Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase

doi:10.1073/pnas.0501432102

Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase

Departments of ^*Pharmacology, ^†Molecular Physiology and Biophysics, and ^§Psychiatry, and Centers for ^∥Human Genetics Research and ^¶Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, TN 37232-8548; and ^‡Department of Neurosciences, Medical University of South Carolina, Charleston, SC 29425

Edited by Susan G. Amara, University of Pittsburgh School of Medicine, Pittsburgh, PA (received for review February 19, 2005)

Abstract

Human serotonin [5-hydroxytryptamine (5-HT)] transporters (hSERT, 5HTT, and SLC6A4) inactivate 5-HT after release and are prominent targets for therapeutic intervention in mood, anxiety, and obsessive-compulsive disorders. Multiple hSERT coding variants have been identified, although to date no comprehensive functional analysis of these variants has been reported. We transfected hSERT or 10 hSERT coding variants and examined total and surface protein expression, antagonist recognition, and transporter modulation by posttranslational, regulatory pathways. Two variants, Pro339Leu and Ile425Val, demonstrated significant changes in surface expression supporting alterations in 5-HT transport capacity (V _max). Regardless of basal transport activity, all SERT variants displayed a capacity for rapid, phorbol ester-triggered down-regulation. Remarkably, five variants (Thr4Ala, Gly56Ala, Glu215Lys, Lys605Asn, and Pro612Ser) demonstrated no capacity for 5-HT uptake stimulation after acute protein kinase G (PKG)/p38 mitogen-activated protein kinase (MAPK) activation. Epstein–Barr virus (EBV)-transformed lymphocytes natively expressing the most common of these variants (Gly56Ala) exhibited a similar loss of 5-HT uptake stimulation by PKG/p38 MAPK activators. HeLa cells transfected with the Gly56Ala variant demonstrated elevated basal phosphorylation and, unlike hSERT, could not be further phosphorylated after 8-bromo cGMP (8BrcGMP) treatments. These studies reveal cellular phenotypes associated with naturally occurring human SERT coding variants and suggest that altered transporter regulation by means of PKG/p38 MAPK-linked pathways may influence risk for disorders attributed to compromised 5-HT signaling.

Footnotes

↵ ** To whom correspondence should be addressed at: Vanderbilt Center for Molecular Neuroscience, Suite 7140, MRBIII, Vanderbilt School of Medicine, Nashville, TN 37232-8548. E-mail: randy.blakely{at}vanderbilt.edu.
Author contributions: H.C.P., J.L.M., D.J.S., and S.R. performed research; H.C.P., J.L.M., D.J.S., S.R., J.S.S., and R.D.B. analyzed data; H.C.P., C.-B.Z., D.J.S., S.R., and R.D.B. designed research; J.L.M., D.J.S., S.R., and J.S.S. contributed new reagents/analytic tools; and H.C.P., C.-B.Z., J.L.M., D.J.S., S.R., R.C.S., W.A.H., J.S.S., and R.D.B. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: 5-HT, 5-hydroxytryptamine; SERT, serotonin transporter; hSERT, human SERT; PKG, protein kinase G; MAPK, mitogen-activated protein kinase; OCD, obsessive-compulsive disorder; TM, transmembrane domain; 8BrcGMP, 8-bromo cGMP; β-PMA, β-phorbol 12-myristate 13-acetate; RTI-55, (3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester.