Browsing by Author "Fan, Jing"

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    Interactions of Rabconnectin-3 with Cav2 calcium channels
    (2019-06-28) Gandini, Maria A; Souza, Ivana A; Fan, Jing; Li, Katherine; Wang, Decheng; Zamponi, Gerald W
    Abstract This study describes the interaction between Cav2 calcium channels and Rabconnectin-3, a di-subunit protein that is associated with synaptic vesicles. Immunostaining reveals that both Rabconnectin-3α (RB-3α) and Rabconnectin-3β (RB-3β) are colocalized in mouse hippocampal neurons. Co-immunoprecipitations from brain tissue is consistent with the formation of a protein complex between RB-3α and RB-3β and both Cav2.2 and the related Cav2.1 calcium channel. The coexpression of either RB-3α or RB-3β with Cav2.2 calcium channels in tsA-201 cells led to a reduction in Cav2.2 current density without any effects on the voltage-dependence of activation or inactivation. Coexpression of both Rabconnectin-3 subunits did not cause an additive effect on current densities. Finally, the presence of Rabconnectin-3 did not interfere with μ-opioid receptor mediated Gβγ modulation of Cav2.2 channels. Altogether, our findings show that Rabconnectin-3 has the propensity to regulate calcium entry mediated by Cav2.2 channels.
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    Signaling complexes involving HCN channels
    (2017) Fan, Jing; Zamponi, Gerald; Turner, Ray; Stys, Peter
    Ih is a hyperpolarization-activated current that is widely distributed in the brain and which contributes to various physiological processes. HCN channels that give rise to Ih are an important target of cellular signals that regulate neuronal responses to external stimuli. In this thesis, I characterize the HCN channel-associated signaling complex and its impact on hippocampal function at molecular and cellular levels. Using whole cell-current recordings in hippocampal primary cultures prepared from wild-type (WT) and cellular prion protein (PrPC) knockout (KO) mouse pups, I found that the absence of PrPC profoundly affected the firing properties of cultured hippocampal neurons. These included an increased number of action potentials (APs) and a decreased spike threshold. By performing whole cell-voltage recordings, a reduced ionic current Ih was observed in KO neurons as indicated by a decreased voltage sag, a hyperpolarizing shift in activation gating and an enhanced input resistance. However, co-IP results did not reveal a molecular complex formed between HCN and PrPC. These results indicate that HCN channels are functionally but not physically associated with PrPC to regulate hippocampal neuronal excitability. Further dissecting on HCN channel-associated signaling in tsA-201 cells revealed that HCN1 and Cav3.2 channels can be associated in a physical complex. The coexpression of HCN1 channels altered the functional properties of Cav3.2 currents, including a reduced Cav3.2 current density, altered channel kinetics and a depolarizing shift in activation gating. Mutual interaction regions in both channels were also determined. Overall, these findings identify HCN channel-associated signaling at molecular and functional levels, including functional interactions between HCN channels and PrPC, and physical interactions between HCN1 and Cav3.2 channels. This study provides a framework for understanding the HCN channel-associated interactions in the context of neuronal excitability.