Frontiers in Physiology | |
Apo state pore opening as functional basis of increased EAAT anion channel activity in episodic ataxia 6 | |
Physiology | |
Daniel Kortzak1  Peter Kovermann1  Jan-Philipp Machtens1  Mariia Suslova1  Christoph Fahlke2  | |
[1] Institute of Biological Information Processing, Molekular-und Zellphysiologie (IBI-1), Forschungszentrum Jülich, Jülich, Germany;null; | |
关键词: neurological diseases; epilepsy; ataxia; glutamate transport; anion channel; kinetic modeling; | |
DOI : 10.3389/fphys.2023.1147216 | |
received in 2023-01-18, accepted in 2023-07-07, 发布年份 2023 | |
来源: Frontiers | |
【 摘 要 】
SLC1A2 and SLC1A3 encode the glial glutamate transporters EAAT2 and EAAT1, which are not only the predominant glutamate uptake carriers in our brain, but also function as anion channels. Two homologous mutations, which predict substitutions of prolines in the center of the fifth transmembrane helix by arginine (P289R EAAT2, P290R EAAT1), have been identified in patients with epileptic encephalopathy (SLC1A2) or with episodic ataxia type 6 (SLC1A3). Both mutations have been shown to impair glutamate uptake and to increase anion conduction. The molecular processes that link the disease-causing mutations to two major alterations of glutamate transporter function remain insufficiently understood. The mutated proline is conserved in every EAAT. Since the pathogenic changes mainly affect the anion channel function, we here study the functional consequences of the homologous P312R mutation in the neuronal glutamate transporter EAAT4, a low capacity glutamate transporter with predominant anion channel function. To assess the impact of charge and structure of the inserted amino acid for the observed functional changes, we generated and functionally evaluated not only P312R, but also substitutions of P312 with all other amino acids. However, only exchange of proline by arginine, lysine, histidine and asparagine were functionally tolerated. We compared WT, P312R and P312N EAAT4 using a combination of cellular electrophysiology, fast substrate application and kinetic modelling. We found that WT and mutant EAAT4 anion currents can be described with a 11-state model of the transport cycle, in which several states are connected to branching anion channel states to account for the EAAT anion channel function. Substitutions of P312 modify various transitions describing substrate binding/unbinding, translocation or anion channel opening. Most importantly, P312R generates a new anion conducting state that is accessible in the outward facing apo state and that is the main determinant of the increased anion conduction of EAAT transporters carrying this mutation. Our work provides a quantitative description how a naturally occurring mutation changes glutamate uptake and anion currents in two genetic diseases.
【 授权许可】
Unknown
Copyright © 2023 Suslova, Kortzak, Machtens, Kovermann and Fahlke.
【 预 览 】
Files | Size | Format | View |
---|---|---|---|
RO202310106438950ZK.pdf | 4461KB | download |