Voltage-sensor activation with a tarantula toxin as cargo.
The opening and closing of voltage-activated Na+, Ca2+ and K+ (Kv) channels underlies electrical and chemical signalling throughout biology, yet the structural basis of voltage sensing is unknown. Hanatoxin is a tarantula toxin that inhibits Kv channels by binding to voltage-sensor paddles, crucial helix-turn-helix motifs within the voltage-sensing domains that are composed of S3b and S4 helices. The active surface of the toxin is amphipathic, and related toxins have been shown to partition into membranes, raising the possibility that the toxin is concentrated in the membrane and interacts only weakly and transiently with the voltage sensors. Here we examine the kinetics and state dependence of the toxin-channel interaction and the physical location of the toxin in the membrane. We find that hanatoxin forms a strong and stable complex with the voltage sensors, far outlasting fluctuations of the voltage sensors between resting (closed) conformations at negative voltages and activated (open) conformations at positive voltages. Toxin affinity is reduced by voltage-sensor activation, explaining why the toxin stabilizes the resting conformation. We also find that when hanatoxin partitions into membranes it is localized to an interfacial region, with Trp 30 positioned about 8.5 A from the centre of the bilayer. These results demonstrate that voltage-sensor paddles activate with a toxin as cargo, and suggest that the paddles traverse no more than the outer half of the bilayer during activation.
Phillips
L Revell
LR
Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, MSC 3701 Bethesda, Maryland 20892-3701, USA.
Milescu
Mirela
M
Li-Smerin
Yingying
Y
Mindell
Joseph A
JA
Kim
Jae Il
JI
Swartz
Kenton J
KJ
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D015640:Ion Channel Gating; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013111:Spider Venoms / Q000378:metabolism*; D013112:Spiders
D008051:Lipid Bilayers; D010455:Peptides; D024642:Potassium Channels, Voltage-Gated; D013111:Spider Venoms; C096469:hanatoxin