Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1.

The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low Km) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low Km of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys160 of LAT1 and Cys110 of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl2 with a Ki of 0.56+/-0.11 microM. The inhibitory effect of HgCl2 for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl2 for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys88 and Cys439, altered amino acid transport. The Vmax was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys439 residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.

Tumor necrosis-factor-alpha (TNF-alpha) induces rapid insertion of Ca2+-permeable alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate (Ca-A/K) channels in a subset of hippocampal pyramidal neurons.

The presence of cell surface Ca2+ permeable alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate (Ca-A/K) channels on subsets of central neurons influences both normal physiological function and vulnerability to excitotoxicity. Factors that regulate the formation and membrane insertion of Ca-A/K channels, however, are poorly understood. Recently, the cytokine tumor necrosis factor-alpha (TNF-alpha) was shown to increase the cell surface expression of an AMPA receptor (AMPAR) subunit (GluR1) and to potentiate vulnerability to AMPAR-mediated injury. In this study, we examined the possibility that TNF-alpha might also increase numbers of functional Ca-A/K channels. In acute hippocampal slice preparations, TNF-alpha appeared to increase Ca-A/K channel numbers in pyramidal neurons (HPNs), as assessed using a histochemical stain based on kainate-induced uptake of Co2+ ions (Co2+ labeling). In dissociated hippocampal neuronal cultures, TNF-alpha exposure (6 nM, 15 min) induced a rapid increase in cell surface levels not only of GluR1, but also of the AMPAR subunit GluR2, on most neurons, without evident new protein synthesis. Furthermore, consistent with the slice studies, fluorescence Ca2+ imaging techniques revealed an increase in numbers of Ca-A/K channels on what appeared to be a subset of HPNs. These observations are the first to provide evidence for the rapid upregulation of neuronal Ca-A/K channels in response to a cytokine or any other soluble factor, and provide a novel mechanism through which TNF-alpha may modulate both synaptic function and neuronal vulnerability.

Heterogeneity of Ca2+-permeable AMPA/kainate channel expression in hippocampal pyramidal neurons: fluorescence imaging and immunocytochemical assessment.

The presence of Ca2+-permeable AMPA/kainate (Ca-A/K) channels on hippocampal pyramidal neurons (HPNs) has been controversial, although they are present on many forebrain GABAergic neurons. We combined high-resolution fluorescence Ca2+ imaging with surface AMPA receptor (AMPAR) subunit immunocytochemistry to examine the expression of functional Ca-A/K channels in dissociated hippocampal neurons at the subcellular level. In GABAergic neurons [identified by glutamate decarboxylase (GAD) immunocytochemistry], focal application of AMPA induced large dendrosomatic intracellular [Ca2+] ([Ca2+]i) rises, consistent with their known strong Ca-A/K channel expression. Surface immunostaining for the AMPAR subunits GluR1 and GluR2 revealed abundant dendritic GluR1 puncta containing little or no GluR2, which, when present, was limited to diffuse staining in the soma and proximal dendrites. In contrast, the majority of HPNs (putatively identified by morphological criteria and lack of GAD labeling) showed little or no AMPA-induced [Ca2+]i rise. Correspondingly, most HPNs showed strong dendritic labeling for both GluR1 and GluR2 that colocalized extensively. A subpopulation of HPNs, however, displayed noticeable [Ca2+]i rises that began and often reached their highest levels in discrete dendritic regions. In these HPNs, levels of GluR1 relative to GluR2 were higher, and GluR1 was often present without overlying GluR2. The present studies, which are the first to directly examine the relationship between the local complement of cell surface AMPAR and the presence of dendritic Ca-A/K channels, clearly indicate that considerable cell surface GluR2 does not preclude the presence of Ca-A/K channels and further show that HPNs display considerable heterogeneity in terms of apparent Ca-A/K channel expression.

Blockade of Ca2+-permeable AMPA/kainate channels decreases oxygen-glucose deprivation-induced Zn2+ accumulation and neuronal loss in hippocampal pyramidal neurons.

Synaptic release of Zn2+ and its translocation into postsynaptic neurons probably contribute to neuronal injury after ischemia or epilepsy. Studies in cultured neurons have revealed that of the three major routes of divalent cation entry, NMDA channels, voltage-sensitive Ca2+ channels (VSCCs), and Ca2+-permeable AMPA/kainate (Ca-A/K) channels, Ca-A/K channels exhibit the highest permeability to exogenously applied Zn2+. However, routes through which synaptically released Zn2+ gains entry to postsynaptic neurons have not been characterized in vivo. To model ischemia-induced Zn2+ movement in a system approximating the in vivo situation, we subjected mouse hippocampal slice preparations to controlled periods of oxygen and glucose deprivation (OGD). Timm's staining revealed little reactive Zn2+ in CA1 and CA3 pyramidal neurons of slices exposed in the presence of O2 and glucose. However, 15 min of OGD resulted in marked labeling in both regions. Whereas strong Zn2+ labeling persisted if both the NMDA antagonist MK-801 and the VSCC blocker Gd3+ were present during OGD, the presence of either the Ca-A/K channel blocker 1-naphthyl acetyl spermine (NAS) or the extracellular Zn2+ chelator Ca2+ EDTA substantially decreased Zn2+ accumulation in pyramidal neurons of both subregions. In parallel experiments, slices were subjected to 5 min OGD exposures as described above, followed 4 hr later by staining with the cell-death marker propidium iodide. As in the Timm's staining experiments, substantial CA1 or CA3 pyramidal neuronal damage occurred despite the presence of MK-801 and Gd3+, whereas injury was decreased by NAS or by Ca2+ EDTA (in CA1).