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

β2-containing nicotinic acetylcholine receptors (nAChRs) and dopamine D 2 receptors (D 2 Rs) cooperate to shape striatal dopamine (DA) output, yet the mechanisms by which nicotinic-dopaminergic crosstalk influences D 2 -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 D 2 -like autoreceptor-mediated inhibition of DA release. NiCh8 binds native α4β2* and α6β2* nAChRs as well as D 2 Rs, 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 D 2 R, 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 D 2 -like autoreceptor-mediated inhibition of DA release and provides a basis for future studies testing the role of D 2 R secondary-pocket engagement.