Intermolecular Association Provides Specific Optical and NMR Signatures for Serotonin at Intravesicular Concentrations

Abstract

Neurotransmitter vesicles contain biomolecules at extraordinarily high concentrations (hundreds of millimoles/liter). Such concentrations can drive intermolecular associations, which may affect vesicular osmolarity and neuronal signaling. Here we investigate whether aqueous serotonin (a monoamine neurotransmitter) forms oligomers at intravesicular concentrations and whether these oligomers have specific spectroscopic signatures that can potentially be used for monitoring neuronal storage and release. We report that, as serotonin concentration is increased from 60μM to 600mM, the normalized fluorescence spectrum of serotonin displays a growing long-wavelength tail, with an isoemissive point at 376nm. The fluorescence decay is monoexponential with a lifetime of 4ns at low concentrations but is multiexponential with an average lifetime of 0.41ns at 600mM. A 600mM serotonin solution has 30% less osmolarity than expected for monomeric serotonin, indicating oligomer formation. The proton NMR chemical shifts move upfield by as much as 0.3ppm at 600mM compared to those at 10mM, indicating a stacking of the serotonin indole moieties. However, no intermolecular crosspeak is evident in the two-dimensional NMR rotating frame Overhauser effect spectroscopy spectrum even at 600mM, suggesting that oligomeric structures are possibly weakly coupled. The appearance of a single peak for each proton suggests that the rate of interconversion between the monomeric and the oligomeric structures is faster than 240Hz. A stopped-flow kinetic experiment also confirms that the rate of dissociation is faster than 100ms. We conclude that serotonin forms oligomers at intravesicular concentrations but becomes monomeric quickly on dilution. NMR signatures of the oligomers provide potential contrast agents for monitoring the activity of serotonergic neurons in vivo.