Raman Spectroscopic Signature Markers of Dopamine-Human Dopamine Transporter Interaction in Living Cells.

Dopamine (DA) controls many psychological and behavioral activities in the central nervous system (CNS) through interactions with the human dopamine transporter (hDAT) and dopamine receptors. The roles of DA in the function of the CNS are affected by the targeted binding of drugs to hDAT; thus, hDAT plays a critical role in neurophysiology and neuropathophysiology. An effective experimental method is necessary to study the DA-hDAT interaction and effects of variety of drugs like psychostimulants and antidepressants that are dependent on this interaction. In searching for obtaining and identifying the Raman spectral signatures, we have used surface enhanced Raman scattering (SERS) spectroscopy to record SERS spectra from DA, human embryonic kidney 293 cells (HEK293), hDAT-HEK293, DA-HEK293, and DA-hDAT-HEK293. We have demonstrated a specific 2D-distribution SERS spectral analytical approach to analyze DA-hDAT interaction. Our study shows that the Raman modes at 807, 839, 1076, 1090, 1538, and 1665 cm-1 are related to DA-hDAT interaction, where Raman shifts at 807 and 1076 cm-1 are the signature markers for the bound state of DA to probe DA-hDAT interaction. On the basis of density function theory (DFT) calculation, Raman shift of the bound state of DA at 807 cm-1 is related to combination of bending modes α(C3-O10-H21), α(C2-O11-H22), α(C7-C8-H18), α(C6-C4-H13), α(C7-C8-H19), and α(C7-C8-N9), and Raman shift at 1076 cm-1 is related to combination of bending modes α(H19-N9-C8), γ(N9-H19), γ(C8-H19), γ(N9-H20), γ(C8-H18), and α(C7-C8-H18). These findings demonstrate that protein-ligand interactions can be confirmed by probing change in Raman shift of ligand molecules, which could be crucial to understanding molecular interactions between neurotransmitters and their receptors or transporters.