The SGK3-dependent trafficking of GluA1 proteins requires functional RAB11 and the generation of PI(3,5)P₂, but is myosin-independent.

<p>(<b>A</b>) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P₂. Significant change (p<0.05) is indicated by * (p = 0.0015). (<b>B</b>) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P₂. Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). (<b>C</b>) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.</p

PIKfyve expression, phosphorylation, and function.

<p>(<b>A</b>) PIKfyve is phosphorylated at Ser318 by SGK3 and at Ser318 by PKB. Purified recombinant GST-tagged wild-type (WT) or S318A mutant (SA) of PIKfyve was subjected to Western blotting. Blots were incubated with rabbit anti-GST antibody (PIKfyve; lower panel), followed by stripping and reprobing with a rabbit anti-PIKfyve antibody specific for phosphoserine 318 (apS318; top panel). (<b>B</b>) RT-PCR demonstrating that PIKfyve is expressed in hippocampus. Lane 1: cDNA from hippocampus, the two primers bind in exons 19 and 20, respectively, and amplify a 315 bp fragment of PIKfyve; lane 2: control reaction to exclude genomic contamination; lane 3: control reaction without reverse transcriptase; lane 4: control reaction without cDNA. (<b>C</b>) The PIKfyve inhibitor YM201636 and SGK inhibitor EMD638683 suppress the upregulating effect of SGK3 on GluA1 currents. GluA1 current amplitudes in oocytes expressing GluA1. Acute injection of purified active SGK3 protein led to an increase in GluA1 currents. The effects of YM201636 and EMD638683 on GluA1 currents were measured in oocytes before and after acute injection of SGK3 protein. Significant change to GluA1 control (p = 0.013) is indicated by *. Numbers of oocytes were n = 7–36.</p

GluA1 function is increased by SGK3 and PIKfyve.

<p>(<b>A</b>) Representative current traces measured in <i>Xenopus</i> oocytes in response to superfusion with 300 µM glutamate plus 100 µM cyclo-thiacide. All currents were measured at −70 mV. Vertical scale-bar, 0,5 µA; horizontal bar, 4 s. (<b>B</b>) GluA1 current amplitudes in oocytes expressing GluA1, or combinations of GluA1 with SGK3, the inactive form of PIKfyve (PIKfyve(S318A)), or wild type PIKfyve. Numbers of oocytes are n = 20–30. Significant changes (p<0.001) are indicated by *** (p = 0.00049; 0.00036; 0.000073, respectively). (<b>C</b>) Representative samples including controls from uninjected oocytes were biotinylated to isolate plasma membrane GluA1, then separated on an SDS gel, Western-blotted and probed with a primary rabbit anti-GluA1 antibody. The GluA1 protein has an apparent molecular weight of ∼105 kDa. (<b>D</b>) Bar graph showing relative abundance of GluA1 plasma membrane protein. The band intensity was quantified by densitometric analysis using the software Scion image.</p

Regulation of GluA1 by SGK3 and PIKfyve is Rab11-dependent.

<p>(<b>A</b>) Representative current traces measured in <i>Xenopus</i> oocytes in response to superfusion with 300 µM glutamate plus 100 µM cyclo-thiacide. All currents were measured at −70 mV. Vertical scale-bar, 0,5 µA; horizontal bar, 4 s. (<b>B</b>) GluA1 current amplitudes in oocytes expressing GluA1, or combinations of GluA1 with Rab11, the dominant negative form of Rab11 (Rab11DN), PIKfyve, or SGK3. Numbers of oocytes are n = 15–30. Significant changes (p<0.001, p<0.05) are indicated by *** (p = 0.00015) and * (p = 0.036), respectively. (<b>C</b>) Representative samples including controls from uninjected oocytes were biotinylated to isolate plasma membrane GluA1, then separated on an SDS gel, Western-blotted and probed with a primary rabbit anti-GluA1 antibody. The GluA1 protein has an apparent molecular weight of ∼105 kDa. (<b>D</b>) Bar graph showing relative abundance of GluA1 plasma membrane protein. The band intensity was quantified by densitometric analysis using the software Scion image.</p

SGK3 mRNA expression in hippocampus after pharmacological NMDA receptor stimulation.

<p>mRNA levels of SGK3 before and after incubation of hippocampal slices with 10 µM NMDA for 30 minutes, determined by quantitative RT-PCR. The expression data from hippocampus were normalized to the expression of the housekeeping gene β-actin. The quantitative RT-PCR revealed a 2.5±0,52 fold increase in SGK3 mRNA in NMDA-treated compared to non-treated hippocampal slices. Number of experiments n = 4.</p

PIKfyve inhibition results in depression of hippocampal basal synaptic transmission.

<p>(<b>A</b>) Basal synaptic transmission was recorded in the CA1 region of hippocampal slices. Control (non-treated) hippocampi, or hippocampi treated with DMSO showed stable evoked responses throughout the 210 min recording period. Treatment with the PIKfyve inhibitor (YM201636) elicited a depression of evoked responses that became evident 40 min after drug application. Line-breaks indicate changes in time-scale. Black bar indicates DMSO or drug application. (<b>B</b>) Analog traces representing evoked potentials obtained in control slices or in slices in the presence of DMSO or YM201636, at the time-points indicated in the graph. Vertical scale-bar, 3 mV; horizontal bar, 3 ms.</p

Postulated model of SGK3-dependent regulation of GluA1.

<p>Upon stimulation of NMDA receptors SGK3 is transcriptionally upregulated. SGK3 in turn phosphorylates/activates PIKfyve, which leads to local production of PI(3,5)P₂ in PIKfyve-containing recycling vesicles. PI(3,5)P₂ in turn stimulates Rab11-dependent plasma membrane-directed trafficking of GluA1-containing vesicles.</p