Expression of Kv1.2 within Kv1.x potassium channel complexes is critical in maintaining appropriate neuronal excitability and determining the threshold for action potential firing. This is attributed to the interaction of Kv1.2 with a hitherto unidentified protein that confers bimodal channel activation gating, allowing neurons to adapt to repetitive trains of stimulation and protecting against hyperexcitability. One potential protein candidate is the sigma‐1 receptor (Sig‐1R), which regulates other members of the Kv1.x channel family; however, the biophysical nature of the interaction between Sig‐1R and Kv1.2 has not been elucidated. We hypothesized that Sig‐1R may regulate Kv1.2 and may further act as the unidentified modulator of Kv1.2 activation. In transiently transfected HEK 293 cells, we found that ligand activation of the Sig‐1R modulates Kv1.2 current amplitude. More importantly, Sig‐1R interacts with Kv1.2 in baseline conditions to influence bimodal activation gating. These effects are abolished in the presence of the auxiliary subunit Kv β 2 and when the Sig‐1R mutation underlying ALS 16 (Sig‐1R‐E102Q), is expressed. These data suggest that Kv β 2 occludes the interaction of Sig‐1R with Kv1.2, and that E102 may be a residue critical for Sig‐1R modulation of Kv1.2. The results of this investigation describe an important new role for Sig‐1R in the regulation of neuronal excitability and introduce a novel mechanism of pathophysiology in Sig‐1R dysfunction.

Summary Ischemic stroke is the second leading cause of death worldwide. Following an ischemic event, neuronal death is triggered by uncontrolled glutamate release leading to overactivation of glutamate sensitive N -methyl- d -aspartate receptor (NMDAR). For gating, NMDARs require not only the binding of glutamate, but also of glycine or a glycine-like compound as a co-agonist. Low glycine doses enhance NMDAR function, whereas high doses trigger glycine-induced NMDAR internalization (GINI) in vitro . Here, we report that following an ischemic event, in vivo , GINI also occurs and provides neuroprotection in the presence of a GlyT1 antagonist (GlyT1-A). Mice pretreated with a GlyT1-A, which increases synaptic glycine levels, exhibited smaller stroke volume, reduced cell death, and minimized behavioral deficits following stroke induction by either photothrombosis or endothelin-1. Moreover, we show evidence that in ischemic conditions, GlyT1-As preserve the vasculature in the peri-infarct area. Therefore, GlyT1 could be a new target for the treatment of ischemic stroke.

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Glycine fulfills several roles in biology including protein synthesis, inhibitory transmission via glycine receptor activation and excitatory transmission through glutamate-sensitive N-methyl-D-aspartate receptors (NMDARs). Low glycine doses enhance NMDAR function while high doses trigger glycine-induced NMDAR internalization (GINI) in vitro. The physiological relevance of GINI has been questioned given that the high-affinity glycine transporter type 1 (GlyT1), located on astrocytes and neurons, maintains synaptic glycine concentrations far below the level that would saturate the glycine binding site (GBS) on NMDARs. Here, we report evidence that GINI occurs also in vivo and is neuroprotective following ischemic insult. Mice pre-treated with a GlyT1 antagonist (GlyT1-A), which increased glycine levels, exhibited smaller stroke volume, reduced cell death, and minimized behavioural deficits following stroke induction by either photothrombosis or endothelin-1. We demonstrate that in a modified in vitro ischemic paradigm, glycine is released at levels surpassing what occurs during ischemia alone. Therefore, glycine accumulates in the synaptic cleft, enhances occupancy of GBS and reaches the set point to trigger GINI. We report that GINI is observed during stroke, in vivo, only in the presence of a GlyT1-A. Moreover, we show evidence of a protective effect on the vasculature in the peri-infarct area. Therefore, these data strongly suggest that GlyT1 is a therapeutic target to prevent cell death following an ischemic event.