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Published on: January 29, 2019
Expression and function of the rat vesicular monoamine transporter 2
Yoav Adam1, Robert H Edwards, Shimon Schuldiner
1Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.
American Journal of Physiology. Cell Physiology
|February 22, 2008
Summary
Researchers expressed and purified rat vesicular monoamine transporter 2 (rVMAT2), enabling studies on monoamine storage. The purified protein demonstrated functional serotonin transport, advancing our understanding of neurotransmitter regulation.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Vesicular monoamine transporters (VMATs) are crucial for storing monoamines in the central nervous system and endocrine cells.
- VMATs function as H+/substrate antiporters and exhibit multidrug transporter characteristics.
- Limited biochemical and structural data exist for VMATs due to expression challenges.
Purpose of the Study:
- To achieve high-level expression of rat VMAT2 (rVMAT2) in a stable cell line.
- To develop novel methods for solubilizing, purifying, and reconstituting active rVMAT2.
- To characterize the functional properties of purified rVMAT2.
Main Methods:
- High-level expression of rVMAT2 in a human embryonic kidney (HEK293) cell line using MPP+ resistance.
- Development of novel solubilization and purification procedures for active rVMAT2.
- Reconstitution of purified rVMAT2 into proteoliposomes for functional transport assays.
Main Results:
- Successfully expressed high levels of rVMAT2 in HEK293 cells.
- Developed procedures for purifying active rVMAT2 and reconstituting it into proteoliposomes.
- Partially purified rVMAT2 bound tetrabenazine; reconstituted rVMAT2 exhibited Deltamu(H+)-driven electrogenic serotonin transport with wild-type affinity and a turnover rate of ~0.4 s(-1).
Conclusions:
- This study presents a successful strategy for the expression, purification, and functional characterization of rVMAT2.
- The developed methods overcome previous limitations in obtaining sufficient quantities of active VMAT protein for biochemical and structural studies.
- The functional data provide insights into the transport mechanism and pharmacological properties of rVMAT2.
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