A nicotinic-dopaminergic hybrid probe enhances D2-like autoreceptor-mediated inhibition of dopamine release.

β2-containing nicotinic acetylcholine receptors (nAChRs) and dopamine D2 receptors (D2Rs) cooperate to shape striatal dopamine (DA) output, yet the mechanisms by which nicotinic-dopaminergic crosstalk influences D2-like autoreceptor-mediated inhibition of DA release remain unclear. Here we use NiCh8, a covalently linked nicotinic-dopaminergic hybrid, as a molecular probe to test whether single-molecule co-engagement can enhance D2-like autoreceptor-mediated inhibition of DA release. NiCh8 binds native α4β2* and α6β2* nAChRs as well as D2Rs, and behaves as a very low-efficacy partial agonist with antagonistic activity at α4β2 nAChRs. In equilibrium slice/synaptosome assays, NiCh8 elicits a modest dihydro-β-erythroidine-sensitive [³H]DA release that is absent in α4/α6 double-knockout (KO) synaptosomes. Strikingly, in superfused striatal synaptosomes NiCh8 potently suppresses basal and nicotine (NIC)-evoked DA outflow at nanomolar concentrations; this inhibition is preserved in β2-KO preparations, abolished by sulpiride, and is not reproduced by the parent pharmacophores (NONI and PAMC) alone or in combination. Structure-guided modeling supports a plausible bitopic binding mode at D2R, in which the dopaminergic fragment is predicted to engage the orthosteric site while the nicotinic fragment may contact a secondary binding region, providing a working structural rationale for the observed inhibitory phenotype. Together, these data identify NiCh8 as a hybrid probe that functionally enhances sulpiride-sensitive D2-like autoreceptor-mediated inhibition of DA release and provides a basis for future studies testing the role of D2R secondary-pocket engagement.