Debra Ann Fadool

Association of Src Tyrosine Kinase with a Human Potassium Channel Mediated by SH3 Domain

The human Kv1.5 potassium channel (hKv1.5) contains proline-rich sequences identical to those that bind to Src homology 3 (SH3) domains. Direct association of the Src tyrosine kinase with cloned hKv1.5 and native hKv1.5 in human myocardium was observed. This interaction was mediated by the proline-rich motif of hKv1.5 and the SH3 domain of Src. Furthermore, hKv1.5 was tyrosine phosphorylated, and the channel current was suppressed, in cells coexpressing v-Src. These results provide direct biochemical evidence for a signaling complex composed of a potassium channel and a protein tyrosine kinase.

Plasma membrane inositol 1,4,5-Trisphosphate-Activated channels mediate signal transduction in lobster olfactory receptor neurons

Inositol 1,4,5-trisphosphate (IP3) selectively evokes an inward (excitatory) current in cultured lobster olfactory receptor neurons (ORNs) and directly activates two types of channels in cell-free patches of plasma membrane from the ORNs. The IP3-activated channels have kinetic properties of odor-activated channels in the ORNs and pharmacological properties of intracellular IP3-activated channels in other systems. An antibody directed against an intracellular, cerebellar IP3 receptor recognizes a protein with a molecular weight similar to the mammalian receptor in the ORNs. The antibody selectively increases odor-evoked inward currents and IP3-activated unitary currents in the ORNs. The data provide further evidence for IP3 as an olfactory second messenger and implicate at least one and possibly two novel plasma membrane IP3 receptors in olfactory transduction.

Awake Intranasal Insulin Delivery Modifies Protein Complexes and Alters Memory, Anxiety, and Olfactory Behaviors

The role of insulin pathways in olfaction is of significant interest with the widespread pathology of diabetes mellitus and its associated metabolic and neuronal comorbidities. The insulin receptor (IR) kinase is expressed at high levels in the olfactory bulb, in which it suppresses a dominant Shaker ion channel (Kv1.3) via tyrosine phosphorylation of critical N- and C-terminal residues. We optimized a 7 d intranasal insulin delivery (IND) in awake mice to ascertain the biochemical and behavioral effects of insulin to this brain region, given that nasal sprays for insulin have been marketed notwithstanding our knowledge of the role of Kv1.3 in olfaction, metabolism, and axon targeting. IND evoked robust phosphorylation of Kv1.3, as well as increased channel protein–protein interactions with IR and postsynaptic density 95. IND-treated mice had an increased short- and long-term object memory recognition, increased anxiolytic behavior, and an increased odor discrimination using an odor habituation protocol but only moderate change in odor threshold using a two-choice paradigm. Unlike Kv1.3 gene-targeted deletion that alters metabolism, adiposity, and axonal targeting to defined olfactory glomeruli, suppression of Kv1.3 via IND had no effect on body weight nor the size and number of M72 glomeruli or the route of its sensory axon projections. There was no evidence of altered expression of sensory neurons in the epithelium. In mice made prediabetic via diet-induced obesity, IND was no longer effective in increasing long-term object memory recognition nor increasing anxiolytic behavior, suggesting state dependency or a degree of insulin resistance related to these behaviors.

Kv1.3 Channel Gene-Targeted Deletion Produces “Super-Smeller Mice” with Altered Glomeruli, Interacting Scaffolding Proteins, and Biophysics

Mice with gene-targeted deletion of the Kv1.3 channel were generated to study its role in olfactory function. Potassium currents in olfactory bulb mitral cells from Kv1.3 null mice have slow inactivation kinetics, a modified voltage dependence, and a dampened C-type inactivation and fail to be modulated by activators of receptor tyrosine signaling cascades. Kv1.3 deletion increases expression of scaffolding proteins that normally regulate the channel through protein-protein interactions. Kv1.3-/- mice have a 1,000- to 10,000-fold lower threshold for detection of odors and an increased ability to discriminate between odorants. In accordance with this heightened sense of smell, Kv1.3-/- mice have glomeruli or olfactory coding units that are smaller and more numerous than those of wild-type mice. These data suggest that Kv1.3 plays a far more reaching role in signal transduction, development, and olfactory coding than that of the classically defined role of a potassium channel-to shape excitability by influencing membrane potential.

Olfaction Under Metabolic Influences

Recently published work and emerging research efforts have suggested that the olfactory system is intimately linked with the endocrine systems that regulate or modify energy balance. Although much attention has been focused on the parallels between taste transduction and neuroendocrine controls of digestion due to the novel discovery of taste receptors and molecular components shared by the tongue and gut, the equivalent body of knowledge that has accumulated for the olfactory system, has largely been overlooked. During regular cycles of food intake or disorders of endocrine function, olfaction is modulated in response to changing levels of various molecules, such as ghrelin, orexins, neuropeptide Y, insulin, leptin, and cholecystokinin. In view of the worldwide health concern regarding the rising incidence of diabetes, obesity, and related metabolic disorders, we present a comprehensive review that addresses the current knowledge of hormonal modulation of olfactory perception and how disruption of hormonal signaling in the olfactory system can affect energy homeostasis.

Neurotrophin modulation of voltage-gated potassium channels in rat through TrkB receptors is time and sensory experience dependent.

The whole‐cell configuration of the patch‐clamp technique, immunoprecipitation experiments and unilateral naris occlusions were used to investigate whether the voltage‐gated potassium channel Kv1.3 was a substrate for neurotrophin‐induced tyrosine phosphorylation and subsequent functional modulation of current properties in cultured rat olfactory bulb (OB) neurons. Membrane proteins of the OB included all three Trk receptor kinases, but the truncated form of the receptor, lacking an intact kinase domain, was the predominant form of the protein for TrkA and TrkC, while TrkB was predominantly found as the full‐length receptor. Acute (15 min) stimulation of OB neurons with bath application of 50 ng ml−1 brain‐derived neurotrophic factor (BDNF), which is a selective ligand for TrkB, caused suppression of the whole‐cell outward current and no changes in the kinetics of inactivation or deactivation. Acute stimulation with either nerve growth factor or neurotrophin‐3 failed to evoke any changes in Kv1.3 function in the OB neurons. Chronic exposure to BDNF (days) caused an increase in the magnitude of Kv1.3 current and speeding of the inactivation and deactivation of the channel. Acute BDNF‐induced activation of TrkB receptors significantly increased tyrosine phosphorylation of Kv1.3 in the OB, as shown using a combined immunoprecipitation and Western blot analysis. With unilateral naris occlusion, the acute BDNF‐induced tyrosine phosphorylation of Kv1.3 was increased in neurons lacking odour sensory experience. In summary, the duration of neurotrophin exposure and the sensory‐dependent state of a neuron can influence the degree of phosphorylation of a voltage‐gated ion channel and its concomitant functional modulation by neurotrophins.

Two Adaptor Proteins Differentially Modulate the Phosphorylation and Biophysics of Kv1.3 Ion Channel by Src Kinase

The Shaker family K+channel protein, Kv1.3, is tyrosine phosphorylated by v-Src kinase at Tyr137 and Tyr449 to modulate current magnitude and kinetic properties. Despite two proline rich sequences and these phosphotyrosines contained in the carboxyl and amino terminals of the channel, v-Src kinase fails to co-immunoprecipitate with Kv1.3 as expressed in HEK 293 cells, indicating a lack of direct Src homology 3- or Src homology 2-mediated protein-protein interaction between the channel and the kinase. We show that the adaptor proteins, n-Shc and Grb10, are expressed in the olfactory bulb, a region of the brain where Kv1.3 is highly expressed. In HEK 293 cells, co-expression of Kv1.3 plus v-Src with Grb10 causes a decrease in v-Src-induced Kv1.3 tyrosine phosphorylation and a reversal of v-Src-induced Kv1.3 current suppression, increase in inactivation time constant (τinact), and disruption of cumulative inactivation properties. Co-expression of Kv1.3 plus v-Src with n-Shc did not significantly alter v-Src-induced Kv1.3 current suppression but reversed v-Src induced increased τinact and restored the right-shifted voltage at half-activation (V 1/2) induced by v-Src. The v-Src-induced shift inV 1/2 and increased τinact was retained when Tyr220, Tyr221, and Tyr304 in the CH domain of n-Shc were mutated to Phe (triple Shc mutant) but was reversed back to control values when either wild-type Shc or the family member Sck, which is not a substrate for Src kinase, was substituted for the triple Shc mutant. Thus the portion of the CH domain that includes Tyr220, Tyr221, and Tyr304 may regulate a shift in Kv1.3 voltage dependence and inactivation kinetics produced by n-Shc in the presence of v-Src. Collectively these data indicate that Grb10 and n-Shc adaptor molecules differentially modulate the degree of Kv1.3 tyrosine phosphorylation, the channels biophysical properties, and the physical complexes associated with Kv1.3 in the presence of Src kinase.

Hyperlipidemic Diet Causes Loss of Olfactory Sensory Neurons, Reduces Olfactory Discrimination, and Disrupts Odor-Reversal Learning

Currently, 65% of Americans are overweight, which leads to well-supported cardiovascular and cognitive declines. Little, however, is known concerning obesitys impact on sensory systems. Because olfaction is linked with ingestive behavior to guide food choice, its potential dysfunction during obesity could evoke a positive feedback loop to perpetuate poor ingestive behaviors. To determine the effect of chronic energy imbalance and reveal any structural or functional changes associated with obesity, we induced long-term, diet-induced obesity by challenging mice to high-fat diets: (1) in an obesity-prone (C57BL/6J) and obesity-resistant (Kv1.3−/−) line of mice, and compared this with (2) late-onset, genetic-induced obesity in MC4R−/− mice in which diabetes secondarily precipitates after disruption of the hypothalamic axis. We report marked loss of olfactory sensory neurons and their axonal projections after exposure to a fatty diet, with a concomitant reduction in electro-olfactogram amplitude. Loss of olfactory neurons and associated circuitry is linked to changes in neuronal proliferation and normal apoptotic cycles. Using a computer-controlled, liquid-based olfactometer, mice maintained on fatty diets learn reward-reinforced behaviors more slowly, have deficits in reversal learning demonstrating behavioral inflexibility, and exhibit reduced olfactory discrimination. When obese mice are removed from their high-fat diet to regain normal body weight and fasting glucose, olfactory dysfunctions are retained. We conclude that chronic energy imbalance therefore presents long-lasting structural and functional changes in the operation of the sensory system designed to encode external and internal chemical information and leads to altered olfactory- and reward-driven behaviors.

Kv1.3 gene-targeted deletion alters longevity and reduces adiposity by increasing locomotion and metabolism in melanocortin-4 receptor-null mice

Objective:Gene-targeted deletion of the voltage-gated potassium channel, Kv1.3, results in ‘super-smeller’ mice that have altered firing patterns of mitral cells in the olfactory bulb, modified axonal targeting to glomerular synaptic units, and behaviorally have an increased ability to detect and discriminate odors. Moreover, the Kv1.3-null mice weighed less than their wild-type counterparts, have modified ingestive behaviors, and are resistant to fat deposition following a moderately high-fat dietary regime. In this study, we investigate whether or not gene-targeted deletion of Kv1.3 (Shaker family member) can abrogate weight gain in a genetic model of obesity, the melanocortin-4 receptor-null mouse (MC4R-null).Design:Mice with double gene-targeted deletions of Kv1.3 and MC4R were generated by interbreeding Kv1.3 (Kv)- and MC4R-null mouse lines to homozygosity. Developmental weights, nose to anus length, fat pad weight, fasting serum chemistry, oxygen consumption, carbon dioxide respiration, locomotor activity and caloric intake were monitored in control, Kv-null, MC4R-null and Kv/MC4R-null mice. Physiological and metabolic profiles were acquired at postnatal day 60 (P60) in order to explore changes linked to body weight at the reported onset of obesity in the MC4R-null model.Results:Gene-targeted deletion of Kv1.3 in MC4R-null mice reduces body weight by decreasing fat deposition and subsequent fasting leptin levels, without changing the overall growth, fasting blood glucose or serum insulin. Gene-targeted deletion of Kv1.3 in MC4R-null mice significantly extended lifespan and increased reproductive success. Basal or light-phase mass-specific metabolic rate and locomotor activity were not affected by genetic deletion of Kv1.3 in MC4R-null mice but dark-phase locomotor activity and mass-specific metabolism were significantly increased resulting in increased total energy expenditure.Conclusions:Gene-targeted deletion of Kv1.3 can reduce adiposity and total body weight in a genetic model of obesity by increasing both locomotor activity and mass-specific metabolism.

Comparison of modulation of Kv1.3 channel by two receptor tyrosine kinases in olfactory bulb neurons of rodents.

Activation of the receptor tyrosine kinase (RTK), insulin (IRK) or neurotrophin B (TrkB), was characterized and compared in olfactory bulb neuron (OBN) cultures from Sprague Dawley rats and sv129 B6 mice. Current suppression attributed to modulation of the delayed rectifier, Kv1.3, a voltage-gated potassium (Kv) channel of the Shaker family, was observed following acute application of the growth factors, insulin or brain-derived neurotrophic factor (BDNF), to mitral cells of either rodent model. Using site-directed mutagenesis of putative tyrosine phosphorylation recognition motifs in the channel, we find that stimulation of Kv1.3 with these growth factors causes multiple phosphorylation, albeit via different residue combinations that are RTK specific.