Mitochondrial Ultrastructure and Glucose Signaling Pathways Attributed to the Kv1.3 Ion Channel

Abstract

Gene-targeted deletion of the potassium channel Kv1.3 (Kv1.3(-∕-)) results in "Super-smeller" mice with a sensory phenotype that includes an increased olfactory ability linked to changes in olfactory circuitry, increased abundance of olfactory cilia, and increased expression of odorant receptors and the G-protein, Golf. Kv1.3(-∕-) mice also have a metabolic phenotype including lower body weight and decreased adiposity, increased total energy expenditure (TEE), increased locomotor activity, and resistance to both diet- and genetic-induced obesity. We explored two cellular aspects to elucidate the mechanism by which loss of Kv1.3 channel in the olfactory bulb (OB) may enhance glucose utilization and metabolic rate. First, using in situ hybridization we find that Kv1.3 and the insulin-dependent glucose transporter type 4 (GLUT4) are co-localized to the mitral cell layer of the OB. Disruption of Kv1.3 conduction via construction of a pore mutation (W386F Kv1.3) was sufficient to independently translocate GLUT4 to the plasma membrane in HEK 293 cells. Because olfactory sensory perception and the maintenance of action potential (AP) firing frequency by mitral cells of the OB is highly energy demanding and Kv1.3 is also expressed in mitochondria, we next explored the structure of this organelle in mitral cells. We challenged wildtype (WT) and Kv1.3(-∕-) male mice with a moderately high-fat diet (MHF, 31.8 % kcal fat) for 4 months and then examined OB ultrastructure using transmission electron microscopy. In WT mice, mitochondria were significantly enlarged following diet-induced obesity (DIO) and there were fewer mitochondria, likely due to mitophagy. Interestingly, mitochondria were significantly smaller in Kv1.3(-∕-) mice compared with that of WT mice. Similar to their metabolic resistance to DIO, the Kv1.3(-∕-) mice had unchanged mitochondria in terms of cross sectional area and abundance following a challenge with modified diet. We are very interested to understand how targeted disruption of the Kv1.3 channel in the OB can modify TEE. Our study demonstrates that Kv1.3 regulates mitochondrial structure and alters glucose utilization; two important metabolic changes that could drive whole system changes in metabolism initiated at the OB.

Keywords: diet-induced obesity; glucose transporter; mitochondria; olfactory bulb; potassium channel.

Figure 1

Kv1.3 and GLUT4 mRNA are expressed predominantly in the glomerular and mitral cell layers of the olfactory bulb. Light photomicrographs containing tissue sections of wildtype (WT^+∕+; left) and Kv1.3-null mice (Kv1.3^−∕−; right) olfactory bulbs labeled with GLUT4 (GLUT4 probe; top) or Kv1.3 (Kv1.3 probe; bottom). Note that GLUT4 mRNA is expressed predominantly in the mitral cell layer (ML) in both WT and Kv1.3^−∕− mice, whereas Kv1.3 mRNA is localized to both the glomerular layer (GL) and ML. GL, glomerular layer; EPL, external plexiform layer; ML, mitral cell layer; GCL, granule cell layer.

Figure 2

GLUT4 is expressed intracellularly and then translocates to the plasma membrane following block of Kv1.3 channel achieved by site-directed mutagenesis of the pore. (A) Model of heterologous co-expression of a non-conducting Kv1.3 channel (W386F Kv1.3) and an extracellular epitope-myc tagged GLUT4 transporter that becomes translocated to the plasma membrane. (B) Western blot of lysates harvested from HEK293 cells transfected with K: Kv1.3 cDNA; W: mutant W386F Kv1.3 cDNA; G: GLUT4-myc cDNA. Blots were probed with myc antibody (anti-myc) to visualize labeling of all GLUT4 in a whole-cell preparation. (C) Bar graph of the normalized pixel immunodensity of the quantified bands for 7 such blots as represented in (B). ^* = one-way analysis of variance (ANOVA), Bonferoni post-hoc test, p ≤ 0.05. Different length vertical lines represent significantly different post-hoc comparison. (D,E) Same as (B,C) but samples were surface immunoprecipitated (IP) with myc antibody (anti-myc) and then labeled with anti-myc to visualize labeling of surface GLUT4. Input (lysates) were labeled with actin antibody (anti-actin) to standardize equal loading.

Figure 3

Kv1.3^−∕− mice have smaller mitochondria in the mitral cell layer of the OB, which do not exhibit an increase in volume when challenged with moderately-high fat diet (MHF). Photomicrographs acquired from the mitral cell layer of the OB of wildtype (+/+) and Kv1.3-null mice (–/–) maintained on MHF vs. CF for 4 months. (A) Low and (B) higher magnification. Bar graph or box blot representing (C) mean number, (D) cross-sectional area, and (E) circularity index of mitochondria collected across 40 fields of view for 3 mice. Data represent mean +∕− s.d. for the bar graph, or mean (line), 25/75% quartile (box) and 5/95% (whiskers) for the box plot. Significantly different, one-way ANOVA, Bonferoni's post-hoc test, ^****p ≤ 0.0001; ^***p ≤ 0.001, ^**p ≤ 0.01; ^*p ≤ 0.05.

Figure 4

Schematic diagram showing cell signaling interactions of plasma membrane Kv1.3 (Kv1.3) and potential interplay with mitochondrial Kv1.3 (mKv1.3). Glycogen-like peptide (GLP-1) and insulin are two signaling hormones that block Kv1.3 current. The channel (red) is known to be a substrate for insulin receptor (IR) kinase (blue) on the N- and C-terminal aspects of the channel protein (Y111-113, Y137, Y479). Insulin-induced phosphorylation (P) of Kv1.3 (blue line) decreases Kv1.3 current amplitude by decreasing Pr_open of the channel. GLUT4 is typically translocated to the membrane upon insulin activation (blue dashed line) but upon block of Kv1.3 conductance, can also be translocated (red dashed line). Metabolism of glucose establishes the H⁺ gradient and the production of ATP via the electron transport chain (ETC). Maintenance of the ionic environment is dependent upon influx of K⁺ through mKv1.3 and mKATP and the balance provided by efflux via the K⁺/H⁺ exchanger. The ATP energy source could be used for the GLP-1R triggered conversion of ATP to cAMP through adenylase cyclase to increase potential PKA activity (?). Metabolic factors that decrease Kv1.3 ion channel activity increase the AP firing frequency in mitral cells that is thought to provide odor quality coding of olfactory information. OMM, outer mitochondria membrane; IMM, inner mitochondria membrane; IMS, inter membrane space.