naive rats. Concordantly, M-channel blocker (XE991)-induced increase in the spontaneous firing rate in LHb neurons was smaller. The protein expression of M-channel subunits, KCNQ2/3 in the LHb was also smaller. Moreover, anxiety levels (tested in open field, marble burying, and elevated plus maze) were higher, which were alleviated by LHb inhibition either chemogenetically or by local infusion of the M-channel opener, retigabine. Intra-LHb infusion of retigabine also reduced ethanol consumption and preference. These findings reveal an important role of LHb M-channels in the expression of AUDs and ADs, and suggest that the M-channels could be a potential therapeutic target for alcoholics.