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.
Tolerance, described as the loss of drug effectiveness over time, is an important component of addiction. The degree of acute behavioral tolerance to alcohol exhibited by a naïve subject can predict the likelihood of alcohol abuse. Thus, the determinants of acute tolerance are important to understand. Calcium-and voltage-gated (BK) potassium channels, consisting of pore forming ␣ and modulatory  subunits, are targets of ethanol (EtOH) action. Here, we examine the role, at the molecular, cellular, and behavioral levels, of the BK 4 subunit in acute tolerance. Single channel recordings in HEK-293 cells show that, in the absence of 4, EtOH potentiation of activity exhibits acute tolerance, which is blocked by coexpressing the 4 subunit. BK channels in acutely isolated medium spiny neurons from WT mice (in which the 4 subunit is well-represented) exhibit little tolerance. In contrast, neuronal BK channels from 4 knockout (KO) mice do display acute tolerance. Brain slice recordings showed tolerance to EtOH's effects on spike patterning in KO but not in WT mice. In addition, 4 KO mice develop rapid tolerance to EtOH's locomotor effects, whereas WT mice do not. Finally, in a restricted access ethanol self-administration assay, 4 KO mice drink more than their WT counterparts. Taken together, these data indicate that the 4 subunit controls ethanol tolerance at the molecular, cellular, and behavioral levels, and could determine individual differences in alcohol abuse and alcoholism, as well as represent a therapeutic target for alcoholism.electrophysiology ͉ knockout mice ͉ striatum ͉ addiction ͉ plasticity A lcohol abuse is the third largest cause of preventable mortality in the world. Tolerance, described as the gradual loss of drug effectiveness over time, is a hallmark of abused drugs. This phenomenon is particularly important in the response to acute alcohol because the degree of tolerance exhibited by a naïve subject can predict the likelihood to develop alcohol abuse (1-4). Thus, identifying the mechanistic and molecular underpinnings of tolerance is essential for understanding the pathophysiology of alcoholism, as well as determining potential therapeutic targets for alcohol abuse. The neurobiology of tolerance is thought to involve several types of adaptation, ranging from alteration in membrane lipid composition (5) to neuroadaptative changes in target proteins (6, 7).In recent years, large conductance calcium-and voltage-gated potassium (BK) channels have emerged as one of the key targets of ethanol action, yet their role in the physiological and behavioral response to alcohol are unknown. Invertebrate studies suggest that BK channels may be important for the development of tolerance to ethanol (8, 9). In mammals, BK channels exist as a complex formed by the association of the pore-forming ␣ subunit with the auxiliary  subunit. The ␣ subunit is encoded by only one gene (slo) with several splice variants (STREX, P27, insertless, etc.), whereas the  subunit is the product of four distinct genes (1-4)...
Supervillin binding to myosin II is crucial for cytokinetic fidelity. This function complements that of anillin in promoting myosin II localization and cleavage furrow ingression during mammalian cell cytokinesis. Interactor analyses show that these proteins act through separate but overlapping pathways.
Cytokinesis requires coordination of cortical myosin II activation with central spindle alignment. Supervillin and anillin are two actin‐ and myosin II‐associated vertebrate proteins implicated in maintenance of the cytokinetic furrow in early cytokinesis, The nature of their cross‐talk is unknown. Point mutations within an evolutionarily conserved sequence in supervillin, residues 99 ‐ 153, eliminate binding to the nonmuscle myosin II heavy chain (MHC), confer dominant‐negative inhibition of cytokinesis, and abrogate the ability of full‐length supervillin to rescue RNAi‐mediated depletion of supervillin. While an observed ~2‐fold increase of anillin in supervillin‐knockdown cells does not eliminate cytokinetic failure, knockdown of both proteins together yields more than additive increases in numbers of multinucleated cells, as compared with depletion of either protein alone. Localizations of supervillin, anillin, MHC (total myosin), and phosphorylated regulatory myosin II light chain (pMRLC, activated myosin) in dividing cells suggest that supervillin and anillin synergize to confine MHC to the furrow. Anillin alone is sufficient to maintain normal levels of pMRLC at the furrow in late anaphase, but supervillin is required for normal pMRLC localization in early anaphase and around the cytokinetic bridge during daughter cell separation. Protein affinity isolations of GFP‐tagged supervillin and anillin indicate that these proteins do not significantly associate with each other although myosin II, actin and other cytoskeletal proteins co‐purify with each bait protein. None of these proteins co‐isolated with GFP alone. Stark differences between specifically co‐isolating kinases, phosphatases, and other signaling proteins reinforce the conclusion that supervillin and anillin regulate cytokinesis through separate pathways. Grant Funding Source: Supported by the National Institutes of Health and the University of Massachusetts Medical School.
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