Kinetic study of interactions between voltage-gated K+-channels and toxins from spider venom
Date Issued
2005
Date
2005
Author(s)
Shih, Yu-Wen
DOI
zh-TW
Abstract
Voltage-gated potassium channels are found in a wide variety of tissues, where their primary role is to respond to the membrane excitation to allow the repolarization phase of an action potential to occur and therefore the K+ ions can efflux. Such channels are normally homotetramers and each subunit contains four voltage-sensing transmembrane segments, namely S1 through S4, whereas S5 and S6 form the pore. Among them, S4 may play the most crucial role in sensing the voltage change. Kv2.1, a member of shab potassium channel family, is one the most commonly applied channels in study of the structural-functional correlation for voltage sensing. Upon binding of hanatoxin, the midpoint of the curve for required gating potential of Kv2.1 can be shifted to the right, which means more difficult to open the channels under the same condition. On the contrary, shaker potassium channels do not show such effect. The secondary structural arrangement of S3C has been, due to such studies, intensively analyzed and the existence of an independent α-helix was then suggested.
It has been accomplished to establish a model and hypothesis describing the molecular details of hanatoxin-binding induced gating. This was based on the 3-D structural analysis with molecular docking and simulations. Recently, research by Mackinnon’s group has led to certain controversial developments in the voltage-sensing theory of Kv channels. Crystal structure of KvAP channel from archaeum Aeropyrum pernix revealed, combined with a series of electrophysiological experiments and sequence comparisons, a novel ‘voltage-sensor paddle’ model, which was anticipated to be applied on eukaryotic Kv channels. However, such revolutionary idea did challenge the conventional “translocation” model and contradict to a few previously acknowledged experimental results. Such discovery has also brought gross impact on our proposed mechanism which was based upon the conventional translocation model in Kv channels.
Therefore, in this thesis, we have performed the kinetic analysis with stopped-flow to examine our previously proposed hypothesis. Such binding study, in combination with related calculations, provides further possibility to consider in a more decent way the discussion of the reasonable conformation and membrane distribution of S3C segment in the toxin-Kv channel interactions.
The binding rate constant kon and release rate constant koff for interactions between hanatoxin/stromatoxin and Kv2.1 S3C segments can be calculated through the kinetic analysis with stopped-flow. Upon utilization of Kv2.1 S3C mutants and Kv1.1 S3C as control experiments, together with the appropriate additions of trifluoroethanol in different concentrations, which allow the refolding of Kv2.1 S3C to occur, it has been indicated that binding of hanatoxin and Kv2.1 S3C may follow the molecular details described in our proposed mechanism. However, the comparison between the hydrophobic and hydrophilic interactions required for binding between hanatoxin and Kv2.1 S3C observed from rationally designed mutants (hydrophobic part v.s. polar part of residues) suggests that both types of interaction are equally crucial for binding. Mutation of either part of residues can result in the abolishment of binding ability for hanatoxin and Kv2.1 S3C. Polar interactions should not be the only dominant factor able to affect such binding as predicted thru simulation study. All together, it is reasonable to comprehend that the S3C residues required for binding with hanatoxin should be located at the boundary of cell membrane, nearby the hydrophilic heads of phospholipids (or interfacial area of external face) with a slight tilting angle. Therefore the conventional ‘translocation’ model may fulfill such requirement better, especially considering the spatial orientations of transmembrane segments around the external crevice. On the other hand, due to the sequence and functional similarity between stromatoxin and hanatoxin, we did expect that the kinetic data for stromatoxin present themselves in a very similar pattern as those of hanatoxin do. However, surprisingly, all the observations tend to draw our attention a totally different way of interpretation. Probably the structural details, rather than general structural feature, of stromatoxin may play pivotal role in the binding behavior. This awaits further investigations. This thesis provides experiments support on our previously proposed molecular mechanism of tarantula toxins binding induced gating in Kv channels to a certain extent. It also leads to some interesting questions open for future study.
Subjects
電壓感應開啟式鉀離子通道
停止流螢光光譜分析儀
電壓感受體
Voltage-gated potassium channel
Stopped-flow spectrofluorimeter
voltage sensor
Type
other
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