MECHANISM OF MAXI-K CHANNEL ACTIVATION BY DEHYDROSOYASAPONIN-I

Dehydrosoyasaponin-I (DHS-I) is a potent activator of high-conductance , calcium-activated potassium (maxi-K) channels. Interaction of DHS-I with maxi-K channels from bovine aortic smooth muscle was studied afte r incorporating single channels into planar lipid bilayers. Nanomolar amounts of intracellular DHS-I caused the appearance of discrete episo des of high channel open probability interrupted by periods of apparen tly normal activity. Statistical analysis of these periods revealed tw o clearly separable gating modes that likely reflect binding and unbin ding of DHS-I. Kinetic analysis of durations of DHS-I-modified modes s uggested DHS-I activates maxi-K channels through a high-order reaction . Average durations of DHS-I-modified modes increased with DHS-I conce ntration, and distributions of these mode durations contained two or m ore exponential components. In addition, dose-dependent increases in c hannel open probability from low initial values were high order with a verage Hill slopes of 2.4-2.9 under different conditions, suggesting a t least three to four DHS-I molecules bind to maximally activate the c hannel. Changes in membrane potential over a 60-mV range appeared to h ave little effect on DHS-I binding. DHS-I modified calcium- and voltag e-dependent channel gating. 100 nM DHS-I caused a threefold decrease i n concentration of calcium required to half maximally open channels. D HS-I shifted the midpoint voltage for channel opening to more hyperpol arized potentials with a maximum shift of -105 mV. 100 nM DHS-I had a larger effect on voltage-dependent compared with calcium-dependent cha nnel gating, suggesting DHS-I may differentiate these gating mechanism s. A model specifying four identical, noninteracting binding sites, wh ere DHS-I binds to open conformations with 10-20-fold higher affinity than to closed conformations, explained changes in voltage-dependent g ating and DHS-I-induced modes. This model of channel activation by DHS -I may provide a framework for understanding protein structures underl ying maxi-K channel gating, and may provide a basis for understanding ligand activation of other ion channels.