Downregulation of the osmolyte transporters SMIT and BGT1 by AMP-activated protein kinase

Abstract

The myoinositol transporter SMIT (SLC5A3) and the betaine/γ-aminobutyric acid (GABA) transporter BGT1 (SLC6A12) accomplish cellular accumulation of organic osmolytes and thus contribute to cell volume regulation. Challenges of cell volume constancy include energy depletion, which compromises the function of the Na(+)/K(+) ATPase leading to cellular Na(+) accumulation and subsequent cell swelling. Energy depletion is sensed by AMP-activated protein kinase (AMPK). The present study explored whether AMPK influences the activity of SMIT and BGT1. To this end, cRNA encoding SMIT or BGT1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1+AMPKβ1+AMPKγ1), of constitutively active (γR70Q)AMPK (AMPKα1+AMPKβ1+(R70Q)AMPKγ1) or of catalytically inactive (αK45R)AMPK ((K45R)AMPKα1+AMPKβ1+AMPKγ1). Substrate-induced current in dual electrode voltage-clamp experiments was taken as measure of osmolyte transport. As a result, in SMIT-expressing, but not in water-injected Xenopus oocytes, myoinositol, added to the extracellular bath, generated a current (I(SMIT)), which was half maximal (K(M)) at ≈7.2μM myoinositol concentration. Furthermore, in BGT1-expressing, but not in water-injected Xenopus oocytes, GABA added to the bath generated a current (I(GABA)), which was half maximal (K(M)) at ≈0.5mM GABA concentration. Coexpression of AMPK and of (γR70Q)AMPK but not of (αK45R)AMPK significantly decreased I(SMIT) and I(GABA). AMPK decreased the respective maximal currents without significantly modifying the respective K(M). In conclusion, the AMP-activated kinase AMPK is a powerful regulator of the organic osmolyte transporters SMIT and BGT1 and thus interacts with cell volume regulation.