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.