Molecular basis of ion channel gating
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Molecular basis of gating behavior and drug binding in voltage-gated Na channels
The pore of voltage-gated ion channels consists of an outer vestibule containing the selectivity filter and an inner cavity or inner vestibule which harbours the gates for channel opening and fast inactivation. These regions are also important sites for drug and toxin interactions: maritime toxins such as tetrodotoxin or μ-conotoxin bind to the outer vestibule whereas the binding site for local anaesthetics, antiarrhythmic agents and anticonvulsive drugs has been mapped to the inner vestibule. Both functional and structural data from K+ channels suggest that outer and inner vestibules communicate with each other via a trajectory of interacting amino acids. By combination of site-directed mutagenesis and analysis of gating behaviour by means of electrophysiological techniques we will define possible sites of functional interaction between the outer and the inner regions of the channel. The student will generate point mutations in the rNav 1.4 channel and express the constructs in mammalian cells and Xenopus laevis oocytes. Finally, the student will test the effect of the mutations on ion permeation and gating behaviour by means of the following techniques: two-electrode voltage-clamp (oocytes), cut-open oocyte, and patch clamp (mammalian cells).

Mutation-induced alterations in drug-binding to voltage-gated Na channels: separation of structure-related and gating-induced changes
The inner channel vestibule contains major binding sites for clinically important Na+ channel blockers (local anaesthetics, antidepressants, antiarrhythmics). The binding affinities of most drugs vary with the conformational state of the channel. Drug binding sites in the channel are commonly defined by means of site-directed mutagenesis. However, most mutations which alter drug-affinity also produce substantial changes in the gating behaviour of the channel. Hence, mutation-induced alterations in drug-binding may result from conformational changes in the binding site of the drug, or from changes in the gating behaviour. We will explore the effect of single and double mutations in the outer and inner vestibule on gating and on drug binding. The changes in binding affinity will then be compared with the respective mutation-induced gating alterations. These multiple comparisons will allow us to separate changes in drug affinity as a result of gating perturbations from changes in drug-affinity due to alterations in the structure of the binding site. The applied techniques are the same as in project 1.