Targeting the Kv1.3 Ion Channel with Peptide Inhibitors and Nanoparticle Bioconjugates: Neuromodulation of the Olfactory Bulb and Its Influence on Whole-Body Metabolism

• Other Authors: Schwartz, Austin Bradley (author)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Neurosciences; Nanotechnology
• Online Access: http://purl.flvc.org/fsu/fd/2018_Su_Schwartz_fsu_0071E_14755

Electrical signaling in the olfactory bulb (OB) is modulated by changes in metabolic state. The voltage-gated potassium channel, Kv1.3, makes up 60 - 80% of the outward current flow in mitral cells (MCs), the primary projection neurons of the OB. The metabolic molecules GLP-1, insulin, and glucose are present in the OB and modulate MC signaling by reducing the activity of Kv1.3. In obesity and diabetes, modulation of MC signaling and Kv1.3 current flow by these molecules is absent. Gene-targeted deletion of Kv1.3 (Kv1.3 -/-) produces a phenotype that encompasses changes in olfactory ability and metabolism. Kv1.3 -/- mice are “supersmellers”, with an enhanced ability to detect and discriminate odors, are leaner than their wild-type counterparts and resistant to diet-induced obesity. These observations suggest metabolism, the OB and Kv1.3 are intimately linked, providing opportunity for therapeutic intervention at the level of the voltage-gated potassium channel. Strategies towards targeting Kv1.3 in the OB, but not other regions of the brain or periphery, are desired. There are several natural modulators of Kv1.3 that can be utilized for therapeutic targeting of the channel. Nedd4-2 is an ubiquitin ligase that mediates ubiquination and degradation of target proteins and can act to regulate Kv1.3 channel density, while the adaptor protein Grb10 can mediate Nedd4-2 activity. Patch-clamp electrophysiology in HEK293 cells, SDS-PAGE, immunoprecipitation, and mutagenesis strategies demonstrated a channel/adaptor/ ligase signalplex. Mutation of the C-terminal, SH3-recognition or ubiquitination sites on Kv1.3 retained the observed co-immunoprecipitation between Nedd4-2/Kv1.3, while the latter prevented a reduction in channel density. A model based on these data is presented for which an atypical interaction may permit Nedd4-2/Kv1.3 interactions that lead to protein degradation and reduced current density, and can be disrupted by Nedd4-2/Grb10 interactions. Venom-derived ion channel inhibitors are a strong alternative to natively expressed Kv1.3 modulators. These inhibitors have strong channel selectivity, potency, and stability; however, tracking delivery to their target can be challenging. Margatoxin (MgTx) is a potent Kv1.3 inhibitor and conjugation to luminescent quantum dots (QDs) can provide a means to track its delivery. Towards this, two approaches were taken. Covalent conjugation of MgTx to QDs produced QD-MgTx, which exhibited a retention of known biophysical properties associated with block of the vestibule of Kv1.3. Towards a more efficient and controlled conjugation, a polyhistidine tagged MgTx (HisMgTxFSU) was produced and conjugated to QDs via polyhistidine-mediated self assembly (QDHisMgTxFSU). Similar to QD-MgTx, QDHisMgTxFSU had a strong ability to inhibit Kv1.3 in HEK293 cells, excite mitral cells of the OB, and label Kv1.3 expressing HEK293 cells. When delivered to the OB via cannula guided delivery, QDHisMgTxFSU failed to label Kv1.3 expressing mitral cells. Therapeutic effects due to delivery of QDHisMgTxFSU, however, were observed. To better understand the role of Kv1.3 in the OB and metabolism, HisMgTxFSU and QDHisMgTxFSU were delivered to the OB of obese mice via cannula-guided delivery and changes in metabolism were measured. Compared to control animals, targeted inhibition of Kv1.3 was found to prevent weight gain and reduce the respiratory quotient (RER). These data provide evidence for the first time that the OB plays a key role in energy regulation and the pathways involved in this regulation are proposed. === A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. === Summer Semester 2018. === July 16, 2018. === Electrophysiology, Ion Channel, Metabolism, Olfactory Bulb, Pharmacology, Quantum Dots === Includes bibliographical references. === Debra Ann Fadool, Professor Directing Dissertation; Thomas Houpt, University Representative; Timothy Cross, Committee Member; Richard Bertram, Committee Member; Yi Zhou, Committee Member.