Abstract
Hyperpolarization-activated cation channels of the HCN gene family are crucial for the regulation of cell excitability. Importantly, these channels play a pivotal role in the control of cardiac and neuronal pacemaker activity. Dysfunction of HCN channels has been associated with human diseases, including cardiac arrhythmia, epilepsy, and neuropathic pain. The properties of three HCN channel isoforms (HCN1, HCN2, and HCN4) have been extensively investigated. By contrast, due to the lack of an efficient heterologous expression system, the functional characteristics of HCN3 were by and large unknown so far. Here, we have used lentiviral gene transfer to overexpress HCN3 in HEK293T cells. HCN3 currents revealed slow activation and deactivation kinetics and were effectively blocked by extracellular Cs+ and the bradycardic agent ivabradine. Cyclic AMP and cGMP had no significant impact on activation kinetics but induced a 5-mV shift of the half-maximal activation voltage (V0.5) to more hyperpolarized potentials. A negative shift of V0.5 induced by cyclic nucleotides is an unprecedented feature within the HCN channel family. The expression of HCN3 in mouse brain was examined by Western blot analysis using a specific antibody. High levels of protein were detected in olfactory bulb and hypothalamus. In contrast, only very low expression was found in cortex. Using reverse transcriptase PCR transcripts of HCN3 were also detected in heart ventricle. In conclusion, the distinct expression pattern in conjunction with the unusual biophysical properties implies that HCN3 may play an unique role in the body.
Highlights
The hyperpolarization-activated cation current, termed Ih or If, is widely expressed in heart cells and neurons
Expression of HCN3 in Mouse Heart and Brain—RT-PCR revealed that HCN3 transcripts are present in the mouse brain and heart ventricle (Fig. 1A)
This band was not observed in non-transfected cells and in cells transfected with HCN1, HCN2, or HCN4, demonstrating the specificity of the anti-HCN3 antibody (Fig. 1B)
Summary
The hyperpolarization-activated cation current, termed Ih or If, is widely expressed in heart cells and neurons. The current is best known for its prime role in the generation of rhythmic activity in cardiac and neuronal pacemaker cells [1, 2]. Recent evidence suggests that Ih is involved in diseases making the channel a promising target for drug therapy. Ih is encoded by a family of four hyperpolarization-activated cyclic nucleotide-gated (HCN11– 4) channels [1, 10]. The functional properties of three members of the HCN channel family (HCN1, HCN2, and HCN4) have been extensively studied using heterologous expression. We show that HCN3 reveals some properties shared by other HCN channel types but is unique among these channels by being rather inhibited than activated by cyclic nucleotides
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