Down-Regulation of the Na⁺-Coupled Phosphate Transporter NaPi-IIa by AMP-Activated Protein Kinase

Abstract

Background/Aims: The Na⁺-coupled phosphate transporter NaPi-IIa is the main carrier accomplishing renal tubular phosphate reabsorption. It is driven by the electrochemical Na⁺ gradient across the apical cell membrane, which is maintained by Na⁺ extrusion across the basolateral cell membrane through the Na⁺/K⁺ ATPase. The operation of NaPi-IIa thus requires energy in order to avoid cellular Na⁺ accumulation and K⁺ loss with eventual decrease of cell membrane potential, Cl^- entry and cell swelling. Upon energy depletion, early inhibition of Na⁺-coupled transport processes may delay cell swelling and thus foster cell survival. Energy depletion is sensed by the AMP-activated protein kinase (AMPK), a serine/threonine kinase stimulating several cellular mechanisms increasing energy production and limiting energy utilization. The present study explored whether AMPK influences the activity of NAPi-IIa. Methods: cRNA encoding NAPi-IIa was injected into Xenopus oocytes with or without additional expression of wild-type AMPK (AMPK^α1-HA+AMPK^β1-Flag+AMPK^γ1-HA), of inactive AMPK^αK45R (AMPK^α1K45R+AMPK^β1-Flag+AMPK^γ1-HA) or of constitutively active AMPK^γR70Q (AMPK^α1-HA+AMPK^β1-Flag+AMPKγ1^R70Q). NaPi-IIa activity was estimated from phosphate-induced current in dual electrode voltage clamp experiments. Results: In NaPi-IIa-expressing, but not in water-injected Xenopus oocytes, the addition of phosphate (1 mM) to the extracellular bath solution generated a current (I_p), which was significantly decreased by coexpression of wild-type AMPK and of AMPK^γR70Q but not of AMPK^αK45R. The phosphate-induced current in NaPi-IIa- and AMPK-expressing Xenopus ooocytes was significantly increased by AMPK inhibitor Compound C (20 µM). Kinetic analysis revealed that AMPK significantly decreased the maximal transport rate. Conclusion: The AMP-activated protein kinase AMPK is a powerful regulator of NaPi-IIa and thus of renal tubular phosphate transport.

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

“…Xenopus oocytes were prepared as previously described [34,35]. cRNA (4.6 ng) encoding either AMPKα1-HA + AMPKβ1-Flag + AMPKγ1-HA (AMPK WT ), or AMPKα1-HA + AMPKβ1-Flag + R70Q AMPKγ1-HA (AMPK γR70Q ) or K45R AMPKα1KD-HA + AMPKβ1-Flag +AMPKγ1-HA (AMPK αK45R ) were injected on the first day and 23 ng of cRNA encoding Cx43-eGFP on the third day after preparation of the Xenopus oocytes [19,36].…”

Regulation of the Na⁺,Cl^- Coupled Creatine Transporter CreaT (SLC6A8) by the Janus Kinase JAK3

“…The control superfusate (ND96-B) contained 93.5 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 and 5 mM HEPES, pH was adjusted to 7.4 by addition of NaOH (2.5 mM) [61]. The flow rate of the superfusion was 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s [62,63]. …”

Regulation of ion channels and transporters by AMP-activated kinase (AMPK)

“…In contrast to its effect on SGLT1, AMPK downregulates a variety of Na + coupled transporters, such as the Na + coupled phosphate transporter NaPi-IIa, 120 the Na + coupled creatine transporter CreaT, 121 the Na + coupled myoinositol transporter SMIT, the NaCl coupled betaine,GABA transporter BGT 122 as well as the Na + coupled amino acid transporters EAAT3 and EAAT4. 123 Inhibition of those transporters decreases the Na + burden for the Na + /K + ATPase and thus energy consumption.…”