Peter Kovermann

A point mutation associated with episodic ataxia 6 increases glutamate transporter anion currents

Episodic ataxia is a human genetic disease characterized by paroxysmal cerebellar incoordination. There are several genetically and clinically distinct forms of this disease, and one of them, episodic ataxia type 6, is caused by mutations in the gene encoding a glial glutamate transporter, the excitatory amino acid transporter-1. So far, reduced glutamate uptake by mutant excitatory amino acid transporter-1 has been thought to be the main pathophysiological process in episodic ataxia type 6. However, excitatory amino acid transporter-1 does not only mediate secondary-active glutamate transport, but also functions as an ion channel. Here, we examined the effects of a disease-associated point mutation, P290R, on glutamate transport, anion current as well as on the subcellular distribution of excitatory amino acid transporter-1 using heterologous expression in mammalian cells. P290R reduces the number of excitatory amino acid transporter-1 in the surface membrane and impairs excitatory amino acid transporter-1-mediated glutamate uptake. Cells expressing P290R excitatory amino acid transporter-1 exhibit larger anion currents than wild-type cells in the absence as well as in the presence of external l-glutamate, despite a lower number of mutant transporters in the surface membrane. Noise analysis revealed unaltered unitary current amplitudes, indicating that P290R modifies opening and closing, and not anion permeation through mutant excitatory amino acid transporter-1 anion channels. These findings identify gain-of-function of excitatory amino acid transporter anion conduction as a pathological process in episodic ataxia. Episodic ataxia type 6 represents the first human disease found to be associated with altered function of excitatory amino acid transporter anion channels and illustrates possible physiological and pathophysiological impacts of this functional mode of this class of glutamate transporters.

Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation

Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride‐sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl−]int to be controlled by two opposing transport processes: chloride is actively accumulated by the Na+‐K+‐2Cl− cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl−]int to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion‐selective channels during glutamate transport, and thus represent a class of ligand‐gated anion channels. Age‐dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT‐1 increased [Cl−]int in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl−]int. Other tested chloride channels or chloride transporters do not contribute to [Cl−]int under our experimental conditions. Neither genetic removal of ClC‐2 nor pharmacological block of K+‐Cl− cotransporter change resting Bergmann glial [Cl−]int in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388–400

CNS Schwann cells display oligodendrocyte precursor-like potassium channel activation and antigenic expression in vitro

Central nervous system (CNS) injury triggers production of myelinating Schwann cells from endogenous oligodendrocyte precursors (OLPs). These CNS Schwann cells may be attractive candidates for novel therapeutic strategies aiming to promote endogenous CNS repair. However, CNS Schwann cells have been so far mainly characterized in situ regarding morphology and marker expression, and it has remained enigmatic whether they display functional properties distinct from peripheral nervous system (PNS) Schwann cells. Potassium channels (K+) have been implicated in progenitor and glial cell proliferation after injury and may, therefore, represent a suitable pharmacological target. In the present study, we focused on the function and expression of voltage-gated K+ channels Kv1–12 and accessory β-subunits in purified adult canine CNS and PNS Schwann cell cultures using electrophysiology and microarray analysis and characterized their antigenic phenotype. We show here that K+ channels differed significantly in both cell types. While CNS Schwann cells displayed prominent KD-mediated K+ currents, PNS Schwann cells elicited KD- and KA-type K+ currents. Inhibition of K+ currents by TEA and Ba2+ was more effective in CNS Schwann cells. These functional differences were not paralleled by differential mRNA expression of Kv1–12 and accessory β-subunits. However, O4/A2B5 and GFAP expressions were significantly higher and lower, respectively, in CNS than in PNS Schwann cells. Taken together, this is the first evidence that CNS Schwann cells display specific properties not shared by their peripheral counterpart. Both Kv currents and increased O4/A2B5 expression were reminiscent of OLPs suggesting that CNS Schwann cells retain OLP features during maturation.

Mutating a conserved proline residue within the trimerization domain modifies Na+ binding to excitatory amino acid transporters and associated conformational changes.

Background: The mutation P290R in the excitatory amino acid transporter 1 (EAAT1) causes episodic ataxia type 6. Results: Voltage clamp fluorometry demonstrates changes in the time course of EAAT3 fluorescence signals by the homologous P259R mutation. Conclusion: P259R alters EAAT3 transport functions by decelerating conformational changes associated with sodium binding. Significance: Studying naturally occurring mutations promises identification of unexpected determinants of transporter functions. Excitatory amino acid transporters (EAATs) are crucial for glutamate homeostasis in the mammalian central nervous system. They are not only secondary active glutamate transporters but also function as anion channels, and different EAATs vary considerably in glutamate transport rates and associated anion current amplitudes. A naturally occurring mutation, which was identified in a patient with episodic ataxia type 6 and that predicts the substitution of a highly conserved proline at position 290 by arginine (P290R), was recently shown to reduce glutamate uptake and to increase anion conduction by hEAAT1. We here used voltage clamp fluorometry to define how the homologous P259R mutation modifies the functional properties of hEAAT3. P259R inverts the voltage dependence, changes the sodium dependence, and alters the time dependence of hEAAT3 fluorescence signals. Kinetic analysis of fluorescence signals indicate that P259R decelerates a conformational change associated with sodium binding to the glutamate-free mutant transporters. This alteration in the glutamate uptake cycle accounts for the experimentally observed changes in glutamate transport and anion conduction by P259R hEAAT3.

Determination of Intracellular Chloride Concentrations by Fluorescence Lifetime Imaging

Fluorescence microscopy with membrane-permeable ion-sensitive fluorophores allows the non-invasive determination of intracellular ion concentrations. Chloride is the major anion of intra- and extracellular fluids influencing a great number of physiological processes. The dysfunction of chloride transporters and channels leads to disturbance in chloride homeostasis that can result in diseases of different parts of the body. The different existing chloride sensitive fluorophores and their usefulness in fluorescence lifetime imaging are put forward in this chapter. Fluorescence lifetime imaging of a chloride sensitive quinolinium dye (MQAE) has been established as an elegant tool to determine intracellular chloride concentrations of different cells in living biological tissue. Details of the experimental procedure are described and two case studies—chloride transport by a chloride transporter (KCC2) across the cell membrane and determination of intracellular chloride concentration in glia cells (EAAT1-positive astrocytes)—are presented.

Impaired K+ binding to glial glutamate transporter EAAT1 in migraine

SLC1A3 encodes the glial glutamate transporter hEAAT1, which removes glutamate from the synaptic cleft via stoichiometrically coupled Na+-K+-H+-glutamate transport. In a young man with migraine with aura including hemiplegia, we identified a novel SLC1A3 mutation that predicts the substitution of a conserved threonine by proline at position 387 (T387P) in hEAAT1. To evaluate the functional effects of the novel variant, we expressed the wildtype or mutant hEAAT1 in mammalian cells and performed whole-cell patch clamp, fast substrate application, and biochemical analyses. T387P diminishes hEAAT1 glutamate uptake rates and reduces the number of hEAAT1 in the surface membrane. Whereas hEAAT1 anion currents display normal ligand and voltage dependence in cells internally dialyzed with Na+-based solution, no anion currents were observed with internal K+. Fast substrate application demonstrated that T387P abolishes K+-bound retranslocation. Our finding expands the phenotypic spectrum of genetic variation in SLC1A3 and highlights impaired K+ binding to hEAAT1 as a novel mechanism of glutamate transport dysfunction in human disease.