Kalirin-7 (Kal7), a multi-functional Rho GDP/GTP exchange factor (GEF) for Rac1 and RhoG, is embedded in the post-synaptic density at excitatory synapses, where it participates in the formation and maintenance of dendritic spines. Kal7 has been implicated in long-term potentiation, fear memories and addiction-like behaviors. Using liquid chromatography and tandem mass spectroscopy, we identified sites phosphorylated by six PSD-localized kinases implicated in synaptic plasticity and behavior, sites phosphorylated when myc-Kal7 was expressed in non-neuronal cells and sites phosphorylated in mouse brain Kal7. A site in the Sec14p domain phosphorylated by calcium/calmodulin dependent protein kinase II, protein kinase A and protein kinase C, was phosphorylated in mouse brain, but not in non-neuronal cells. Phosphorylation in the spectrin-like repeat region was more extensive in mouse brain than in non-neuronal cells, with a total of 20 sites identified. Sites in the pleckstrin homology domain and in the linker region connecting the GEF domain to the PDZ binding motif were heavily phosphorylated in both non-neuronal cells and in mouse brain and affected GEF activity. We postulate that the kinase convergence and divergence observed in Kal7 identify it as a key player in integration of the multiple inputs that regulate synaptic structure and function.

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Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). a4b2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encode the a4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants (a4R336C, a4P451L, and a4R487Q) to the common variant to determine their effects on a4b2 nAChR pharmacology. We examined [ 3 H]epibatidine binding, interacting proteins, and phosphorylation of the a4 nAChR subunit with liquid chromatography and tandem mass spectrometry (LC-MS/ MS) in HEK 293 cells and voltage-clamp electrophysiology in Xenopus laevis oocytes. We observed significant effects of the a4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified a4b2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in X. laevis oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these a4 rare variants, as well as shifts in receptor function after incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of human a4b2 nAChRs, resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common a4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.