Ultra-Slow Inactivation in Sodium Channels

Voltage-gated Na+ channels are pore forming macromolecules which play a central role in physiology: they enable transmission of depolarizing impulses between cells, which provides the basis for the coordination of higher processes like locomotion or cognition. The proper function of Na+ channels requires (i.) selectivity for permeation of Na+ ions (i.e. excluding other ions from entering the pore) and (ii.) gating, i.e. the opening and closing of their ion-permeable pores in response to the relevant biological signal, namely a change in transmembrane voltage. The exact mechanism by which selectivity of permeation and gating occurs is currently poorly understood. During the study of the adult rat skeletal muscle Na+ channel we found that certain mutations of the lysine 1237 residue in the domain III of the outer pore region, which is essential for the Na+ selectivity of the channel, produced substantial changes in channel gating. Specifically, the channels entered a nonconducting state ("inactivation") from which recovery was very slow. We refer to this state as "ultra-slow inactivation". Binding to the pore of a partially blocking peptide reduced the number of channels entering into the ultra-slow inactivation state. These findings most likely indicate that ultra-slow inactivation occurs by a structural rearrangement of the outer pore of the channel. Large molecules entering the pore can interfere with this molecular motion and thereby prevent entry into the ultra-slow inactivated state. The goal of this project is to further elucidate the mechanism of ultra-slow inactivation. We will systematically introduce mutations at various positions of the outer channel vestibule in order to identify the sites which are essential for the prevention of ultra-slow inactivation. Furthermore, we will investigate the mechanism by which pore blocking peptides can interfere with the ultraslow inactivated state. These studies will help us to shed light on molecular motions associated with ion permeation in Na+ channels.

Voltage-gated Na+ channels are pore-forming, membrane-associated macromolecules, which regulate the flow of Na+ ions across the cell membrane. These molecular switches enable the conduction of excitatory impulses between cells and thereby form the basis of the function of the heart, skeletal muscle and nervous system. The proper function of these channels requires accurate opening and closing of the pore as well as the transient development of a state of inexcitability, which is referred to as "inactivated" state. Inactivation is of great significance for the proper transduction of electrical signals between cells. Defective inactivation of Na+ channels is associated with a number of diseases, such as certain forms of epilepsy, skeletal muscle weakness and cardiac arrhythmias. There are a number of inactivated states which are defined by their time course of development and removal. Whereas "fast inactivation" develops on a time scale of milliseconds, "slow" inactivated states require up to several minutes for full development. The molecular basis for fast inactivation is a closure of the intracellular channel pore by a "lid". The molecular basis of slow inactivated states are not clearly defined. In this project we could show that a specific slow inactivated state, called "ultra-slow inactivation" is produced by a closure of the intracellular pore of the channel. This closure occurs more likely if the lid for fast inactivation is disabled. Hence, fast inactivation protects the channel from collapse during ultra-slow inactivation. We also found that certain drugs (e.g. local anesthetics) which are able to enter the inner vestibule of the channel inhibit the closure of the inner pore by a foot-in-the-door mechanism. On the other hand, more extracellular parts of the channel, which are involved in the control of ion flow through the channel ("selectivity filter") are also able to modulate ultra-slow inactivation: The activation of the channel is associated with movements of the outer pore, which produce a collapse of the inner channel pore. Conversely, the introduction of positive charge at a defined location in the outer vestibule can inhibit the closure of the inner vestibule. Furthermore, unnaturally large ions, which are forced to flow through the channel, interact with a specific site at the border between outer and inner vestibule thereby promoting the collapse of the inner vestibule. These results of the project allow new insights into the complex function of this ionic channel and provide important information for the development of novel pharmaceutic agents for the treatment of diseases of heart, skeletal muscle and nervous system.