Journal: The Journal of Neuroscience

Article Title: Recessive Loss-of-Function Mutation in the Pacemaker HCN2 Channel Causing Increased Neuronal Excitability in a Patient with Idiopathic Generalized Epilepsy

doi: 10.1523/JNEUROSCI.3727-11.2011

Figure Lengend Snippet: Transfection of human HCN2 into neonatal rat neurons. Single confocal sections of neuronal cultures cotransfected with the EYFP plasmid and with the empty pcDNA 3.1 plasmid (control) or the HCN2-containing pcDNA 3.1 plasmid as indicated. The HCN2 signal (red) was detected in HCN2-transfected cells also expressing EYFP (green) but not in control cells, suggesting that the signal from endogenous HCN2 channels was below detection level. Nuclei counterstained with DAPI (blue). Scale bar, 20 μm.

Article Snippet: Neonatal rat cortical neuron cultures Neocortical neurons were isolated from postnatal day 3 (P3) CD rat pups (Charles River).

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: The Journal of Neuroscience

Article Title: Recessive Loss-of-Function Mutation in the Pacemaker HCN2 Channel Causing Increased Neuronal Excitability in a Patient with Idiopathic Generalized Epilepsy

doi: 10.1523/JNEUROSCI.3727-11.2011

Figure Lengend Snippet: Properties of Ih from wt and E515K mutant HCN2 channels expressed in rat neonatal cortical neurons. A, Normalized current traces recorded with a double-step protocol to −75/−135 mV from a holding potential of −35 mV, showing that mutant channels activate at more negative voltages than wt channels. B, Plots of mean Ih activation curves of wt, wt/E515K, and E515K channels; a large negative shift of the E515K curve is apparent, while wt and wt/E515K curves essentially superimpose. Half-activation voltages (V1/2) and slope factors (s) from Boltzmann curve fitting were −74.0 ± 3.1 and 8.7 ± 0.7 mV (wt, n = 6), −75.5 ± 1.1 and 7.6 ± 1.1 mV (wt/E515K, n = 4), and −98.6 ± 2.6 and 14.7 ± 1.1 mV (E515K, n = 4). C, Mean activation time constant curves of wt (n = 12), wt/E515K (n = 7), and E515K channels (n = 11) showing superimposition of the first two and a shift to more negative voltages of the latter; curves drawn through points. D, Mean isochronal I-V curves, normalized to cell capacitance, for Ih recorded from cells transfected with empty plasmid, reflecting endogenous current (control, n = 11) and with wt (n = 14), wt/E515K (n = 9), and E515K channels (n = 18) upon application of 3 s steps in the range of −55 to −135 mV from a holding potential of −35 mV. Note that current densities from wt and wt/E515K channels are similar in the whole voltage range, and that the density from E515K channels approaches wt and wt/E515K densities at high negative voltages.

Article Snippet: Neonatal rat cortical neuron cultures Neocortical neurons were isolated from postnatal day 3 (P3) CD rat pups (Charles River).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation