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Figure 5 | Neural Development

Figure 5

From: Distinct perinatal features of the hyperpolarization-activated non-selective cation current Ih in the rat cortical plate

Figure 5

Developmental expression change of HCN subunits. (A) Quantitative real-time PCR of rat neocortical tissue for developmental stages E20, P0 and P30 revealed age-dependent changes in HCN3 and HCN4 mRNA expression levels, whereas the expression of HCN1 and HCN2 remained stable. Data were normalized to respective HPRT expression. Experiments were repeated three times and error bars represent SEM. (B) A family of pharmacologically isolated Ih at E20 recorded from a holding voltage of −40 mV and activation potentials between −40 and −130 mV confirmed a functional expression of Ih already at E20. (C) Western blot analysis from neocortical lysates for HCN1-HCN4 proteins at P0 and P30. For all blots we used anti-beta-actin as loading control and the corresponding subunits (HCN1-HCN4) over-expressed in HEK293 cells as positive controls (ctrl, right lanes). HCN1, HCN2 and HCN4 membrane protein fraction probes were treated with peptide:N-glycosidase F (PNGase F) in order to remove N-linked glyocsylation. HCN1 was detected in both, P0 and P30 protein extracts, but at higher molecular weights at P30. Deglycosylation at P30 shifted the band to lower molecular weights. However, no shift was observed at P0. Note that the bands at P0 still do not match the bands of deglyocsylated P30 protein. HCN2 was not detectable at P0 but at P30. Deglyocosylation of protein extracts shifted the top band to a lower molecular weight in the P30 samples. HCN4 was only detectable at P0 and the upper band was partially reduced by PNGase F treatment. HCN3 was present in membrane extracts of P0 neocortex tissue whereas no protein was detectable in P30 membrane fractions.

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