开题报告内容：（包括拟研究或解决的问题、采用的研究手段及文献综述，不少于2000字）
1.Introduction
Alcohol use disorder (AUD) rank third on the list of preventable causes of morbidity and mortality in the United States, affecting over 18 million people, causing over 100 000 deaths annually and costing in excess of $220 billion. This exceeds the costs of other leading preventable causes of death such as cigarette smoking and physical inactivity. Presently, the only pharmacotherapeutic agents approved by the United States Food and Drug Administration (FDA) for the treatment of AUDs are disulfiram, naltrexone,and acamprosate. These drugs attempt to deter alcohol intake by blocking its metabolism or by targeting the neurochemical and neuropeptide systems in the downstream cascades leading to craving and dependence. However, their success rate even when combined with psychotherapy, has been limited with approximately 70% of patients relapsing back into heavy drinking within one year. A critical barrier is the lack of information regarding the molecular target(s) by which ethanol (EtOH) exerts its pharmacological activity. Some reviews highlight findings implicating P2X4receptors (P2X4Rs) as a target for the development of therapeutics to treat AUDs.
Ion channels are important targets of ethanol action. Ligand-gated ion channels (LGICs) are widely held to play an important role in ethanol-induced behaviors and drinking. Research in this area has focused on investigating the effects of ethanol on two large “superfamilies” of LGICs:(1) The nicotinic acetylcholine receptor superfamily(cys-loop) with members including nicotinic acetylcholine receptors(nAChRs), 5-hydroxytryptaminetype 3 receptors(5-HT3Rs), gamma;-aminobutyric acid type-A receptors (GABAARs) and glycine receptors(GlyRs) and (2 ) the glutamate superfamily.
P2XRs are a family of ligand-gated ion channels (LGICs) activated by extracellular ATP. Of the P2XR subtypes, P2X4Rs are expressed the most abundantly in the CNS. Converging evidence suggests that P2X4Rs are involved in the development and progression of AUD. First, in vitro studies report that pharmacologically relevant EtOH concentrations can negatively modulate ATP-activated currents. Second, P2X4Rs in the mesocorticolimbic dopamine system are thought to play a role in synaptic plasticity and are located ideally to modulate brain reward systems. Third, alcohol preferring(P) rats have lower functional expression of the p2rx4 gene than alcohol-non preferring(NP) rats suggesting an inverse relationship between alcohol intake and P2X4R expression. Similarly, whole brain p2rx4 expression has been shown to relate inversely to innate 24h alcohol preference across 28 strains of rats. Fourth, mice lacking the p2rx4 gene drink more EtOH than wildtype controls
To date,P2X4Rs are the most ethanol-sensitive P2XR subtype tested. Findings indicate that intoxicating and anesthetic ethanol concentrations desensitize the response of P2X4R-expressing cell lines to ATP exposure, altering the ability of these receptors to modulate neurotransmission. Initial voltage clamp studies in the late 1990rsquo;s, testing rat recombinant homomeric P2X4Rs expressed in Xenopus oocytes found that ethanol caused concentration-dependent inhibition of ATP-induced currents with the IC50 determined at 58mM. Caution must be taken when interpreting the IC50 value in this study as it was determined by testing a range of ethanol concentrations from 1to 500 mM.Later studies using two-electrode voltage clamp confirmed the inhibitory effects of ethanol in rat P2X4Rs,but focused on lower concentrations(25–200mM). The extent of ethanol inhibition was 50–60% at 200mM. Notably,the inhibitory effects of ethanol on ATP-gated P2X4R function were not dependent on the expression or recording system as similar results were found when tested in frog oocytes, transfected HEK293cells,and lentivirally transduced mouse hippocampal neurons. Ethanol-induced P2X4R inhibition may provide some insight regarding the role of P2X4Rs in ethanol intake.
Recent investigations from two-electrode voltage clamp and patch clamp studies demonstrate that ethanol inhibition of ATP- activated currents in P2X4Rs is not dependent on membrane potential(from minus;60 to 20mV),and ethanol does not change the reversal potential of ATP-activated current . This work argues that ethanol does not affect the receptor function directly,but rather act as a negative allosteric modulator as illustrated by its ability to shift the ATP concentration response curve to the right and increase the EC50 value significantly. Earlier investigations suggested that ethanol might inhibit P2X4Rs by decreasing the apparent affinity of the binding site for ATP.However,recent studies using patch clamp and a HEK293 expression system provided more insights into the intricate mechanisms of ethanol action inP2X4Rs . In this latter study, the authors reported that there was no difference in the magnitude of ethanol inhibition at a wide range of ATP concentrations, which contradicted the previous findings that a more prominent effect was present at the lower end of the tested ethanol concentrations. The observation that there was no change in channel deactivation supported the recent findings. These studies also demonstrated that ethanol interaction with P2X4Rs is rapid with very fast rates of association and dissociation. Moreover, the findings suggested a use-dependent mechanism for the action of ethanol consistent with properties of an open-channel blocker. Interestingly, in contrast to classic open- channel blockers, ethanol did not stabilize the open/desensitized states of the channel and did not change the ion permeability ratio. These properties are consistent with a low-affinity modulator “binding,” which is a dynamic series of on and off bounces in the putative binding sites or interaction pockets.
Glycine receptors (GlyRs) and g-aminobutyric acid type A receptors (GABAARs) are the primary inhibitory ligand-gated ion channels (LGICs) in the brain that have been implicated in causing many acute and chronic behavioral effects of ethanol, including tolerance, dependence, reward, anxiolysis, motor ataxia, impaired cognition, sedation, and aggression. Prior studies identified extracellular loop 2 of GlyRs and GABAARs as a site of ethanol action, and found that structural modifications in this region profoundly influence receptor sensitivity to ethanol. This initial work found that replacing loop 2 of a1 GlyR and g2 GABAAR subunits with loop 2 of the more ethanol sensitive d GABAARs significantly increased ethanol sensitivity of the resultant receptor, and identified important physical-chemical properties of loop 2 that alter receptor sensitivity to ethanol and agonist.
Recently it is demonstrated that IVM significantly reduces ethanol intake in both male and female mice across several models of self-administration. Pre-clinical evidence has demonstrated the ability of IVM to reduce ethanol consumption. However, this effect is somewhat limited in magnitude. Despite its lipophilic nature, IVM does not readily achieve high brain concentration, most likely because it is a good substrate for the P-gp transporter. At the present time, there is an ongoing effort to identify and/or develop new compounds that enhances IVMrsquo;s retention in the CNS while maintaining its ability to reduce alcohol intake and maintain its high tolerability and broad safety profile. It is thought that modification of the IVM structure to reduce its P-gp substrate recognition and alteration of its interaction with a target edbrain receptor should positively impact the drugrsquo;s ability to reduce ethanol intake.
2.Objectives
