Glucagon-like Peptide 1-Mediated Modulation of Kv1.3 Activity
Landi Conde, Daniel Roberto (author)
Fadool, Debra Ann (professor directing thesis)
Vincis, Roberto (committee member)
Stagg, Scott (committee member)
Lenhert, Steven John (committee member)
Bertram, R. (Richard) (committee member)
Florida State University (degree granting institution)
College of Arts and Sciences (degree granting college)
Department of Biological Science (degree granting department)
There is a well-established relationship between the olfactory and the endocrine systems. At the center of this relationship sits the olfactory bulb, which acts as a relay station for the sense of smell. In fact, neurons in the olfactory epithelium and the olfactory bulb express a myriad of metabolic receptors. Mitral cells are the major projection neurons in the olfactory bulb, and they express receptors for important metabolic factors such as glucose, insulin, and glucagon-like peptide 1 (GLP-1). GLP-1 is a gut hormone that aids in the regulation of blood glucose by promoting the release of insulin following a meal. The main source of GLP-1 is the intestinal L-cells, but it is also released from neurons in the central nervous system. Mitral cells in the olfactory bulb express the receptor for GLP-1 (GLP-1R). The GLP-1R is a G-protein-coupled receptor that is mechanistically coupled to adenylyl cyclase, which catalyzes the conversion of ATP into cAMP, leading to the activation of cAMP-dependent Protein Kinase A (PKA). GLP-1R action increases neuronal excitability by reducing the hyperpolarizing currents in mitral cells through an unknown mechanism. The main voltage-gated potassium channel in mitral cells is Kv1.3. Since the effect of GLP-1 on mitral cell excitability is lost in Kv1.3-/- cells, it is hypothesized that GLP-1R activation leads to an inhibition of Kv1.3 activity. My project focuses on describing the signaling pathway that leads to Kv1.3 inhibition upon GLP-1 stimulation. Previous reports conclude that GLP-1R activation in multiple tissues leads to a partial inhibition of potassium channels, usually via phosphorylation. To test this, I transfected Kv1.3 and the GLP-1R in a heterologous expression system (HEK293) and performed both electrophysiological and biochemical assays. GLP-1 stimulation leads to an overall decrease in currents generated by Kv1.3 without affecting the channel's kinetic properties for activation, inactivation, or deactivation. This effect is lost in mutant versions of Kv1.3 that are resistant to Serine phosphorylation. In parallel, I performed an immunoprecipitation of Kv1.3 from transfected cells following GLP-1 stimulation. As revealed by immunoblotting, Kv1.3 protein extracted from cells co-expressing the GLP-1R show a marked increase in Serine/Threonine phosphorylation when compared to the control. Taken together, my results indicate that GLP-1R activation does indeed lead to Serine phosphorylation of Kv1.3, causing a partial inhibition of its activity without apparent changes in its kinetic properties. An exception would be the triple mutant S3A-Kv1.3, wherein the half-point to maximal activation (V1/2) shows a depolarizing shift without a reduction in the generated currents. Because of this, my results point to a role for S105 in the activation of Kv1.3 that is independent of ionic conductance. GLP-1-induced inhibition, however, is not as strong as the one previously observed in mitral cells. I hypothesize that the phosphorylation is mediated by cAMP-dependent PKA activation, but I discuss the possibility of different pathways affecting Kv1.3 that are part of the complex reaction cascade triggered by GLP-1. In conclusion, GLP-1-mediated phosphorylation of Kv1.3 might lead to the disincorporation of Kv1.3 channels from the membrane, effectively reducing the hyperpolarizing currents in the cell without affecting the kinetic properties of channel activity. My results point to possible therapeutic targets that may contribute to the modulation of mitral cell activity and, through it, olfactory ability and metabolic regulation.
GLP-1, glucagon-like peptide 1, Kv1.3, metabolism, olfaction, potassium channel modulation
March 23, 2022.
A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science.
Includes bibliographical references.
Debra Ann Fadool, Professor Directing Thesis; Roberto Vincis, Committee Member; Scott Stagg, Committee Member; Steve Lenhert, Committee Member; Richard Bertram, Committee Member.
Florida State University