Mark W. Fleck

Antagonism of α3β4 nicotinic receptors as a strategy to reduce opioid and stimulant self-administration

The iboga alkaloid ibogaine and the novel iboga alkaloid congener 18-methoxycoronaridine are putative anti-addictive agents. Using patch-clamp methodology, the actions of ibogaine and 18-methoxycoronaridine at various neurotransmitter receptor ion-channel subtypes were determined. Both ibogaine and 18-methoxycoronaridine were antagonists at alpha 3 beta 4 nicotinic receptors and both agents were more potent at this site than at alpha 4 beta 2 nicotinic receptors or at NMDA or 5-HT(3) receptors; 18-methoxycoronaridine was more selective in this regard than ibogaine. In studies of morphine and methamphetamine self-administration, the effects of low dose combinations of 18-methoxycoronaridine with mecamylamine or dextromethorphan and of mecamylamine with dextromethorphan were assessed. Mecamylamine and dextromethorphan have also been shown to be antagonists at alpha 3 beta 4 nicotinic receptors. All three drug combinations decreased both morphine and methamphetamine self-administration at doses that were ineffective if administered alone. The data are consistent with the hypothesis that antagonism at alpha 3 beta 4 receptors is a potential mechanism to modulate drug seeking behavior. 18-Methoxycoronaridine apparently has greater selectivity for this site than other agents and may be the first of a new class of synthetic agents acting via this novel mechanism to produce a broad spectrum of anti-addictive activity.

Dopamine Directly Modulates GABAA Receptors

Dopamine is a critical neuromodulator that governs movement and motivation. Although dopamine is only thought to target GPCRs in vertebrates (D1-5), it can activate a Cys-loop pentameric ligand-gated ion channel in C. elegans (LGC-53). This channel shares homology with mammalian GABAA receptors, among other Cys-loop channels. The GABAA β3 subunit in particular is important for mediating tonic GABA signaling in dopamine-projecting striatal medium spiny neurons. We found 30% residue identity between ligand-binding domains of LGC-53 and β3, and hypothesized that β3-containing GABAA receptors may be directly modulated by DA. To address this, we used immunolabeling with specific anti-β3 antibodies and whole-cell patch clamp electrophysiology in cultured rat striatal neurons and transfected HEK 293 cells. We determined that 79 ±3% of cultured cells endogenously expressed β3, and 94% of neurons gave tonic-level GABA-evoked currents blocked by phasic concentrations of dopamine (0.1-10 mM). Inhibition was recapitulated in HEK 293 cells transfected with α1β3, α1β3δ or α1β2γ2 subunits, but we instead observed potentiation in α1β3γ2 and α5β3γ2. Surprisingly, dopamine (1 mM) evoked rapid currents in α1β2γ2, α1β3γ2 and α5β3γ2 in the absence of GABA. In α1β3(H267A)γ2 (α1Zβ3γ2) receptors insensitive to trace Zn2+ inhibition, we found that other biogenic amines evoked comparatively smaller currents than DA. When the ratio of α1 was increased or we mutated a critical Zβ3 tyrosine residue (Y62L), relative dopamine responses decreased. Furthermore, dopamine activity was retained but GABA activity was drastically reduced in Zβ3γ2 receptors, while Zβ3 or γ2 alone did not elicit currents. Finally, dopamine currents were picrotoxin- but not bicuculline- or gabazine-sensitive. Taken together, dopamine at phasic levels is a GABAA allosteric modulator that may inhibit tonic GABA in prototypical α-containing GABAA receptors. However, at β/γ-containing receptors that do not need α subunits, dopamine acts as a positive allosteric modulator.

Ethanol alters calcium signaling in axonal growth cones.

Calcium (Ca2+) channels are sensitive to ethanol and Ca2+ signaling is a critical regulator of axonal growth and guidance. Effects of acute and chronic exposure to ethanol (22, 43, or 87 mM) on voltage-gated Ca2+ channels (VGCCs) in whole cells, and KCl-induced Ca2+ transients in axonal growth cones, were examined using dissociated hippocampal cultures. Whole-cell patch-clamp analysis in neurons with newly-formed axons (Stage 3) revealed that rapidly inactivating, low-voltage activated (LVA) and non-inactivating, high-voltage activated (HVA) currents were both inhibited in a dose-dependent manner by acute ethanol, with relatively greater inhibition of HVA currents. When assessed by Fluo-4-AM imaging, baseline fluorescence and Ca2+ response to ethanol in Stage 3 neurons was similar compared to neurons without axons, but peak Ca2+ transient amplitudes in response to bath-applied KCl were greater in Stage 3 neurons and were decreased by acute ethanol. The amplitude of Ca2+ transients elicited specifically in axonal growth cones by focal application of KCl was also inhibited by acute exposure to moderate-to-high concentrations of ethanol (43 or 87 mM), whereas a lower concentration (22 mM) had no effect. When 43 or 87 mM ethanol was present continuously in the medium, KCl-evoked Ca2+ transient amplitudes were also reduced in growth cones. In contrast, Ca2+ transients were increased by continuous exposure to 22 mM ethanol. Visualization using a fluorescent dihydropyridine analog revealed that neurons continuously exposed to ethanol expressed increased amounts of L-type Ca2+ channels, with greater increases in axonal growth cones than cell bodies. Thus, acute ethanol reduces Ca2+ current and KCl-induced Ca2+ responses in whole cells and axonal growth cones, respectively, and chronic exposure is also generally inhibitory despite apparent up-regulation of L-type channel expression. These results are consistent with a role for altered growth cone Ca2+ signaling in abnormal neuromorphogenesis associated with fetal alcohol spectrum disorders.

Histamine-gated ion channels in mammals?

There is ample pharmacological and physiological evidence for yet unidentified histamine receptors in mammalian brain that are linked to a Cl(-) conductance. In invertebrates, two histamine-gated chloride channels (HisCl α1 and α2) are already well known. HisCl channels are members of the Cys-loop receptor superfamily of ligand-gated ion channels and are closely related to the mammalian GABA(A) and glycine receptors (GlyR). Indeed, they share particularly strong homology within the ligand binding and ion channel domains. Here we discuss the possibility that mammalian HisCl channels might exist among the known GABA(A) or GlyR subunits. Studies published to date support this hypothesis, including evidence for direct histamine gating of GABA(A) β homomers, histamine potentiation of GABA(A) αβ and αβγ heteromeric receptors, and GABA(A) receptor blockade by some antihistamines. We explore what is known about the binding-site structure, function and pharmacology of invertebrate HisCl channels and other histamine binding sites to support and inform a broader search for HisCl channels among the mammalian GABA(A) and GlyR subunits. The discovery and identification of HisCl-like channels in mammals would not only enhance understanding of inhibitory signaling and histamine function in the mammalian brain, but also provide new avenues for development of therapeutic compounds targeting this novel histamine site. This commentary is therefore intended to foster consideration of a novel and potentially important target of histamine and histaminergic drugs in the CNS.