Brain Insulin Receptor Causes Activity-Dependent Current Suppression in the Olfactory Bulb Through Multiple Phosphorylation of Kv1.3

Abstract

Insulin and insulin receptor (IR) kinase are found in abundance in discrete brain regions yet insulin signaling in the CNS is not understood. Because it is known that the highest brain insulin-binding affinities, insulin-receptor density, and IR kinase activity are localized to the olfactory bulb, we sought to explore the downstream substrates for IR kinase in this region of the brain to better elucidate the function of insulin signaling in the CNS. First, we demonstrate that IR is postnatally and developmentally expressed in specific lamina of the highly plastic olfactory bulb (OB). ELISA testing confirms that insulin is present in the developing and adult OB. Plasma insulin levels are elevated above that found in the OB, which perhaps suggests a differential insulin pool. Olfactory bulb insulin levels appear not to be static, however, but are elevated as much as 15-fold after a 72-h fasting period. Bath application of insulin to cultured OB neurons acutely induces outward current suppression as studied by the use of traditional whole-cell and single-channel patch-clamp recording techniques. Modulation of OB neurons is restricted to current magnitude; IR kinase activation does not modulate current kinetics of inactivation or deactivation. Transient transfection of human embryonic kidney cells with cloned Kv1.3 ion channel, which carries a large proportion of the outward current in these neurons, revealed that current suppression was the result of multiple tyrosine phosphorylation of Kv1.3 channel. Y to F single-point mutations in the channel or deletion of the kinase domain in IR blocks insulin-induced modulation and phosphorylation of Kv1.3. Neuromodulation of Kv1.3 current in OB neurons is activity dependent and is eliminated after 20 days of odor/sensory deprivation induced by unilateral naris occlusion at postnatal day 1. IR kinase but not Kv1.3 expression is downregulated in the OB ipsilateral to the occlusion, as demonstrated in cryosections of right (control) and left (sensory-deprived) OB immunolabeled with antibodies directed against these proteins, respectively. Collectively, these data support the hypothesis that the hormone insulin acts as a multiply functioning molecule in the brain: IR signaling in the CNS could act as a traditional growth factor during development, be altered during energy metabolism, and simultaneously function to modulate electrical activity via phosphorylation of voltage-gated ion channels.

Deletion of voltage‐gated channel affects glomerular refinement and odorant receptor expression in the mouse olfactory system

“…Because we cannot detect expression of Kv1.3 in the main olfactory epithelium by either SDS-PAGE or immunocytochemistry (Fig. 6) and because expression is predominantly, but not exclusively, localized to the mitral cells (Fadool et al, 2000;Colley et al, 2007,Marks and, the increased frequency of action potential firing centrally in the olfactory bulb may be modulating OR protein expression, as well as altering typically singular axonal projections to be supernumerary, the latter of which appear to be OR-type dependent. An alternative hypothesis, which our data cannot eliminate, is that deletion of Kv1.3 may be affecting electrical events at even higher areas of olfactory processing in the piriform cortex (Kues and Wunder, 1992;Colley et al, 2007) or events in the dentate gyrus of the hippocampus (Kues and Wunder, 1992) or the hypothalamus (Tucker et al, 2007).…”

“…1RR-SS). We additionally used cloned Kv1.3 heterologously expressed in HEK 293 cells as a migration standard to demonstrate specificity of the αKv1.3 antibody, which was previously well characterized in native OB by immunocy-tochemistry and Western analysis (Fadool et al, 2000;Tucker and Fadool, 2002;Fadool et al, 2004;Marks and Fadool, 2007). …”

The voltage-gated potassium channel Kv1.3 regulates peripheral insulin sensitivity

“…Channel activity is up-regulated by serum-glucocorticoid-activated kinase, one of the main mediators of aldosterone's action at the renal distal tubule (17). Protein kinase C increases (18) and tyrosine kinase inhibits Kv1.3 channel activity (19). Kv1.3 is highly expressed in the olfactory bulb (20), and it appears to mediate a large fraction of the outward current detected in these neurons, which also express the insulin receptor (IR).…”

“…Kv1.3 is highly expressed in the olfactory bulb (20), and it appears to mediate a large fraction of the outward current detected in these neurons, which also express the insulin receptor (IR). The available data indicate that insulin down-regulates Kv1.3 activity in the olfactory bulb (19,21) through the phosphorylation of multiple tyrosine residues in Kv1.3 by IR kinase.…”

Sexual dimorphism and developmental expression of signal‐transduction machinery in the vomeronasal organ

“…Exposure times were adjusted to account for differences in reactivity across antisera. Films were scanned with a Hewlett-Packard Photosmart Scanner (model 106-816) and analyzed with Quantiscan software (Biosoft, Cambridge, United Kingdom) as described previously (Fadool et al, 2000b). …”