Fig. 1.

Effects of A-769662 on Kv2.1 function in HEK293 cells and phosphorylation of Kv2.1 by AMPK in cell-free assays. (A) HEK293 cells stably expressing rat Kv2.1 (8) were incubated with A-769662 (100 μM) ± compound C (40 μM) for 20 min. Activation is indicated by filled symbols and continuous lines; inactivation is indicated by open symbols and dashed lines. Data points are mean ± SEM (n = 5–15). Curves were obtained by fitting to the sigmoidal Boltzmann equation. (B) Phosphorylation of Kv2.1 by AMPK in cell-free assays. Proteins were immunoprecipitated from HEK293 cells stably expressing rat Kv2.1 using anti-Kv2.1 or control Ig (IgG), incubated with [γ-³²P]ATP ± purified AMPK, and SDS gels were analyzed by protein staining (Upper) or autoradiography (Lower). (C) Alignment of the recognition motif for AMPK with sequences around sites on ACC1, ACC2, HMG-CoA reductase (HMGR), and Kv2.1. Basic and hydrophobic residues involved in recognition by AMPK are marked by bold type and/or underlining.