The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK is a powerful regulator of ion transport. The study explored whether SPAK similarly regulates nutrient transporters, such as the Na+-coupled glucose transporter SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of wild-type SPAK, constitutively active T233ESPAK, WNK-insensitive T233ASPAK or catalytically inactive D212ASPAK, and electrogenic glucose transport determined by dual-electrode voltage-clamp experiments. Moreover, Ussing chamber was employed to determine the electrogenic glucose transport in intestine from wild-type mice (spak wt/wt) and from gene-targeted mice carrying WNK-insensitive SPAK (spak tg/tg). In SGLT1-expressing oocytes, but not in water-injected oocytes, the glucose-dependent current (I g) was significantly decreased following coexpression of wild-type SPAK and T233ESPAK, but not by coexpression of T233ASPAK or D212ASPAK. Kinetic analysis revealed that SPAK decreased maximal I g without significantly modifying the glucose concentration required for halfmaximal I g (K m). According to the chemiluminescence experiments, wild-type SPAK but not D212ASPAK decreased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I g, which was similar in the absence and presence of SPAK, suggesting that SPAK did not accelerate the retrieval of SGLT1 protein from the cell membrane but rather down-regulated carrier insertion into the cell membrane. Intestinal electrogenic glucose transport was significantly lower in spak wt/wt than in spak tg/tg mice. In conclusion, SPAK is a powerful negative regulator of SGLT1 protein abundance in the cell membrane and thus of electrogenic glucose transport.
The Journal of Membrane Biology – Springer Journals
Published: Aug 27, 2014
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