REVERSIBLE PROTEIN-PHOSPHORYLATION REGULATES THE ACTIVITY OF THE SLOW-VACUOLAR ION-CHANNEL

Protein storage vacuoles (PSVs) within barley (Hordeum vulgare) aleuro ne cells contain abundant K, Ca, Mg and P reserves. These minerals are transported from the PSV and are used to support growth of the embryo . In this study, the regulation of transport through slow-vacuolar (SV ) ion channels in the tonoplast of barley aleurone PSVs was examined u sing the patch-clamp technique. Okadaic acid (OA), an inhibitor of pro tein phosphatase types 1 and 2A, reduced whole-vacuole SV currents by 60%. This inhibition by OA was overcome by exogenous calcineurin. Addi ng ATP (200 mu M) to the bath solution as a substrate for kinase activ ity decreased SV channel activity by 70%. This reduction in activity w as prevented by the kinase inhibitor H-7. From these data, it is concl uded that protein phosphorylation can inhibit SV channel activity, and that both the protein kinase and protein phosphatase involved in this regulation are present at the PSV tonoplast. Whole-vacuolar SV curren ts were significantly higher when 2 mM ATP was used to bathe PSVs than with 200 mu M ATP Calmodulin-like domain protein kinase (CDPK) at eit her ATP concentration increased SV channel activity by similar to 150% , implying that protein phosphorylation can also stimulate SV channel activity. When PSVs were treated with the ATP analog AMP-PNP SV channe l activity was not reduced. Hence, ATP hydrolysis is not essential for sustained SV channel activity. A model in which SV channel activity i s regulated by protein phosphorylation at two sites is presented.