Negative regulation of the creatine transporter SLC6A8 by SPAK and OSR1.

ABSTRACT

BACKGROUND/AIMS: Transport regulation involves several kinases including SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether SPAK and/or OSR1 participate in the regulation of the creatine transporter CreaT (SLC6A8), which accomplishes Na+ coupled cellular uptake of creatine in several tissues including kidney, intestine, heart, skeletal muscle and brain. METHODS: cRNA encoding SLC6A8 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 and catalytically inactive (D164A)OSR1. Transporter activity was determined from creatine (1 mM) induced current utilizing dual electrode voltage clamp. RESULTS: Coexpression of wild-type SPAK and of (T233E)SPAK, but not of (T233A)SPAK or of (D212A)SPAK was followed by a significant decrease of creatine induced current in SLC6A8 expressing oocytes. Coexpression of SPAK significantly decreased maximal transport rate. Coexpression of wild-type OSR1, (T185E)OSR1 and (T185A)OSR1 but not of (D164A)OSR1 significantly negatively regulated SLC6A8 activity. OSR1 again decreased significantly maximal transport rate. CONCLUSIONS: Both, SPAK and OSR1, are negative regulators of the creatine transporter SLC6A8.