Traditionally prescribed for mood disorders, tricyclic antidepressants (TCAs) have shown promising therapeutic effects on chronic neuralgia and irritable bowel syndrome. However, the mechanism by which these atypical effects manifest is unclear. Among the proposed mechanisms is the well-known pain-related inhibitory G-protein coupled receptor (GiPCR), namely, the opioid receptor (OR). Here, we confirmed that TCA indeed stimulates OR and regulates the gating of TRPC4, a downstream signaling of the Gi-pathway. In an ELISA to quantify the amount of intracellular cAMP, a downstream product of OR/Gi-pathway, treatment with amitriptyline (AMI) showed a decrease in [cAMP]i similar to that of the μOR agonist. Next, we explored the binding site of TCA by modeling the previously revealed ligand-bound structure of μOR. A conserved aspartate residue of ORs was predicted to participate in salt bridge interaction with the amine group of TCAs, and in aspartate-to-arginine mutation, AMI did not decrease the FRET-based binding efficiency between the ORs and Gαi2. As an alternative way to monitor the downstream signaling of Gi-pathway, we evaluated the functional activity of TRPC4 channel, as it is well known to be activated by Gαi. TCAs increased the TRPC4 current through ORs, and TCA-evoked TRPC4 activation was abolished by an inhibitor of Gαi2 or its dominant-negative mutant. As expected, TCA-evoked activation of TRPC4 was not observed in the aspartate mutants of OR. Taken together, OR could be proclaimed as a promising target among numerous binding partners of TCA, and TCA-evoked TRPC4 activation may help to explain the nonopioid analgesic effect of TCA.

In DRAM products, the copper interconnections with larger grain size are preferred for lower electrical resistance and better circuit performance. We studied the copper properties which are the evolution of the grain size, distribution of the grain, and the metal line texture formation during self-annealing. And we were able to evaluate the thermal budget in the DRAM process by performing thermal excursion stress test. We found out that the condition with self-annealing time affects copper grain size and stress migration. It has been measured by the EBSD analysis system and TOF-TEM. Compared with the conventional copper anneal process which has no time delay, the self-annealing process with time delay showed the more bamboo microstructure at dram damascene process. In addition, we observed that the self-annealing process helped enhanced thermal stress stability, which is caused by lower hillock deformation to copper top surface after a batch furnace at 400°C, N2 ambient for three times.