Mutation in glutamate transporter homologue GltTk provides insights into pathologic mechanism of episodic ataxia 6

Abstract

Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an arginine in the glutamate transporter EAAT1. In vitro studies showed that this mutation leads to a reduction in the substrates transport and an increase in the anion conductance. It was hypothesised that the structural basis of these opposed functional effects might be the straightening of transmembrane helix 5, which is kinked in the wild-type protein. In this study, we present the functional and structural implications of the mutation P208R in the archaeal homologue of glutamate transporters GltTk. We show that also in GltTk the P208R mutation leads to reduced aspartate transport activity and increased anion conductance, however a cryo-EM structure reveals that the kink is preserved. The arginine side chain of the mutant points towards the lipidic environment, where it may engage in interactions with the phospholipids, thereby potentially interfering with the transport cycle and contributing to stabilisation of an anion conducting state.

Molecular insights into disease-associated glutamate transporter (EAAT1 / SLC1A3) variants using in silico and in vitro approaches

“…This substantially reduces the affinity of UCPH-101 for this site, as observed by the loss of the original binding pose in the docking results (Figure 6G). Interestingly, other EAAT1 Pro mutations have been shown not to revert the kink, as opposed to original hypotheses (Colucci et al, 2023).…”

Apo state pore opening as functional basis of increased EAAT anion channel activity in episodic ataxia 6

“…Colucci et al recently reported the single-particle cryo-EM structure of the archaeal glutamate transporter homologue, Glt Tk , carrying the homologous P208R mutation ( Colucci et al, 2023 ). They found the structure of the fifth transmembrane helix preserved in mutant Glt Tk and the inserted arginine to project into the membrane.…”

Custom Design of a Humidifier Chamber for In Meso Crystallization

“…The protein was purified as described previously . Briefly, C-terminally 8-His-tagged Glt Tk in a pBAD24-derived plasmid was expressed in Escherichia coli MC1061.…”

“…The protein was purified as described previously. 14 Briefly, C-terminally 8-His-tagged Glt Tk in a pBAD24-derived plasmid was expressed in Escherichia coli MC1061. Cells were grown in LB medium with 100 μg/mL ampicillin at 37 °C and 200 rpm and induced with 0.05% L-arabinose for 3 h upon reaching 0.8 OD 600 .…”

“…To test our setup, we performed in meso crystallization trials with a glutamate transporter homologue Glt Tk , − which is widely used as a study model for investigations on solute carrier family 1 (SLC1) of transporters alongside another archaeal homologue Glt Ph . − Structurally, Glt Tk is a homotrimer, with every monomer consisting of a rigid scaffold domain that also serves as an oligomerization interface and an elevator domain, that undergoes conformational change during substrate transport. The transport domain of Glt Tk binds three sodium ions and a substrate aspartate molecule, occluding it by two helical hairpin elements, HP1 and HP2 .…”