Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system and is removed from the synaptic cleft by sodium-dependent glutamate transporters. To date, five distinct glutamate transporters have been cloned from animal and human tissue: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (refs 1-5). GLAST and GLT-1 are localized primarily in astrocytes, whereas EAAC1 (refs 8, 9), EAAT4 (refs 9-11) and EAAT5 (ref 5) are neuronal. Studies of EAAT4 and EAAC1 indicate an extrasynaptic localization on perisynaptic membranes that are near release sites. This localization facilitates rapid glutamate binding, and may have a role in shaping the amplitude of postsynaptic responses in densely packed cerebellar terminals. We have used a yeast two-hybrid screen to identify interacting proteins that may be involved in regulating EAAT4--the glutamate transporter expressed predominately in the cerebellum--or in targeting and/or anchoring or clustering the transporter to the target site. Here we report the identification and characterization of two proteins, GTRAP41 and GTRAP48 (for glutamate transporter EAAT4 associated protein) that specifically interact with the intracellular carboxy-terminal domain of EAAT4 and modulate its glutamate transport activity.

Excitatory amino-acid carrier 1 (EAAC1) is a high-affinity Na+-dependent L-glutamate/D,L-aspartate cell-membrane transport protein. It is expressed in brain as well as several non-nervous tissues. In brain, EAAC1 is the primary neuronal glutamate transporter. It has a polarized distribution in cells and mainly functions perisynaptically to transport glutamate from the extracellular environment. In the kidney it is involved in renal acidic amino-acid re-absorption and amino-acid metabolism. Here we describe the identification and characterization of an EAAC1-associated protein, GTRAP3-18. Like EAAC1, GTRAP3-18 is expressed in numerous tissues. It localizes to the cell membrane and cytoplasm, and specifically interacts with carboxy-terminal intracellular domain of EAAC1. Increasing the expression of GTRAP3-18 in cells reduces EAAC1-mediated glutamate transport by lowering substrate affinity. The expression of GTRAP3-18 can be upregulated by retinoic acid, which results in a specific reduction of EAAC1-mediated glutamate transport. These studies show that glutamate transport proteins can be regulated potently and that GTRAP can modulate the transport functions ascribed to EAAC1. GTRAP3-18 may be important in regulating the metabolic function of EAAC1.

Mutations in SPTBN2, the gene encoding β-III spectrin, cause spinocerebellar ataxia type 5 in humans (SCA5), a neurodegenerative disorder resulting in loss of motor coordination. How these mutations give rise to progressive ataxia and what the precise role β-III spectrin plays in normal cerebellar physiology are unknown. We developed a mouse lacking full length β-III spectrin and found that homozygous mice reproduced features of SCA5 including gait abnormalities, tremor, deteriorating motor coordination, Purkinje cell loss and cerebellar atrophy (molecular layer thinning). In vivo analysis reveals an age-related reduction in simple spike firing rate in surviving β-III−/− Purkinje cells while in vitro studies show these neurons to have reduced spontaneous firing, smaller sodium currents and dysregulation of glutamatergic neurotransmission. Our data suggest an early loss of EAAT4- (protein interactor of β-III spectrin) and subsequent loss of GLAST-mediated uptake may play a role in neuronal pathology. These findings implicate a loss of β-III spectrin function in SCA5 pathogenesis and indicate there are at least two physiological effects of β-III spectrin loss that underpin a progressive loss of inhibitory cerebellar output, namely an intrinsic Purkinje cell membrane defect due to reduced sodium currents and alterations in glutamate signaling.

Cerebral palsy is commonly attributed to perinatal asphyxia. However, Schnekenberg et al. describe here four individuals with ataxic cerebral palsy likely due to de novo dominant mutations associated with increased paternal age. Therefore, patients with cerebral palsy should be investigated for genetic causes before the disorder is ascribed to asphyxia.