Dual expression of constitutively active Gαs-protein-coupled receptors differentially establishes the resting activity of the cAMP-gated HCN2 channel in a single compartment.

The hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) channel is a major subtype of the HCN channel family expressed in the nervous system that sets the membrane potential, regulates cell excitability and senses changes in the extracellular environment. Neurons express various Gαs-protein-coupled receptors (GPCRs), many of which show ligand-independent constitutive activity. These membrane-bound proteins are expressed in various subcellular compartments of neurons. Therefore, some proportion of HCN2 channels opens in response to the basal cAMP pool size produced by constitutively active GPCRs. Here, we employed an exogenous HEK293 expression system and voltage-clamp patch-clamp recordings to investigate basal HCN2 channel activity in the presence of two GPCRs with diverse basal activities in a single compartment. We utilized the ß2-adrenoceptor (ß2AR) together with odorant receptors (ORs), as both GPCR families are known to show strong basal activity. Consequently, ß2AR alone strongly enhanced the activity of HCN2 channels, and co-expression of ORs further diversified the HCN2 channel activity, which was totally abolished by an adenylate cyclase inhibitor. Thus, we conclude that the dual expression of constitutively active GPCRs establishes the diverse range of the basal cAMP pool size in resting cells through mutual additive or suppressive interactions, even in the absence of external stimulation.

Paradoxical activation of c-Src as a drug-resistant mechanism.

ATP-competitive inhibitors have been developed as promising anti-cancer agents. However, drug-resistance frequently occurs, and the underlying mechanisms are not fully understood. Here, we show that the activation of c-Src and its downstream phosphorylation cascade can be paradoxically induced by Src-targeted and RTK-targeted kinase inhibitors. We reveal that inhibitor binding induces a conformational change in c-Src, leading to the association of the active form c-Src with focal adhesion kinase (FAK). Reduction of the inhibitor concentration results in the dissociation of inhibitors from the c-Src-FAK complex, which allows c-Src to phosphorylate FAK and initiate FAK-Grb2-mediated Erk signaling. Furthermore, a drug-resistant mutation in c-Src, which reduces the affinity of inhibitors for c-Src, converts Src inhibitors into facilitators of cell proliferation by enhancing the phosphorylation of FAK and Erk in c-Src-mutated cells. Our data thus reveal paradoxical enhancement of cell growth evoked by target-based kinase inhibitors, providing potentially important clues for the future development of effective and safe cancer treatment.