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CELLULAR AND MOLECULAR

Regulation of Choline Transporter Surface Expression and Phosphorylation by Protein Kinase C and Protein Phosphatase 1/2A

Department of Anatomy and Neurobiology, University of Kentucky Medical Center, Lexington, Kentucky (J.G., S.A.); and Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee (S.M.F., R.D.B.)

The Na⁺/Cl^–-dependent, hemicholinium-3-sensitive choline transporter (CHT) provides choline for acetylcholine biosynthesis. Recent studies show that CHT contains canonical protein kinase C (PKC) serine and threonine residues. We examined the ability of PKC and serine/threonine protein phosphatase 1/2A (PP1/PP2A) to regulate CHT function, surface expression, and phosphorylation. In mouse crude striatal and hippocampal synaptosomes, PKC activators -phorbol 12-myristate 13-acetate (-PMA) and -phorbol 12,13-dibutyrate produced time- and concentration-dependent reductions in CHT function. PP1/PP2A inhibitors okadaic acid (OKA) and calyculin A (CL-A) produced a time- and concentration-dependent decrease in CHT function. However, tautomycin (PP1 inhibitor) and cyclosporin A (PP2B inhibitor) failed to alter CHT-mediated choline uptake. Choline transport kinetic studies following -PMA, OKA, and CL-A treatment revealed a reduction in the maximal choline transport velocity (V_max) with no change in K_m for choline. These modulators also produced no change in the total levels of CHT protein in the crude hippocampal and striatal synaptosomes; however, surface biotinylation studies using the membrane-impermeant N-hydroxysuccinimide-biotin in crude synaptosomes following treatment with -PMA, OKA, and CL-A indicate significant reductions of CHT levels in biotinylated fractions. Pretreatment with OKA alone, but not -PMA, significantly augmented the phosphorylation level of CHT proteins. Our findings suggest that neuronal PKC and PP1/PP2A activity may establish the level of function and surface expression of CHT. These studies also provide the first evidence that CHT is a phosphoprotein and that the basal PP1/PP2A activity may have a dominant role in controlling the levels of CHT phosphorylation.

Address correspondence to: Dr. Subbu Apparsundaram, Anatomy and Neurobiology, 306 Whitney-Hendrickson Building, 800 Rose Street, University of Kentucky Medical Center, Lexington, KY 40536-0098. E-mail: subbu{at}uky.edu

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