SPAK Dependent Regulation of Peptide Transporters PEPT1 and PEPT2

Background/Aims: Adenosine 5'-monophosphate (AMP)-activated protein kinase (Ampk) modulates a wide array of cellular functions and regulates various ion channels and transporters. In failing human hearts an increased Ampkα1 activity was observed. The present study aimed to uncover the impact of Ampkα1 on cardiac electrical remodeling. Methods: Gene-targeted mice lacking functional Ampkα1 (Ampkα1-/-) and corresponding wild-type mice were exposed to pressure overload by “transverse aortic constriction” (TAC). In vivo electrophysiology was performed with a single catheter technique, myocardial conduction velocities and conduction characteristics investigated in isolated hearts, transcript levels quantified by RT-PCR and protein abundance determined by Western blotting. Moreover, connexin 43 (Cx43) was expressed in Xenopus oocytes with or without coexpression of wild-type or mutant AMPK and Cx43 protein abundance quantified utilizing confocal microscopy. Results: TAC treatment increased Ampkα1 protein expression in cardiac tissue from wild-type mice. TAC further increased left ventricular conduction inhomogeneity and triggered conduction blocks, effects blunted in the Ampkα1^-/- mice. TAC treatment decreased Cx43 protein abundance in cardiac tissue, an effect significantly blunted in the Ampkα1^-/- mice. TAC treatment did not modify Cx43 mRNA levels but increased ubiquitination of Cx43 protein, an effect mitigated by Ampkα1 deficiency. As shown in Xenopus oocytes, Cx43 cell membrane protein abundance was significantly downregulated by wild-type AMPK^WT and constitutively active AMPK^γR70Q, but not by catalytically inactive AMPK^αK45R. Conclusion: Ampkα1 stimulates ubiquitination of the gap junction protein Cx43, thereby contributing to gap junction remodeling following pressure overload.

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“…The eGFP-tagged Cx43 construct was sequenced to confirm the insert and tag in frame. cRNA was synthesized as described previously [32,33]. …”

Section: Methodsmentioning

confidence: 99%

AMP-Activated Protein Kinase α1 Regulates Cardiac Gap Junction Protein Connexin 43 and Electrical Remodeling Following Pressure Overload

Alesutan

Voelkl²,

Stöckigt³

et al. 2015

Cell Physiol Biochem

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“…The flow rate of the superfusion was approx. 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s [81,82,83]. For kinetic analysis the glutamate induced-current (I Glu ) was plotted against the respective glutamate concentration ( s ) and maximal current (I max ) as well as concentration required for halfmaximal current ( k m ) calculated using the equation I Glu = I max ∙s/(k m + s).…”

Section: Methodsmentioning

confidence: 99%

Up-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by ß-Klotho

Warsi

Abousaab²,

Läng

2015

Neurosignals

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Background/Aims: Klotho, a transmembrane protein expressed in chorioid plexus of the brain, kidney, and several other tissues, is required 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. At least in part by exerting β-glucuronidase activity, soluble klotho regulates several ion channels and carriers. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The present study explored the effect of Klotho protein on the excitatory glutamate transporters EAAT1 (SLC1A3) and EAAT2 (SLC1A2), Na⁺ coupled carriers clearing excitatory amino acids from the synaptic cleft and thus participating in the regulation of neuronal excitability. Methods: cRNA encoding EAAT1 or EAAT2 was injected into Xenopus laevis oocytes and glutamate (2 mM)-induced inward current (I_Glu) taken as measure of glutamate transport. Measurements were made without or with prior 24 h treatment with soluble ß-Klotho protein (30 ng/ml) in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Results: I_Glu was observed in EAAT1 and in EAAT2 expressing oocytes but not in water injected oocytes. In both, EAAT1 and EAAT2 expressing oocytes I_Glu was significantly increased by treatment with soluble ß-Klotho protein, an effect reversed by DSAL. Treatment with ß-klotho protein increased significantly the maximal transport rate without significantly modifying the affinity of the carriers. Conclusion: ß-Klotho up-regulates the excitatory glutamate transporters EAAT1 and EAAT2 and thus participates in the regulation of neuronal excitation.

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“…Xenopus oocytes were prepared as previously described [41,42]. cRNA encoding K v1.3 (2.5 ng) was injected on the same or the second day of Xenopus oocytes preparation [43,44].…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Voltage Gated K<sup>+</sup> Channel K<sub>v1.3</sub> by Recombinant Human Klotho Protein

Almilaji

Honisch

Liu

et al. 2014

Kidney Blood Press Res

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5,279

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Background/Aims: Klotho, a protein mainly produced in the kidney and released into circulating blood, contributes to the negative regulation of 1,25(OH)₂D₃ formation and is thus a powerful regulator of mineral metabolism. As β-glucuronidase, alpha Klotho protein further regulates the stability of several carriers and channels in the plasma membrane and thus regulates channel and transporter activity. Accordingly, alpha Klotho protein participates in the regulation of diverse functions seemingly unrelated to mineral metabolism including lymphocyte function. The present study explored the impact of alpha Klotho protein on the voltage gated K⁺ channel K_v1.3. Methods: cRNA encoding K_v1.3 (KCNA3) was injected into Xenopus oocytes and depolarization induced outward current in K_v1.3 expressing Xenopus oocytes determined utilizing dual electrode voltage clamp. Experiments were performed without or with prior treatment with recombinant human Klotho protein (50 ng/ml, 24 hours) in the absence or presence of a β-glucuronidase inhibitor D-saccharic acid-1,4-lactone (DSAL, 10 µM). Moreover, the voltage gated K⁺ current was determined in Jcam lymphoma cells by whole cell patch clamp following 24 hours incubation without or with recombinant human Klotho protein (50 ng/ml, 24 hours). K_v1.3 protein abundance in Jcam cells was determined utilising fluorescent antibodies in flow cytometry. Results: In K_v1.3 expressing Xenopus oocytes the K_v1.3 currents and the protein abundance of K_v1.3 were both significantly enhanced after treatment with recombinant human Klotho protein (50 ng/ml, 24 hours), an effect reversed by presence of DSAL. Moreover, treatment with recombinant human Klotho protein increased K_v currents and K_v1.3 protein abundance in Jcam cells. Conclusion: Alpha Klotho protein enhances K_v1.3 channel abundance and K_v1.3 currents in the plasma membrane, an effect depending on its β-glucuronidase activity.

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SPAK Dependent Regulation of Peptide Transporters PEPT1 and PEPT2

Cited by 26 publications

References 58 publications

AMP-Activated Protein Kinase α1 Regulates Cardiac Gap Junction Protein Connexin 43 and Electrical Remodeling Following Pressure Overload

AMP-Activated Protein Kinase α1 Regulates Cardiac Gap Junction Protein Connexin 43 and Electrical Remodeling Following Pressure Overload

Up-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by ß-Klotho

Regulation of the Voltage Gated K<sup>+</sup> Channel K<sub>v1.3</sub> by Recombinant Human Klotho Protein

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