Regulation of the Na&lt;sup&gt;+&lt;/sup&gt;,Cl&lt;sup&gt;-&lt;/sup&gt; Coupled Creatine Transporter CreaT (SLC6A8) by the Janus Kinase JAK3

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Section: Discussionsupporting

confidence: 51%

Down-Regulation of Store-Operated Ca2+ Entry and Na+ Ca2+ Exchange in MCF-7 Breast Cancer Cells by Pharmacological JAK3 Inhibition

Yan¹,

Zhang²,

Hosseinzadeh³

et al. 2016

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“…Xenopus oocytes were prepared as previously described [30][31][32]. 2.5 ng cRNA encoding KCNA5 and 10 ng of cRNA encoding wild-type SGK3, constitutively active S419D SGK3 , inactive K191N SGK3 or wild type Nedd4-2 were injected on the same day after preparation of the oocytes.…”

Section: Voltage Clamp In Xenopus Oocytesmentioning

confidence: 99%

SGK3 Sensitivity of Voltage Gated K+ Channel Kv1.5 (KCNA5)

Ahmed

Fezai²,

Uzcátegui³

et al. 2016

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Background: The serum & glucocorticoid inducible kinase isoform SGK3 is a powerful regulator of several transporters, ion channels and the Na⁺/K⁺ ATPase. Targets of SGK3 include the ubiquitin ligase Nedd4-2, which is in turn a known regulator of the voltage gated K⁺ channel K_v1.5 (KCNA5). The present study thus explored whether SGK3 modifies the activity of the voltage gated K⁺ channel KCNA5, which participates in the regulation of diverse functions including atrial cardiac action potential, activity of vascular smooth muscle cells, insulin release and tumour cell proliferation. Methods: cRNA encoding KCNA5 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild-type SGK3, constitutively active ^S419DSGK3, inactive ^K191NSGK3 and/or wild type Nedd4-2. Voltage gated K⁺ channel activity was quantified utilizing dual electrode voltage clamp. Results: Voltage gated current in KCNA5 expressing Xenopus oocytes was significantly enhanced by wild-type SGK3 and ^S419DSGK3, but not by ^K191NSGK3. SGK3 was effective in the presence of ouabain (1 mM) and thus did not require Na⁺/K⁺ ATPase activity. Coexpression of Nedd4-2 decreased the voltage gated current in KCNA5 expressing Xenopus oocytes, an effect largely reversed by additional coexpression of SGK3. Conclusion: SGK3 is a positive regulator of KCNA5, which is at least partially effective by abrogating the effect of Nedd4-2.

“…For generation of cRNA [43,44], constructs were used encoding wild-type mTOR [45], EAAT1 [46] and EAAT2 [47][48][49]. The constructs were sequenced to verify the correctness of the plasmid [50].…”

Section: Constructsmentioning

confidence: 99%

Up-Regulation of the Excitatory Amino Acid Transporters EAAT1 and EAAT2 by Mammalian Target of Rapamycin

Abousaab¹,

Uzcátegui²,

Elsir³

et al. 2016

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Background:The excitatory amino-acid transporters EAAT1 and EAAT2 clear glutamate from the synaptic cleft and thus terminate neuronal excitation. The carriers are subject to regulation by various kinases. The EAAT3 isoform is regulated by mammalian target of rapamycin (mTOR). The present study thus explored whether mTOR influences transport by EAAT1 and/or EAAT2. Methods: cRNA encoding wild type EAAT1 (SLC1A3) or EAAT2 (SLC1A2) was injected into Xenopus oocytes without or with additional injection of cRNA encoding mTOR. Dual electrode voltage clamp was performed in order to determine electrogenic glutamate transport (I EAAT ). EAAT2 protein abundance was determined utilizing chemiluminescence. Results: Appreciable I EAAT was observed in EAAT1 or EAAT2 expressing but not in water injected oocytes. I EAAT was significantly increased by coexpression of mTOR. Coexpression of mTOR increased significantly the maximal I EAAT in EAAT1 or EAAT2 expressing oocytes, without significantly modifying affinity of the carriers. Moreover, coexpression of mTOR increased significantly EAAT2 protein abundance in the cell membrane. Conclusions: The kinase mTOR up-regulates the excitatory amino acid transporters EAAT1 and EAAT2.