SPAK Sensitive Regulation of the Epithelial Na&lt;sup&gt;+&lt;/sup&gt; Channel ENaC

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Background/Aims: β-Klotho, a transmembrane protein expressed in several tissues including the brain and the kidney, is critically important for inhibition of 1,25(OH)₂D₃ formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. Soluble klotho is a β-glucuronidase participating in the regulation of several ion channels and carriers. The present study explored the effect of β-Klotho protein on the peptide transporters PEPT1 and PEPT2. Methods: cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes and glycine-glycine (2 mM)-induced inward current (I_Gly) taken as measure of glycine-glycine transport. Measurements were made without or with prior 24 h treatment with soluble β-Klotho protein (30 ng/ml) in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Ussing chamber experiments were employed to determine electrogenic peptide transport across intestinal epithelia of klotho deficient (kl^-/-) and corresponding wild type (kl^+/+) mice. Results: I_Gly was observed in PEPT1 and in PEPT2 expressing oocytes but not in water injected oocytes. In both, PEPT1 and PEPT2 expressing oocytes I_Gly was significantly decreased by treatment with soluble β-Klotho protein. As shown for PEPT1, β-klotho protein decreased significantly the maximal transport rate without significantly modifying the affinity of the carrier. The effect of β-Klotho on PEPT1 was reversed by DSAL. Intestinal I_Gly was significantly larger in kl^-/- than in kl^+/+ mice. Conclusion: β-Klotho participates in the regulation of the peptide transporters PEPT1 and PEPT2.

Section: Ussing Chamber Experimentsmentioning

confidence: 99%

β-Klotho as a Negative Regulator of the Peptide Transporters PEPT1 and PEPT2

Abousaab

Warsi²,

Salker³

et al. 2016

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“…Normalized test-pulse peak currents were plotted versus pre-pulse voltage and fitted to the Boltzmann equation yielding V 1/2 for channel activation and inactivation. The oocytes were maintained at 17°C in ND96A, a storage solution containing (in mM): 88.5 NaCl, 2 KCl, 1 MgC1 2 , 1.8 CaC1 2 , 5 HEPES (pH 7.5), 5 sodium pyruvate (C 3 H 3 NaO 3 ), Gentamycin (100 mg/l), Tetracycline (50 mg/l), Ciprofloxacin (1.6 mg/l), Theophylline (90 mg/l) [33][34][35][36]. The voltage clamp experiments were performed at room temperature 3 days after the first injection [37][38][39].…”

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.

“…The final solutions were titrated to pH 7.4 using NaOH. The voltage clamp experiments [57][58][59] were performed at room temperature 6 days after injection. Two-electrode voltage-clamp recordings were performed at a holding potential of -70 mV.…”

Section: Voltage Clamp In Xenopus Oocytesmentioning

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.