The Mechanism by which ATP Binding Opens IKs Channels

Abstract

In cardiac myocytes KCNQ1 and KCNE1 form the IKs channel that is important in terminating action potentials. KCNQ1 has a common Kv channel membrane topology with the central pore domain (PD, S5-S6) and the voltage-sensing domain (VSD, S1-S4). We have demonstrated that ATP directly binds to the cytosolic C-terminus of KCNQ1 to regulate IKs channel function. Here we study if ATP regulates VSD movements or pore opening. using inside-out patch clamp techniques, we found that the steady state G-V relationship and current kinetics of the WT IKs channel are not changed by [ATP]. Consistent with these results, mutations in the putative ATP binding site reduce ATP sensitivity of the current amplitude but do not alter G-V relationship or activation kinetics. Furthermore, using voltage clamp fluorometry, we measured ionic current and VSD movements of the WT and mutant channels in which ATP binding is eliminated; and our results show that florescence signals remain the same, while the ionic current is completely absent in the mutant channels due to the lack of ATP binding. These results suggest that ATP does not regulate VSD movements but is required for pore opening by directly pulling the activation gate to open from the binding site located downstream from S6. Supporting this mechanism, a mutation located between S6 and the putative ATP binding site is found to alter the coupling between ATP binding and channel opening by reducing ATP sensitivity and shifting the G-V relationship to more positive voltages at a given [ATP]; however, increasing [ATP] shifts the G-V back toward negative voltages. The effects of this mutation can be explained by an allosteric model for the coupling between ATP binding and channel opening in which the mutation reduces the allosteric factor for the coupling.