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Thr136Ile polymorphism of human vesicular monoamine transporter-1 (SLC18A1 gene) influences its transport activity in vitro.

Khalifa A, Watson-Siriboe A, Shukry S, Chiu W, Nelson M, Geng Y, Fischer-Stenger K, Porter J, Stewart J.
Journal Article 2012; 33(5): 546-551 PubMed PMID: 23090274 Citation  Keywords:  Animals, Biological Transport:genetics, Bipolar Disorder:genetics, COS Cells, Cercopithecus aethiops, Humans, In Vitro Techniques, Isomerism, Polymorphism, Single Nucleotide, Protein Structure, Tertiary, Schizophrenia:genetics, Serotonin:metabolism, Trans.   

OBJECTIVES: Although single nucleotide polymorphisms of the human vesicular monoamine transporter 1 (hVMAT1) gene SLC18A1 have been associated with neuropsychiatric disorders, there is limited information on the function of naturally occurring hVMAT1 variant proteins. This study evaluated transport activity of full length hVMAT1 isoform-a (NP_003044.1) with a threonine (Thr) or isoleucine (Ile) at amino acid 136 and hVMAT1 isoform-b (NP_00135796.1) with a 136-Thr and deletion of 32 amino acids in the central region of the protein. Genetic studies have previously linked the 136-Thr to bipolar disorder.

METHODS: Expression vectors with hVMAT1 DNA coding for isoform variants were transfected into COS-1 cells. Expression of immunoreactive proteins was assessed by Western blotting, and function was assayed by ATP-dependent transport of radiolabeled serotonin and concentration-dependent inhibition by reserpine.

RESULTS: Immunoreactive isoform-a proteins were observed as a major doublet (68-71 Kd) and a minor 39 Kd protein. The major isoform-b protein was 47 Kd with minor 57 and 115 Kd proteins. Isoform-b had no detectable transport activity, despite a large amount of immunoreactive protein. Transport activity of isoform-a with 136-Thr was 20-50% lower than with 136-Ile in time course studies (2.5-5 min) and in additional 5 min assays repeated with 5-6 transfections per variant. Kinetic analyses indicated a lower transport Vmax of isoform-a with 136-Thr but no significant differences in the transport Km or reserpine IC50.

CONCLUSIONS: Deletion of amino acids 307-338 in hVMAT1 isoform-b abolishes transport activity, and a 136-Thr partially reduces activity of isoform-a....

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