Browsing by Subject "Sodium Channels"
Now showing 1 - 3 of 3
- Results Per Page
- Sort Options
Item Naᵥ1.8+ Visceral Afferent Neurons: Roles in Metabolism and Inflammation(2013-06-05) Udit, Swalpa 1985-; Scherer, Philipp; Hooper, Lora V.; Mangelsdorf, David J.; Zinn, Andrew R.; Elmquist, JoelThe increasing prevalence of obesity and its related comorbidities are major health issues facing western societies. Obesity involves a long-term, chronic perturbation of energy balance, in which food intake is not properly matched to calories expended. It is also closely linked with low-grade inflammation characterized by increased levels of circulating inflammatory cytokines and acute-phase reactants, concomitant with the activation of a network of inflammatory signaling pathways. As vagal afferents are a neuronal population that can rapidly respond to both food-related stimuli as well as inflammatory agents and cytokines, I hypothesized that intact vagal afferent function may be required for proper regulation of energy balance and particularly diet-induced inflammation. The sensory component of the vagus nerve has long been considered the main neural relay by which nutritional signals from the gut reach brain sites maintaining homeostasis. To study the function of this neuronal population, we generated a genetic mouse model with specific ablation of sensory vagal neurons by driving expression of diphtheria toxin (DTA) in a Cre-dependent manner through crossing the DTA mouse, which expresses DTA upon Cre activity, with the well-characterized Naᵥ1.8-Cre mouse, which expresses Cre recombinase under the control of Naᵥ1.8, a voltage gated sodium channel present only in peripheral sensory neurons, including 80% of vagal sensory afferent neurons Metabolic phenotyping of these ablated mice in comparison to control littermates did not reveal differences in body weight or food intake on chow diet. However, input from vagal afferents does appear to be important for linking ingested nutrients to acute changes in energy expenditure. This work also suggests that vagal afferents may be involved in checking inflammation to certain stimuli, possibly dietary high fat. As activity of vagal afferents has been shown to be decreased in obesity, dysfunction in this group of neurons could contribute to the diet induced inflammation associated with obesity.Item Slow Inactivation of Sodium Channels: Structural Clues and Disease Associations(2009-06-19) Webb, Jadon Ray; Cannon, Stephen C.Voltage 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 outerItem Voltage-Gated Sodium Channel Activity in Mouse Skeletal Muscle Fibers: Normal Gating and Defects Associated with Periodic Paralysis Mutants(2010-11-02) Fu, Yu; Cannon, Stephen C.Mutations in SCN4A, the gene encoding the skeletal muscle Na+ channel (NaV1.4) α-subunit, cause several disorders related to skeletal muscle excitability. The functional consequences of these NaV1.4 mutations have been extensively characterized in heterologous expression systems. These studies have significantly advanced our understanding of the pathophysiology of these disorders. The in vivo functional consequences on channel activity, however, have yet to be defined. Animal models are now available in genetically engineered mice, which provide an opportunity to examine channel function in mature skeletal muscle. We optimized a two-electrode voltage clamp protocol to improve the fidelity of Na+ current recording from acutely dissociated intact muscle fibers. Computer simulation, incorporating measured capacitance and ionic current densities, was used to confirm sufficient voltage control and distortion-free Na+ currents. The gating properties of endogenous Na+ currents were measured and compared between two mouse strains, C57BL/6 and 129-E. The most dramatic finding was a hyperpolarized shift in the voltage dependence of activation (-25 mV) and fast inactivation (-18 mV) as compared to the studies in HEK293 cells expressing NaV1.4 plus the accessory β1-subunit. A possible contribution from NaV1.5 channels in the mouse muscle preparation was excluded by RT-PCR and TTX sensitivity. There was no significant difference in voltage dependence of fast gating between C57BL/6 and 129-E. The entry rate into slow inactivation was slower for Na+ channel in 129-E fibers; while the recovery from slow inactivation was similar between two mouse stains. Two NaV1.4 missense mutations associated with divergent clinical phenotypes - NaV1.4-M1592V in hyperkalemic periodic paralysis (HyperPP) and NaV1.4-R663H (homolog of human R669H) in hypokalemic periodic paralysis (HypoPP) - were characterized with voltage-clamp recordings in fully differentiated fibers from knock-in mutant mice. The NaV1.4-M1592V mutation produced gain-of-function defects, with the major changes being a slightly increased persistent current and moderately disrupted slow inactivation. In contrast, the HypoPP knock-in mutant R663H resulted in loss-of-function changes, due to an enhancement of inactivation, both fast and slow, and impaired activation. These observations provide important validation of prior findings using heterologous expression systems and yield quantitative information on the severity of the gating defects in mammalian skeletal muscles.