Functional consequences of SLC1A3 mutations associated with episodic ataxia 6

The episodic ataxias (EA) are a group of inherited neurological diseases characterized by paroxysmal cerebellar incoordination. There exist nine forms of episodic ataxia with distinct neurological symptoms and genetic origins. Episodic ataxia type 6 (EA6) differs from other EA forms in long attack duration, epilepsy and absent myokymia, nystagmus, and tinnitus. It has been described in seven families, and mutations in SLC1A3, the gene encoding the glial glutamate transporter EAAT1, were reported in each family. How these mutations affect EAAT1 expression, subcellular localization, and function, and how such alterations result in the complex neurological phenotype of EA6 is insufficiently understood. We here compare the functional consequences of all currently known mutations by heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole‐cell patch clamp recordings of EAAT1 transport and anion currents. We observed impairments of multiple EAAT1 properties ranging from changes in transport function, impaired trafficking to increased protein expression. Many mutations caused only slight changes illustrating how sensitively the cerebellum reacts on impaired EAAT1 functions.

such as ubiquitin-protein ligase 4, and mutations in the fibroblast growth factor 14 have been recently associated to the newly defined subtype EA9. So far, causal genes have not yet been identified for EA3,4,and 7. There are other EA syndromes that have been associated to mutations in the ion channel genes KCNA2 and SCN2A (Corbett et al., 2016;Maksemous, Smith, Sutherland, Sampaio, & Griffiths, 2018;Schwarz et al., 2016Schwarz et al., , 2019, but thus far they have not been designated as specific EA-subtypes. Episodic ataxia type 6 (EA6) combines paroxysmal cerebellar incoordination with epilepsy and migraine-like headache and differs from other EA forms in the occurrence of long-lasting attacks of ataxia and epilepsy and absent myokymia, nystagmus, and tinnitus . Thus far, EA6 has been reported in only seven families, and was associated with mutations in SLC1A3, encoding the glial excitatory amino acid transporter 1 (EAAT1), in all the cases (Figure 1; Choi, Jen, et al., 2017; F I G U R E 1 Localizations of EA6-associated mutations in hEAAT1. de Vries et al., 2009;Iwama et al., 2017;Jen, Wan, Palos, Howard, & Baloh, 2005;Pyle et al., 2015).
Here, we study the functional consequences of the remaining six known EA6-associated SLC1A3 mutations using heterologous expression in mammalian cells, confocal imaging, biochemistry, and whole-cell patch clamping. A mutation predicting p.M128R (Ref Seq NM_004172.4: c.383T>G: p.Met128Arg) was reported in a 10-yearold girl with disease onset at 11 months and episodes of truncal ataxia and strabismus (Iwama et al., 2017). In another family, three patients, who experienced ataxia episodes associated with nausea, photophobia, vertigo, and speech and vision problems from early childhood, and one unaffected family member carry a SLC1A3 mu- in two unaffected and two affected members of one family, who suffered from unsteadiness and dizziness with a late onset at age 55 years (Choi, Jen, et al., 2017;. A SLC1A3 mutation resulting in expression of p.T318A hEAAT1 (RefSeq NM_004172.4: c.952A>G: p.Thr318Ala) was reported in patients with ataxia, dizziness, and dysarthria, and the p.A329T mutation (RefSeq NM_004172.4: c.985G>A: p.Ala329Thr) was found in a patient carrying additional CACNA1A mutations and suffering from ataxia, dizziness, and epileptic seizures .
EAATs are trimeric proteins with three subunits associating via immobile trimerization domains (Figure 1). They are prototypical elevator transporter: each subunit exhibits a mobile transport domain that contains all substrate-binding sites and that shuttle substrates from outside to inside or vice versa via combined 16 Å vertical movement and 37°rotation (Reyes, Ginter, & Boudker, 2009 et al., 2005), all other known mutations are localized in the transport domain (Figure 1a,b;Canul-Tec et al., 2017). Figure 1c demonstrates the conservation of affected residues within human EAATs. were determined by multiplying relative total protein amounts ( Figure 2d) and percentages of complex glycolysated proteins ( Figure 2e).

| Electrophysiology
Whole-cell patch-clamp recordings were performed using an EPC-10 amplifier controlled by PatchMaster software (HEKA
3.2 | EA6-associated mutations exert multiple effects on hEAAT1 glutamate uptake hEAAT1 transports glutamate stoichiometrically coupled to three Na + and one H + in exchange with one K + (Zerangue & Kavanaugh, 1996). Each uptake cycle is associated with the net transport of two positive charges, and glutamate transport can thus be quantified using electrophysiological techniques by subtracting current amplitudes measured before glutamate application from amplitudes obtained in the presence of the substrate. Since EAATs also function as anion channels, permeable anions have to be replaced with nonpermeable anions to measure uptake currents in isolation (Wadiche, Amara & Kavanaugh, 1995). Figure 3a shows representative sub- showed glutamate uptake levels that were increased in parallel to its higher expression level in HEK293T cells (Figure 3d). Figure S2 depicts plots of glutamate uptake currents versus capacitances of cells expressing WT or mutant transporters and a comparison of glutamate transport current densities that were obtained from the slopes of these relationships. Individual mutants exert similar effects on uptake currents and uptake current densities, demonstrating that the expression levels rather than cell sizes determine the amplitudes of the whole-cell currents.

| Anion currents of WT and mutant hEAAT1
EAAT anion channels open upon lateral movement of the transport domain from intermediate translocation states (Machtens et al., 2015). The kinetics of EAAT anion currents are thus tightly linked to transitions within the glutamate uptake cycle (Fahlke et al., 2016;Otis & Kavanaugh, 2000), and changes in anion current kinetics provide insights into possible disease-associated alterations within the hEAAT1 uptake cycle. We studied hEAAT1 anion currents under two different ionic conditions. Whereas the transporter can move through the whole transport cycle in cells intracellularly dialyzed with a KNO 3 -based solution (Figure 4a), the use of NaNO 3based solutions restricts the accessible states to the so-called Na +hemicycle ( Figure 5a).

| DISCUSSION
Episodic ataxia is characterized by attacks of incoordination, imbalance, and inter-attack weakness, often associated with progressive ataxia, epilepsy, dystonia, or hemiplegic migraine . The spectrum of neurological symptoms indicates a cerebellar origin, and indeed all known episodic ataxia disease genes encode cerebellar proteins. Approximately 2/3 of all cases with episodic ataxia carry mutations in KCNA1 (EA1) or CACNA1A (EA2) . Mutations in CACNB4 (Escayg et al., 2000) were associated with EA5 and in SLC1A3 with EA6. Since all proteins encoded by EA disease genes likely contribute to synaptic transmission in the cerebellum, EA6-associated SLC1A3 mutations were initially assumed to modify glutamatergic synaptic transmission via reduced glutamate reuptake capability.
Our group studied the functional consequences of the p.P290R mutation in SLC1A3, which was reported for the first and most severe case of EA6 (Jen et al., 2005), using heterologous expression and cellular electrophysiology (Hotzy et al., 2013;Winter et al., 2012).
We found that p.P290R decreases glutamate transport rates, but increased absolute open probabilities of EAAT1 anion channels. We proposed that gain-of-function of p.P290R hEAAT1 anion channels decreases anion concentrations in Bergmann glia (Untiet et al., 2017) and hypothesized that the resulting increase in driving force of GATs (GABA transporters) might reduce import of GABA and thus inhibitory synaptic transmission in the EA6 patient (Winter et al., 2012). However, a transgenic animal that is heterozygous for the p.P290R mutation (Slc1a P290R/+ ) demonstrated a rather distinct disease mechanism. The p.P290R knock-in animals exhibited Bergmann glia degeneration between P10 and P20, presumably because of enhanced Cl − efflux, cell shrinkage, and subsequent apoptosis . The lack of Bergmann glia results impairs glutamate re-uptake, modifies synaptic transmission in the cerebellum, and causes generalized cerebellar degeneration in a certain percentage of transgenic animals. Thus, p.P290R causes a dramatic reduction in cerebellar glutamate uptake, albeit primarily affecting EAAT1 anion channel activity.
We here studied the functional consequences of the remaining six published EA6 mutations using similar approaches we originally applied to p.P290R (Winter et al., 2012). We compared the effects of proximately twofold. We cannot conclude with certainty that hEAAT1 expression will be similarly increased in patients. However, such change in transporter biogenesis would correspond to anion current enhancements in Bergmann glia of heterozygous patients, which is roughly equivalent to the changes we observed in the knockin animal model Slc1a3 P290R/+ . The glutamate independent of p.V393I hEAAT1 anion currents in cells dialyzed with Na + -based solutions ( Figure 5) suggests that Na + -bound translocation is severely impaired, and such an alteration would also explain the reduced normalized glutamate transport (Figure 3d).
Moreover, normalized glutamate transport by p.V393I hEAAT1 ( Figure 3d) is lower than that for WT hEAAT1 and might contribute to reduced glutamate uptake in patients carrying the corresponding SLC1A3 mutation.
The consequences of p.C186S, p.A329T, and p.R499Q in patients carrying the causal SLC1A3 mutations are difficult to predict. The p.C186S mutations was already evaluated using radiotracer flux measurements in COS7 cells transfected with WT or mutant EAAT1 and found to decrease individual uptake rates by 18% (de Vries et al., 2009). In our experiments, we observed a slightly increased complex glycosylation ( Figure 2) and a decreased normalized glutamate uptake capability ( Figure 3). The remaining mutations, that is, The mutations analyzed in this study caused less pronounced changes in transport functions than those observed for p.P290R (Winter et al., 2012), and symptoms were milder than that for the p.P290R patient (Jen et al., 2005). However, among the remaining EA6 mutations we did not observe a clear correlation of the functional impairments of hEAAT1 in our experiments and the reported clinical symptoms. The p.M128R mutation had the most pronounced effects on hEAAT1 function, that is, loss-of-function of homotrimetric transporters and dominant negative effects in coexpression studies.
The p.M128R variant was described in a patient who suffered from episodes of truncal ataxia, strabismus, intentional tremor, and slurred speech (Iwama et al., 2017). Another mutation, p.T318A, had opposite effects on hEAAT1 functions. It enhanced hEAAT1 expression resulting in larger glutamate uptake and anion currents, but caused more severe symptoms, that is, ataxia, dizziness, dysarthria, gazeevoked nystagmus, rebound upbeat nystagmus, and cognitive impairment . Both, p.C186S and p.V393I caused only mild reductions in hEAAT1glutamate uptake and anion currents.
Patients with these mutations exhibited similar clinical symptoms (Choi, Jen, et al., 2017;de Vries et al., 2009), however, the disease onset was very late (in the sixth decade of life) for patients with p.V393I. The variants p.A329T and p.R499Q had the least severe effects on hEAAT1 function, and the p.A329T mutation was found in a patient with an additional CACNA1A mutation , which might also contribute to the disease phenotype.
For p.A329T ataxia, dizziness, and gaze-evoked nystagmus seizure were reported, whereas p.R499Q was only associated with speech disturbance and upper limb clumsiness. Episodic ataxia 6 is a rare disease, and only few EA6-associated SLC1A3 mutations have been reported, with small numbers of affected patients per family. In several cases, the family members who carry the same SLC1A3 mutations that was found in the EA6 patients do not suffer from episodic ataxia. We tested six missense mutations that were reported in patients with episodic ataxia in heterologous expressions systems. We found distinct alterations of hEAAT1 function and/or expression for each of the SLC1A3 mutations and demonstrated that episodic ataxia 6 can be associated with impaired as well as with enhanced hEAAT1 glutamate transport and/or anion current. The variants p.M128R and p.V393I caused changes in hEAAT1 transport and/or anion channel activity. The p.T381A mutation enhanced both hEAAT1 uptake and anion currents. For the remaining three, normal current amplitudes at increased expression levels suggested impaired glutamate uptake and anion currents. Taken together, genetic and functional data illustrated the importance of modifying genetic or environmental factors in determining the disease phenotype of SLC1A3-associated diseases. Our findings suggested that even small alterations can result in clinically significant alteration in cerebellar function, demonstrating how sensibly the cerebellum reacts on even slight alterations of hEAAT1 function.

ACKNOWLEDGMENTS
We are grateful to Yulia Kolobkova and Miriam Engels for helpful discussions, and also Arne Franzen and Petra Thelen for their excellent technical assistance. This study was supported by the German (E-RARE network Treat-ION, BMBF 01GM1907C to C. F.). Open access funding enabled and organized by Projekt DEAL.

CONFLICTS OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are openly available in GitHub at https://github.com/dkortzak/episodic_ataxia_6.