Abstracts

Rationale: Altered neuronal excitability in the epileptic brain is often associated with changes in the molecular composition and surface expression of ion channels. Among these, the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels mediate the sub-threshold current Ih and therefore contribute significantly to the regulation of intrinsic excitability of neurons. Four HCN isoforms (HCN1-4) can assemble in different combinations to yield homo- or hetero- tetrameric channels. Importantly, the molecular composition of the channels determines their biophysical properties and therefore influences their precise roles in specific brain regions and sub-cellular compartments. Seizure-induced heteromerization of HCN1/2 is a likely contributing mechanism to altered Ih in epilepsy (Brewster et al. 2005; Zha et al. 2008), yet very little is known about the cellular and molecular pathways that govern the assembly and membrane expression of heteromric HCN channels. Methods: To directly study heteromeric HCN1/2 channels on the cell surface, we employed the sensitized-emission Förster Resonance Energy Transfer (FRET) technique, combined with Total Internal Reflection Fluorescence (TIRF) microscopy in live cells. This was achieved by heterologous co-expression of HCN1 and HCN2 channel constructs, fused to either the YFP variant mCitrine or the CFP variant mCerulean at their N' terminus. Results: Co-expression of fluorophore-fused HCN1 and HCN2 channels in HEK293 and CHO cells resulted in a positive FRET signal (p<0.001, n=38; p<0.01,n=31), indicating HCN1/2 heteromerization in these cells. In contrast, a control group where mCitrine-fused HCN channels were co-expressed with un-fused mCerulean (n=66,32) resulted only in negligible signal, supporting the specificity of the HCN1/2 FRET signal. To study HCN channel heteromerization in neurons, we utilized TIRF/FRET imaging in primary hippocampal neurons. In these pilot experiments, positive FRET signal was detected along the somata and dendrites of neurons, indicating the presence of heteromeric HCN1/2 channels. Simultaneous TIRF imaging of HCN1 and HCN2 channels was accomplished, thus providing for the first time a tool to study isoform-specific dynamic properties of HCN channel surface expression. Conclusions: These results provide a first-time visualization of heteromeric HCN1/2 channels in live cells, demonstrating both intracellular and surface expression patterns. The FRET/TIRF approach emerges as a beneficial tool for further inquiries into the cellular pathways that govern heteromerization and isoform-specific targeting of HCN channels to the cell membrane, which is tightly controlled in a variety of physiological and pathological contexts. Supported by NIH grants R37 NS35439; T32 NS045540