Anomalous Calcium Dependent Binding of Calmodulin to KCNQ2 Potassium Channels

Abstract

Often, the free concentration of the ubiquitous Ca2+ sensor calmodulin (CaM) is limiting, probably below 50 nM in excitable cells, because most of it is bound to targets inside the cell. However, it is generally assumed that the binding characteristics do not depend on CaM concentration, and it has been generally studied at much larger concentrations. We show here that this assumption does not hold true. We have found that Ca2+ favors or reduces the association between KCNQ2 potassium channels and CaM depending on the free CaM concentration, shifting abruptly between negative and positive cooperativity when the concentration of CaM is below or above ∼26 nM, respectively. With CaM concentrations below 26 nM there was a ∼four-fold decrease in the binding affinity in the presence of Ca2+, shifting the Kd from ∼4.7 to ∼22 nM. In contrast, with CaM concentrations above 26 nM there was a ∼two-fold increase in the binding affinity in the presence of Ca2+, shifting the Kd from ∼54 nM to ∼22 nM. In addition, when 12.5 nM CaM was used in the assay, the EC50 for Ca2+ binding changed from ∼302 nM to ∼608 nM or ∼645 nM in the presence of molar excess of the KCNQ2 or KCNQ3 CaM binding sites, respectively. In contrast, using 100 nM CaM in the assay, the affinity for Ca2+ binding increased slightly in the presence of molar excess of the KCNQ2 binding protein, lowering the EC50 to ∼287 nM. The anomalous CaM-dependent behavior of CaM adds complexity to the integration of calcium signaling inside the cell.