2-Nitroimipramine: a photoaffinity probe for the serotonin uptake/tricyclic binding site complex

Area of Science:

• Neuroscience
• Biochemistry
• Pharmacology

Background:

• The serotonin transporter (SERT) is a key target for antidepressants.
• Photoaffinity probes are used to identify protein targets of drug binding.

Purpose of the Study:

• To identify the specific protein(s) labeled by the photoaffinity probe [3H]2-Nitroimipramine ([3H]2-NI) in tissues expressing the serotonin uptake system.
• To characterize the binding properties of [3H]2-NI to its target protein.

Main Methods:

• Photoaffinity labeling of membrane homogenates from human platelets, rat brain, and rat liver with [3H]2-NI.
• Analysis of incorporated radioactivity using SDS-polyacrylamide gel electrophoresis and fluorography.
• Competition assays with selective and non-selective serotonin uptake inhibitors.
• Scatchard analysis of binding data.
• Functional assays measuring serotonin uptake in intact platelets after cross-linking with 2-NI.

Main Results:

• [3H]2-NI preferentially and covalently incorporated into a 30-kd protein in all tissues examined.
• Labeling of the 30-kd band was specifically inhibited by serotonin uptake inhibitors.
• Serotonin itself modulated the labeling of 30-kd and 35-kd bands.
• Scatchard analysis indicated high-affinity, saturable binding to the 30-kd protein.
• Cross-linking with 2-NI reduced serotonin uptake Vmax but not Km in platelets.

Conclusions:

• The 30-kd protein labeled by [3H]2-NI is likely the serotonin transporter.
• [3H]2-NI serves as an effective photoaffinity probe for identifying and characterizing the serotonin transporter.
• The results challenge existing models of allosteric interactions at serotonergic synapses.