Alcoholism and Alternative Splicing of Candidate Genes

Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism

Alternative splicing of the NMDAR1 subunit affects modulation by calcium.

Four splice variants of the NR1 receptor subunit, characterized by the presence or absence of cassettes encoding inserts of 21 (Insert 1) and 37 (Insert 2) amino acids were expressed in Xenopus oocytes and studied using voltage-clamp techniques. In 1.8 mM Ca2+, a slow inward current (Islow), which peaked 20 s after exposure to NMDA was evident when Insert I was present, but not when absent. However, in elevated external Ca2+ medium a similar Islow was observed in variants missing Insert I. The Ca2+ dependency of Islow reflected a requirement for intracellular accumulation of Ca2+. The divalent ion permeability of Insert I containing and Insert 1 lacking receptor channels expressed alone, as well as in heteromeric assemblies with NR2A and NR2B, was similar for all combinations tested. Thus, the lower Ca2+ dependency for Islow in oocytes expressing Insert I was not due to higher calcium entry. Islow was less sensitive to blockers of ICl(Ca) than were endogenous calcium-activated chloride currents (ICl(Ca)). Also, Islow was not abolished in Cl(-)-free external medium, when voltage was manipulated such that Islow was outward-going. Thus, Islow, while containing a component due to activation of endogenous ICl(Ca), is primarily due to current flowing through the receptor ion channel. Development of Islow was unaffected by PKC or PKA inhibitors. The modulation of the Ca2+ dependency of Islow by Insert I occurs in a range of Ca2+ concentrations which are physiologically relevant, and may provide an important means of modulation of glutamate transmission under normal and pathological conditions

Homomeric assemblies of NMDAR1 splice variants are sensitive to ethanol.

Studies have shown that the neuronal NMDA receptor is a target for the actions of ethanol. Recently, a number of subunits of the NMDA receptor have been cloned and functionally expressed in various combinations. We have expressed four splice variants of the NMDAR1 subunit in Xenopus oocytes, and find that homomeric assemblies of this subunit, in the absence of other subunits, exhibit ethanol sensitivity comparable to that seen in neurons. In the presence of calcium, the reduction of total current was greatest in the NMDAR1-LL splice variant, and was significantly less in the NMDAR1-SS variant. The increased sensitivity of NMDAR1-LL may be attributed to a particularly sensitive slow current 'hump' which is more pronounced in NMDAR1-LL than in NMDAR1-SS. The reduction of NMDA-evoked current by ethanol was significantly different when calcium was replaced by barium in the external medium. In this case, the slow current hump was significantly reduced, current through NMDAR1-LL was less reduced by ethanol, and the percent reduction of NMDAR1-LL and NMDAR1-SS currents was similar. NMDA-evoked currents in heteromeric receptors formed by coinjection of the mouse epsilon-1 subunit with the NMDAR1 splice variants responded to ethanol similarly to homomeric assemblies

Sustained ethanol inhibition of native AMPA receptors on medial septum/diagonal band (MS/DB) neurons

1. The direct impact of ethanol on native, non-NMDA glutamate receptors was examined in acutely isolated MS/DB neurons from rat. The impact of ethanol functional tolerance and physical dependence on non-NMDA receptor function was also determined. 2. Non-NMDA receptors were defined pharmacologically as predominantly the AMPA subtype, because both AMPA- or kainate-activated currents were blocked by GYKI 52466, a selective AMPA receptor antagonist. The relative magnitude of potentiation of AMPA-activated currents by 10 or 100 μM cyclothiazide was consistent with recombinant AMPA flop-subtype receptors. Finally, the selective kainate receptor agonist, SYM 8021, induced little current in MS/DB neurons. 3. AMPA receptor currents when activated by kainate were sensitive to ethanol, showing inhibition of ∼5–50% when 10–300 mM ethanol and kainate were briefly co-applied (3 s). Ethanol (100 mM) also inhibited both the initial transient peak and sustained currents activated by AMPA. Inhibition was sustained during continuous ethanol superfusions of 5 min, suggesting a lack of acute tolerance to ethanol-induced AMPA receptor blockade. 4. Rapid application of 3–3000 μM kainate activated concentration-dependent currents in MS/DB neurons from Control and Ethanol Dependent animals that were not significantly different. Also, direct ethanol inhibition (300 mM) of kainate-activated currents was not reduced by ethanol dependence, suggesting a lack of functional tolerance. 5. These results suggest that native AMPA receptors on MS/DB neurons are inhibited by pharmacologically-relevant concentrations of ethanol. However, these receptors, unlike NMDA receptors, do not undergo adaptation with sustained ethanol exposure sufficient to induce physical dependence

Mutations at F637 in the NMDA receptor NR2A subunit M3 domain influence agonist potency, ion channel gating and alcohol action

Background and purpose: NMDA receptors are important molecular targets of ethanol action in the CNS. Previous studies have identified a site in membrane-associated domain 3 (M3) of the NR1 subunit and two sites in M4 of the NR2A subunit that influence alcohol action; the sites in NR2A M4 also regulate ion channel gating. The purpose of this study was to determine whether mutations at the site in the NR2A subunit corresponding to the NR1 M3 site influence alcohol action and ion channel gating. Experimental approach: We investigated the effects of mutations at phenylalanine (F) 637 of the NR2A subunit using whole-cell and single-channel patch-clamp electrophysiological recording in transiently-transfected HEK 293 cells. Key results: Mutations at F637 in the NR2A subunit altered peak and steady-state glutamate EC50 values, maximal steady-state to peak current ratios (Iss:Ip), mean open time, and ethanol IC50 values. Differences in glutamate potency among the mutants were not due to changes in desensitization. Ethanol IC50 values were significantly correlated with glutamate EC50 values, but not with maximal Iss:Ip or mean open time. Ethanol IC50 values were linearly and inversely related to molecular volume of the substituent. Conclusions and implications: These results demonstrate that NR2A(F637) influences NMDA receptor affinity, ion channel gating, and ethanol sensitivity. The changes in NMDA receptor affinity are likely to be the result of altered ion channel gating. In contrast to the cognate site in the NR1 subunit, the action of ethanol does not appear to involve occupation of a critical volume at NR2A(F637)