Philip M. Newton

Protein Kinase Cϵ Regulates γ-Aminobutyrate Type A Receptor Sensitivity to Ethanol and Benzodiazepines through Phosphorylation of γ2 Subunits

Ethanol enhances γ-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABAA receptors, especially at low concentrations achieved during social drinking. We postulated that the ϵ isoform of protein kinase C (PKCϵ) regulates the ethanol sensitivity of GABAA receptors, as mice lacking PKCϵ show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCϵ mutant to selectively inhibit the catalytic activity of PKCϵ. We used this mutant and PKCϵ-/- mice to determine that PKCϵ phosphorylates γ2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at α1β2γ2 receptors, which is the most abundant GABAA receptor subtype in the brain. Our findings indicate that PKCϵ phosphorylation of γ2 regulates the response of GABAA receptors to specific allosteric modulators, and, in particular, PKCϵ inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.

The biology of protein kinase C.

This review gives a basic introduction to the biology of protein kinase C, one of the first calcium-dependent kinases to be discovered. We review the structure and function of protein kinase C, along with some of the substrates of individual isoforms. We then review strategies for inhibiting PKC in experimental systems and finally discuss the therapeutic potential of targeting PKC. Each aspect is covered in summary, with links to detailed resources where appropriate.

Deletion of N-type calcium channels alters ethanol reward and reduces ethanol consumption in mice

N-type calcium channels are modulated by acute and chronic ethanol exposure in vitro at concentrations known to affect humans, but it is not known whether N-type channels are important for behavioral responses to ethanol in vivo. Here, we show that in mice lacking functional N-type calcium channels, voluntary ethanol consumption is reduced and place preference is developed only at a low dose of ethanol. The hypnotic effects of ethanol are also substantially diminished, whereas ethanol-induced ataxia is mildly increased. These results demonstrate that N-type calcium channels modulate acute responses to ethanol and are important mediators of ethanol reward and preference.

Amygdala protein kinase C epsilon controls alcohol consumption

Alcoholism is a progressive disorder that involves the amygdala. Mice lacking protein kinase C epsilon (PKCɛ) show reduced ethanol consumption, sensitivity and reward. We therefore investigated whether PKCɛ signaling in the amygdala is involved in ethanol consumption. Local knockdown of PKCɛ in the amygdala reduced ethanol consumption and preference in a limited‐access paradigm. Further, mice that are heterozygous for the PKCɛ allele consume less ethanol compared with wild‐type mice in this paradigm. These mice have a >50% reduction in the abundance of PKCɛ in the amygdala compared with wild‐type mice. We conclude that amygdala PKCɛ is important for ethanol consumption in mice.

Acute Functional Tolerance to Ethanol Mediated by Protein Kinase Cɛ

A low level of response to ethanol is associated with increased risk of alcoholism. A major determinant of the level of response is the capacity to develop acute functional tolerance (AFT) to ethanol during a single drinking session. Mice lacking protein kinase C epsilon (PKCɛ) show increased signs of ethanol intoxication and reduced ethanol self-administration. Here, we report that AFT to the motor-impairing effects of ethanol is reduced in PKCɛ (−/−) mice when compared with wild-type littermates. In wild-type mice, in vivo ethanol exposure produced AFT that was accompanied by increased phosphorylation of PKCɛ and resistance of GABAA receptors to ethanol. In contrast, in PKCɛ (−/−) mice, GABAA receptor sensitivity to ethanol was unaltered by acute in vivo ethanol exposure. Both PKCɛ (−/−) and PKCɛ (+/+) mice developed robust chronic tolerance to ethanol, but the presence of chronic tolerance did not change ethanol preference drinking. These findings suggest that ethanol activates a PKCɛ signaling pathway that contributes to GABAA receptor resistance to ethanol and to AFT. AFT can be genetically dissociated from chronic tolerance, which is not regulated by PKCɛ and does not alter PKCɛ modulation of ethanol preference.

Increased response to morphine in mice lacking protein kinase C epsilon

The protein kinase C (PKC) family of serine–threonine kinases has been implicated in behavioral responses to opiates, but little is known about the individual PKC isozymes involved. Here, we show that mice lacking PKCɛ have increased sensitivity to the rewarding effects of morphine, revealed as the expression of place preference and intravenous self‐administration at very low doses of morphine that do not evoke place preference or self‐administration in wild‐type mice. The PKCɛ null mice also show prolonged maintenance of morphine place preference in response to repeated testing when compared with wild‐type mice. The supraspinal analgesic effects of morphine are enhanced in PKCɛ null mice, and the development of tolerance to the spinal analgesic effects of morphine is delayed. The density of μ‐opioid receptors and their coupling to G‐proteins are normal. These studies identify PKCɛ as a key regulator of opiate sensitivity in mice.

The substrates and binding partners of protein kinase Cε

The epsilon isoform of protein kinase C (PKCepsilon) has important roles in the function of the cardiac, immune and nervous systems. As a result of its diverse actions, PKCepsilon is the target of active drug-discovery programmes. A major research focus is to identify signalling cascades that include PKCepsilon and the substrates that PKCepsilon regulates. In the present review, we identify and discuss those proteins that have been conclusively shown to be direct substrates of PKCepsilon by the best currently available means. We will also describe binding partners that anchor PKCepsilon near its substrates. We review the consequences of substrate phosphorylation and discuss cellular mechanisms by which target specificity is achieved. We begin with a brief overview of the biology of PKCepsilon and methods for substrate identification, and proceed with a discussion of substrate categories to identify common themes that emerge and how these may be used to guide future studies.

A Blocker of N- and T-type Voltage-Gated Calcium Channels Attenuates Ethanol-Induced Intoxication, Place Preference, Self-Administration, and Reinstatement

There is a clear need for new therapeutics to treat alcoholism. Here, we test our hypothesis that selective inhibitors of neuronal calcium channels will reduce ethanol consumption and intoxication, based on our previous studies using knock-out mice and cell culture systems. We demonstrate that pretreatment with the novel mixed N-type and T-type calcium channel antagonist 1-(6,6-bis(4-fluorophenyl)hexyl)-4-(3,4,5-trimethoxybenzyl)piperazine (NP078585) reduced ethanol intoxication. NP078585 also attenuated the reinforcing and rewarding properties of ethanol, measured by operant self-administration and the expression of an ethanol conditioned place preference, and abolished stress-induced reinstatement of ethanol seeking. NP078585 did not affect alcohol responses in mice lacking N-type calcium channels. These results suggest that selective calcium channel inhibitors may be useful in reducing acute ethanol intoxication and alcohol consumption by human alcoholics.

Turnaround time and market capacity in contract cheating

Contract cheating is the process whereby students auction off the opportunity for others to complete assignments for them. It is an apparently widespread yet under-researched problem. One suggested strategy to prevent contract cheating is to shorten the turnaround time between the release of assignment details and the submission date, thus making it difficult for students to make arrangements with contractors. Here, we outline some characteristics of the current market for contract cheating and demonstrate that short turnaround times are unlikely to prevent contract cheating because requested turnaround times for university-level assignments completed via contract cheating are already short (average 5 days). In addition, for every contractor awarded a job, there are an average of 10 others offering to complete it within the specified time suggesting that there is abundant excess capacity in the market.

Increased sensitivity to the aversive effects of ethanol in PKCε null mice revealed by place conditioning.

: Determining the intracellular signaling pathways that mediate the rewarding effects of ethanol may help identify drug targets to curb excessive alcohol consumption. Mice lacking the epsilon isoform of protein kinase C (PKCepsilon) voluntarily consumed less ethanol than wild-type mice in two-bottle choice and operant self-administration assays. Decreased consumption may reflect either increased or decreased sensitivity to the rewarding effects of ethanol. Alternatively, decreased voluntary consumption may reflect a change in sensitivity to the aversive effects of ethanol. The authors used place conditioning to determine that PKCepsilon null mice have an increased sensitivity to the aversive effects of ethanol but a decreased sensitivity to the rewarding effects of ethanol. Together these data suggest that PKCepsilon null mice voluntarily consume less ethanol because they derive less reward and are more sensitive to the aversive effects of ethanol.