Journal: Journal of neurophysiology

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

doi:

Figure Lengend Snippet: Activation of IR kinase induces suppression of Kv1.3. A: human embryonic kidney (HEK 293) cell cotransfected with Kv1.3 and insulin receptor kinase (IR) was voltage-clamped in the cell-attached configuration at –90 mV and stepped in 10-mV increments to 0 mV. Left, recorded using control bath solution. Middle, same cell after 20 min of bath-applied insulin (0.1 μg/ml). Right, cell-attached patch from a HEK 293 cell cotransfected with Kv1.3 and IR containing a truncated β subunit (IRtrunc; see methods) after an identical application of insulin under the same voltage protocol. For statistical comparisons, see Table 1. B: current–voltage plot of HEK 293 cells transfected as in A. Patches were voltage-clamped at –80 mV and stepped in 20-mV increments to +40 mV. ■ = Kv1.3 + IR control, ● = Kv1.3 + IR Insulin, ▲ = Kv1.3 + IR(trunc) Insulin. Inset: plot of the mean peak current magnitude of these patches as measured at the 140-mV depolarization step. Open bar, Kv1.3 + IR Control; solid bar, Kv1.3 + IR Insulin; crosshatched bar, Kv1.3 + IR(trunc) Insulin; *, significantly different paired t-test. C: HEK 293 cells cotransfected with Kv1.3 plus IR and sequentially double-labeled with Kv1.3 (1:200) and IRβ (1:100) as viewed at ×40 by confocal microscopy. No fluorescent signal is observed in the absence of primary antiserum. Arrows denote cells immunocytochemically labeled for both proteins, indicating the uptake of both cDNA constructs into single cells.

Article Snippet: Polyclonal antibody directed against the β subunit of the human insulin receptor was purchased from Upstate Biotechnology.

Techniques: Activation Assay, Control, Transfection, Labeling, Confocal Microscopy, Construct