Evidence for the involvement of dynamin and other GTPases in the trafficking of the choline cotransporter.

摘要

The sodium-dependent, high affinity, hemicholinium-3 (HC-3) sensitive choline transporter (ChCoT) supplies choline for acetylcholine synthesis and is rate liming to cholinergic function (Birks and MacIntosh, 1961). This transporter has an obligation to cotransport sodium. Thus the mechanisms which regulate it are of great interest. The endocytosis of the ChCoT has been shown to be both dynamin (Ferguson & Blakely, 2004) and clathrin dependent (Riberio et. al., 2003). In studies reported herein, we used transient permeabilization of Limulus brain hemi-slices to assess first the GTPase requirements for ChCoT trafficking. Hemi-slices were treated with 100ng/ml streptolysin-O (SLO) in calcium free buffered Limulus saline. Tissues were resealed and washed in Chao's for 60 min. Hemi-slices were assessed for surface integrity by measuring [3H]choline (Ch) uptake and [3H]HC-3 binding. Uptake and binding were unaltered. Transiently permeabilized hemi-slices were treated with the non hydrolysable GTP analog, 5'-Guanylylimido-phosphate (GPP), and either [3H]Ch uptake or [3H]HC-3 binding was determined. GPP caused a dose dependent increased [3H]Ch uptake which reached a maximum of 70% at 100 muM, GPP. A similar result was observed for [3H]HC-3 binding. Additionally, the antecedent exposure to 120 mM KCl in Chao's combined with 100 mum GPP resulted in an additive increased uptake of approximately 173% suggesting two separate up-regulation mechanisms.;Secondly, we determined the role of the large GTPase, dynamin, in ChCoT trafficking. SLO transiently permeabilized brain hemi-slices were treated with a dynamin antibody and high affinity choline uptake was determined. The dynamin antibody caused a dose dependent increase in [3H]Ch uptake, with a maximal increase of 56% at an antibody concentration of 0.1mug/ml. Exposure to the dynamin antibody in combination with 100 muM GPP resulted in a 70% increase in choline uptake, a value previously observed with 100 muM GPP alone. Thus, the maximum effect of the dynamin antibody was approximately 80% of that achieved for total GTPases. We conclude that dynamin is the principal GTPase involved in the retrieval of the ChCoT from the membrane. However, other GTPases are involved also. Further studies are needed to determine which specific GTPases, in addition to dynamin, are involved in this retrieval mechanism.