Abstract
We have recently identified a novel 63 amino acid residue three-finger toxin (called Tx7335) from eastern green mamba snake (Dendroaspis angusticeps) which interacts with KcsA and induces an increase in frequency and duration of individual channel openings when added to the outside of the channel. The toxin exerts this activating effect both on wild-type KcsA as well as on an agitoxin2-sensitive mutant form of the channel, indicating a mode of action and binding site that are different from the classic pore-blocker toxins. We are currently using NMR spectroscopy to unravel the structural underpinnings of this mechanism of action. High yield of purified 15N labeled KcsA and excellent NMR spectral quality have been achieved. Currently the characterization of toxin binding using 1H15N correlation spectra of 15N labeled KcsA in the absence and presence of toxin is ongoing. Experiments are conducted in different membrane mimetics including DMPC/DHPC bicelles and DPC or DM micelles at different pH, temperature and salt concentration. Some chemical shifts and peak intensity changes upon toxin addition have been observed. Continuing NMR structural studies will further elucidate the mechanism of how Tx7335 interacts with KcsA and shed light on the conformational and dynamic changes of C-type inactivation in KcsA and on a novel mechanism of ion channel regulation.
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