Mutations associated with epileptic encephalopathy modify EAAT2 anion channel function.

OBJECTIVE: Mutations in the gene solute carrier family member 1A2 (SLC1A2) encoding the excitatory amino acid transporter 2 (EAAT2) are associated with severe forms of epileptic encephalopathy. EAAT2 is expressed in glial cells and presynaptic nerve terminals and represents the main l-glutamate uptake carrier in the mammalian brain. It does not only function as a secondary active glutamate transporter, but also as an anion channel. How naturally occurring mutations affect these two transport functions of EAAT2 and how such alterations cause epilepsy is insufficiently understood. METHODS: Here we studied the functional consequences of three disease-associated mutations, which predict amino acid exchanges p.Gly82Arg (G82R), p.Leu85Pro (L85P), and p.Pro289Arg (P289R), by heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole-cell patch-clamp recordings of EAAT2 l-glutamate transport and anion current. RESULTS: G82R and L85P exchange amino acid residues that contribute to the formation of the EAAT anion pore. They enlarge the pore diameter sufficiently to permit the passage of l-glutamate and thus function as l-glutamate efflux pathways. The mutation P289R decreases l-glutamate uptake, but increases anion currents despite a lower membrane expression. SIGNIFICANCE: l-glutamate permeability of the EAAT anion pore is an unexpected functional consequence of naturally occurring single amino acid substitutions. l-glutamate efflux through mutant EAAT2 anion channels will cause glutamate excitotoxicity and neuronal hyperexcitability in affected patients. Antagonists that selectively suppress the EAAT anion channel function could serve as therapeutic agents in the future.

The expanding toolbox to study the LRRC8-formed volume-regulated anion channel VRAC.

The volume-regulated anion channel (VRAC) is activated upon cell swelling and facilitates the passive movement of anions across the plasma membrane in cells. VRAC function underlies many critical homeostatic processes in vertebrate cells. Among them are the regulation of cell volume and membrane potential, glutamate release and apoptosis. VRAC is also permeable for organic osmolytes and metabolites including some anti-cancer drugs and antibiotics. Therefore, a fundamental understanding of VRAC's structure-function relationships, its physiological roles, its utility for therapy of diseases, and the development of compounds modulating its activity are important research frontiers. Here, we describe approaches that have been applied to study VRAC since it was first described more than 30 years ago, providing an overview of the recent methodological progress. The diverse applications reflecting a compromise between the physiological situation, biochemical definition, and biophysical resolution range from the study of VRAC activity using a classic electrophysiology approach, to the measurement of osmolytes transport by various means and the investigation of its activation using a novel biophysical approach based on fluorescence resonance energy transfer.

Increased glutamate transporter-associated anion currents cause glial apoptosis in episodic ataxia 6.

Episodic ataxia type 6 is an inherited neurological condition characterized by combined ataxia and epilepsy. A severe form of this disease with episodes combining ataxia, epilepsy and hemiplegia was recently associated with a proline to arginine substitution at position 290 of the excitatory amino acid transporter 1 in a heterozygous patient. The excitatory amino acid transporter 1 is the predominant glial glutamate transporter in the cerebellum. However, this glutamate transporter also functions as an anion channel and earlier work in heterologous expression systems demonstrated that the mutation impairs the glutamate transport rate, while increasing channel activity. To understand how these changes cause ataxia, we developed a constitutive transgenic mouse model. Transgenic mice display epilepsy, ataxia and cerebellar atrophy and, thus, closely resemble the human disease. We observed increased glutamate-activated chloride efflux in Bergmann glia that triggers the apoptosis of these cells during infancy. The loss of Bergmann glia results in reduced glutamate uptake and impaired neural network formation in the cerebellar cortex. This study shows how gain-of-function of glutamate transporter-associated anion channels causes ataxia through modifying cerebellar development.

Both Orai1 and TRPC1 are Involved in Excessive Store-Operated Calcium Entry in Striatal Neurons Expressing Mutant Huntingtin Exon 1.

It has been previously reported that N-terminus of mutant huntingtin (product of the 1st exon) is sufficient to cause a Huntington's disease (HD) pathological phenotype. In view of recent data suggesting that improper regulation of store-operated calcium (SOC) channels is involved in neurodegenerative processes, we investigated influence of expression of the mutant huntingtin N-terminal fragment (Htt138Q-1exon) on SOC entry (SOCE) in mouse neuroblastoma cells (Neuro-2a) and in primary culture of medium spiny neurons (MSNs) isolated from mice. The results show that SOCE in these cells is enhanced upon lentiviral expression of the Htt138Q-1exon. Moreover, we demonstrated that RNAi-mediated knockdown of TRPC1, Orai1, or STIM1 proteins leads to dramatic reduction of abnormal SOCE in both Neuro-2a and MSNs, expressing Htt138Q-1exon. Thus, we concluded that abnormal SOCE in these cells is maintained by both TRPC1- and Orai1-containing channels and required STIM1 for its activation. Furthermore, EVP4593 compound previously tested as a potential anti-HD drug in a Drosophila screening system has proved to be capable of reducing SOCE to the normal level in MSNs expressing the Htt138Q-1exon.