Figure 7.

Calcineurin regulation of Kv4.2 surface expression in hippocampal neurons and COS7 cells. A, COS7 cells are cotransfected with Kv4.2 and AKAP79, and treated with CaN inhibitor FK506 (4 μm, 30 min) followed with Ht31 control peptide or Ht31 (10 μm, 15 min). Surface proteins were labeled with NHS-SS-Biotin and probed with mouse anti-Kv4.2 (1:2000). Cells with FK506 treatment had 20% decrease of surface Kv4.2 expression in the Ht31 control group. In contrast, the cells treated with both FK506 and Ht31 show no significant changes compared with the Ht31 control. GAPDH served as a loading control. β-Actin is labeled as negative surface control. B, Pooled data normalized to total Kv4.2 expression shows that FK506 decreases surface Kv4.2 expression by ∼40% (n = 5, p < 0.05) and no difference between control and FK506 + Ht31 treatment (n = 5, p > 0.05). Error bars represent SEM. C, In electrophysiological recordings from cultured hippocampal neurons, peak A-current density was significantly decreased by FK506 (4 μm, 20 min) treatment (n = 9) compared with control peptide alone (n = 14, p < 0.05). This decrease was also found in neurons cotreated with FK506 and Ht31 peptide (n = 13) compared with Ht31 treatment alone (n = 11). As with the biochemical results, cells treated with both FK506 and Ht31 show no significant changes compared with the Ht31 control peptide in the absence of FK506. Error bars represent SEM. Calibration: 200 pA, 200 ms. p < 0.05.