AP-3-dependent Mechanisms Control the Targeting of a Chloride Channel (ClC-3) in Neuronal and Non-neuronal Cells

Abstract

Adaptor protein (AP)-2 and AP-3-dependent mechanisms control the sorting of membrane proteins into synaptic vesicles. Mouse models deficient in AP-3, mocha, develop a neurological phenotype of which the central feature is an alteration of the luminal synaptic vesicle composition. This is caused by a severe reduction of vesicular levels of the zinc transporter 3 (ZnT3). It is presently unknown whether this mocha defect is restricted to ZnT3 or encompasses other synaptic vesicle proteins capable of modifying synaptic vesicle contents, such as transporters or channels. In this study, we identified a chloride channel, ClC-3, whose level in synaptic vesicles and hippocampal mossy fiber terminals was reduced in the context of the mocha AP-3 deficiency. In PC-12 cells, ClC-3 was present in transferrin receptorpositive endosomes, where it was targeted to synapticlike microvesicles (SLMV) by a mechanism sensitive to brefeldin A, a signature of the AP-3-dependent route of SLMV biogenesis. ClC-3 was packed in SLMV along with the AP-3-targeted synaptic vesicle protein ZnT3. Co-segregation of ClC-3 and ZnT3 to common intracellular compartments was functionally significant as revealed by increased vesicular zinc transport with increased ClC3 expression. Our work has identified a synaptic vesicle protein in which trafficking to synaptic vesicles is regulated by AP-3. In addition, our findings indicate that ClC-3 and ZnT3 reside in a common vesicle population where they functionally interact to determine vesicle luminal composition.

Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles

“…This decrease was due to a redistribution of the kinase rather than global changes in brain kinase expression, because kinase content was not detectably modified in P1-P2 brain fractions ( Figure 6D). The effects of the mocha mutation on the subcellular distribution of PI4KII␣ are similar to those reported for ZnT3 and for another synaptic vesicle protein whose traffic is regulated by AP-3: chloride channel 3 (ClC3) (Salazar et al, 2004a). ZnT3 and ClC3 targeting defects also are manifested as a decreased content of these proteins in a subset of nerve terminals in situ (Salazar et al, 2004a), such as in nerve terminals of the hippocampus (Salazar et al, 2004a;Seong et al, 2005).…”

“…Immunostaining of wild-type and mocha brain hippocampal sections revealed a reduction in PI4KII␣ immunoreactivity in the mossy fiber nerve terminals of the hilus (Figure 7, A-D) and CA3 region (Figure 7, E-H). These nerve terminals were previously shown to be enriched in ZnT3 (Palmiter et al, 1996) and ClC-3 (Stobrawa et al, 2001) in wild-type, but not in mocha mice (Salazar et al, 2004a). The decreased kinase immunostaining in mossy nerve terminals was not due to a "global" change in nerve terminal number or structure because VAMPII distribution and abundance were not affected by the mocha mutation (Figure 7, I and J).…”

“…ZnT3 and ClC3 targeting defects also are manifested as a decreased content of these proteins in a subset of nerve terminals in situ (Salazar et al, 2004a), such as in nerve terminals of the hippocampus (Salazar et al, 2004a;Seong et al, 2005). Therefore, we explored whether the PI4KII␣ content in mouse brain nerve terminals was similarly affected by the mocha mutation.…”

“…Immunoperoxidase staining on brain sections has been described in detail previously (Salazar et al, 2004a). All stainings were performed in duplicate in at least two independent experiments using two different monospecific affinitypurified phosphatidylinositol-4-kinase type II␣ antibodies.…”

“…All stainings were performed in duplicate in at least two independent experiments using two different monospecific affinitypurified phosphatidylinositol-4-kinase type II␣ antibodies. (Salazar et al, 2004a) were transfected with pEGFP-PI4KII␣, and the pEF6/V5-His plasmid encoding the blasticidin-resistant gene (Invitrogen). Double transfectant clones were selected with 10 g/ml blasticidin and 0.8 mg/ml G418.…”