Slow Inactivation of Sodium Channels: Structural Clues and Disease Associations

dc.contributor.advisorCannon, Stephen C.en
dc.creatorWebb, Jadon Rayen
dc.description.abstractVoltage gated sodium channels underlie the rapid upstroke of action potentials in electrically excitable mammalian tissues. A cardinal feature of Na+ channels is their ability to rapidly inactivate to a refractory state during membrane depolarization, in a process known as 'fast inactivation'. During sustained membrane depolarization or prolonged busts of discharges, channels can further inactivate to non-conducting states collectively referred to as 'slow inactivation'. Fast inactivation occurs by occlusion of the inner pore by the intracellular III-IV Loop, and defects in fast inactivation gating are known to underlie certain forms of myotonia, periodic paralysis, epilepsy, and cardiac arrhythmias. The mechanism of slow inactivation and its relevance to human disease, on the other hand, are much less understood. The primary aim of this thesis was to characterize the mechanism of sodium channel slow inactivation, and also to further define its role in disease. In Chapter 1, an overview of sodium channel structure and gating is provided as background for understanding the rational and interpretation of the experimental studies. The experiments in Chapter 2 characterized the gating of a sodium channel mutation (P1158S) associated with temperature-sensitive periodic paralysis. This disease mutation caused a robust defect in slow inactivation, in accordance with an emerging model that associates defective slow inactivation with increased susceptibility to paralytic attacks. Additionally, the slow inactivation gating defects were elicited by cold temperature, analogous to the temperature-dependent provocation of paralysis. This finding further strengthens the association between defective slow inactivation gating and a specific disease phenotype. Chapter 3 explores the interaction of the sodium channel Beta-1 subunit and slow inactivation, which is incompletely characterized especially in mammalian cell expression systems. I found that co-expression of wild-type Beta-1 significantly depolarized the voltage-dependence of steady-state slow inactivation and also reduced the number of channels occupying the slow state (IS) after a long depolarizing conditioning pulse, but did not affect the kinetics of slow inactivation.. To understand which region(s) of Beta-1 are important for modulation of slow inactivation, two mutant constructs were tested. A point mutation in the extracellular N-terminus associated with epilepsy (C121W) disrupts a critical disulfide bond in an Ig-like fold and abolished the ability of Beta-1 to modulate slow inactivation. Conversely, truncation of the short cytoplasmic C-terminus did not alter the effects of Beta-1 on slow inactivation. These observations parallel the structure-function relations that have been established for Beta-1 modulation of fast inactivation. Interestingly, however, I used a mutant fast-inactivation deficient alpha-subunit to show that the Beta-1 effect on slow inactivation was independent of coupling to fast inactivation. In Chapter 4, the interaction of slow inactivation and alkali metal cations is explored. External cations have been shown to influence slow inactivation, but little is known about the location and mechanism of this interaction. To address this, I examined the interaction of Group IA alkali metal cations with slow inactivation in rat Nav1.4 channels expressed in HEK293t cells. Slow inactivation was significantly impeded by external, but not internal Na+ and Li+ cations in the buffer solutions. External K+, Rb+, and Cs+, on the other hand, caused little effect compared to sucrose (cation-free) buffer. Cation effects on slow inactivation were found to be very low affinity and were not dependent on the ability of cations to permeate deep into the channel. Indeed, Na+ interaction occurred at a shallow apparent electrical distance of 0.15 relative to the outside of the channel, and was affected by mutagenesis in the outeren
dc.format.digitalOriginborn digitalen
dc.subjectSodium Channelsen
dc.subjectMuscle Proteinsen
dc.subjectProtein Subunitsen
dc.titleSlow Inactivation of Sodium Channels: Structural Clues and Disease Associationsen
dc.type.materialTexten School of Biomedical Sciencesen Southwestern Medical Centeren of Philosophyen


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