Mrp1 is essential for sphingolipid signaling to p-glycoprotein in mouse blood-brain and blood-spinal cord barriers.

At the blood-brain and blood-spinal cord barriers, P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to central nervous system (CNS) pharmacotherapy. Recently, we showed that signaling through tumor necrosis factor-α (TNF-α), sphingolipids, and sphingosine-1-phosphate receptor 1 (S1PR1) rapidly and reversibly reduced basal P-glycoprotein transport activity in the rat blood-brain barrier. The present study extends those findings to the mouse blood-brain and blood-spinal cord barriers and, importantly, identifies multidrug resistance-associated protein 1 (Mrp1, Abcc1) as the transporter that mediates S1P efflux from brain and spinal cord endothelial cells. In brain and spinal cord capillaries isolated from wild-type mice, TNF-α, sphingosine, S1P, the S1PR agonist fingolimod (FTY720), and its active, phosphorylated metabolite, FTY720P, reduced P-glycoprotein transport activity; these effects were abolished by a specific S1PR1 antagonist. In brain and spinal cord capillaries isolated from Mrp1-null mice, neither TNF-α nor sphingosine nor FTY720 reduced P-glycoprotein transport activity. However, S1P and FTY720P had the same S1PR1-dependent effects on transport activity as in capillaries from wild-type mice. Thus, deletion of Mrp1 alone terminated endogenous signaling to S1PR1. These results identify Mrp1 as the transporter essential for S1P efflux from the endothelial cells and thus for inside-out S1P signaling to P-glycoprotein at the blood-brain and blood-spinal cord barriers.

Importance of glycosylation on function of a potassium channel in neuroblastoma cells.

The Kv3.1 glycoprotein, a voltage-gated potassium channel, is expressed throughout the central nervous system. The role of N-glycans attached to the Kv3.1 glycoprotein on conducting and non-conducting functions of the Kv3.1 channel are quite limiting. Glycosylated (wild type), partially glycosylated (N220Q and N229Q), and unglycosylated (N220Q/N229Q) Kv3.1 proteins were expressed and characterized in a cultured neuronal-derived cell model, B35 neuroblastoma cells. Western blots, whole cell current recordings, and wound healing assays were employed to provide evidence that the conducting and non-conducting properties of the Kv3.1 channel were modified by N-glycans of the Kv3.1 glycoprotein. Electrophoretic migration of the various Kv3.1 proteins treated with PNGase F and neuraminidase verified that the glycosylation sites were occupied and that the N-glycans could be sialylated, respectively. The unglycosylated channel favored a different whole cell current pattern than the glycoform. Further the outward ionic currents of the unglycosylated channel had slower activation and deactivation rates than those of the glycosylated Kv3.1 channel. These kinetic parameters of the partially glycosylated Kv3.1 channels were also slowed. B35 cells expressing glycosylated Kv3.1 protein migrated faster than those expressing partially glycosylated and much faster than those expressing the unglycosylated Kv3.1 protein. These results have demonstrated that N-glycans of the Kv3.1 glycoprotein enhance outward ionic current kinetics, and neuronal migration. It is speculated that physiological changes which lead to a reduction in N-glycan attachment to proteins will alter the functions of the Kv3.1 channel.

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels.

Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.

Novel Kv3 glycoforms differentially expressed in adult mammalian brain contain sialylated N-glycans.

The N-glycan pool of mammalian brain contains remarkably high levels of sialylated N-glycans. This study provides the first evidence that voltage-gated K+ channels Kv3.1, Kv3.3, and Kv3.4, possess distinct sialylated N-glycan structures throughout the central nervous system of the adult rat. Electrophoretic migration patterns of Kv3.1, Kv3.3, and Kv3.4 glycoproteins from spinal cord, hypothalamus, thalamus, cerebral cortex, hippocampus, and cerebellum membranes digested with glycosidases were used to identify the various glycoforms. Differences in the migration of Kv3 proteins were attributed to the desialylated N-glycans. Expression levels of the Kv3 proteins were highest in cerebellum, whereas those of Kv3.1 and Kv3.3 were much lower in the other 5 regions. The lowest level of Kv3.1 was expressed in the hypothalamus, whereas the lowest levels of Kv3.3 were expressed in both thalamus and hypothalamus. The other regions expressed intermediate levels of Kv3.3, with spinal cord expressing the highest. The expression level of Kv3.4 in the hippocampus was slightly lower than that in cerebellum, and was closely followed by the other 4 regions, with spinal cord expressing the lowest level. We suggest that novel Kv3 glycoforms may endow differences in channel function and expression among regions throughout the central nervous system.

Complex oligosaccharides are N-linked to Kv3 voltage-gated K+ channels in rat brain.

Neuronal Kv3 voltage-gated K(+) channels have two absolutely conserved N-glycosylation sites. Here, it is shown that Kv3.1, 3.3, and 3.4 channels are N-glycosylated in rat brain. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced faster migrating immunobands than those of undigested membranes. Additionally, partial PNGase F digests showed that both sites are occupied by oligosaccharides. Neuraminidase treatment produced a smaller immunoband shift relative to PNGase F treatment. These results indicate that both sites are highly available and occupied by N-linked oligosaccharides for Kv3.1, 3.3, and 3.4 in rat brain, and furthermore that at least one oligosaccharide is of complex type. Additionally, these results point to an extracytoplasmic S1-S2 linker in Kv3 proteins expressed in native membranes. We suggest that N-glycosylation processing of Kv3 channels is critical for the expression of K(+) currents at the surface of neurons, and perhaps contributes to the pathophysiology of congenital disorders of glycosylation.