ABSTRACT
Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) in the nervous system are implicated in a variety of neuronal functions including learning and memory, regulation of vigilance states and pain. Dysfunctions or genetic loss of these channels have been shown to cause human diseases such as epilepsy, depression, schizophrenia, and Parkinson's disease. The physiological functions of HCN1 and HCN2 channels in the nervous system have been analyzed using genetic knockout mouse models. By contrast, there are no such genetic studies for HCN3 channels so far. Here, we use a HCN3-deficient (HCN3-/-) mouse line, which has been previously generated in our group to examine the expression and function of this channel in the CNS. Specifically, we investigate the role of HCN3 channels for the regulation of circadian rhythm and for the determination of behavior. Contrary to previous suggestions we find that HCN3-/- mice show normal visual, photic, and non-photic circadian function. In addition, HCN3-/- mice are impaired in processing contextual information, which is characterized by attenuated long-term extinction of contextual fear and increased fear to a neutral context upon repeated exposure.
ABSTRACT
RATIONALE: The hyperpolarization-activated current I(h) that is generated by hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) plays a key role in the control of pacemaker activity in sinoatrial node cells of the heart. By contrast, it is unclear whether I(h) is also relevant for normal function of cardiac ventricles. OBJECTIVE: To study the role of the HCN3-mediated component of ventricular I(h) in normal ventricular function. METHODS AND RESULTS: To test the hypothesis that HCN3 regulates the ventricular action potential waveform, we have generated and analyzed a HCN3-deficient mouse line. At basal heart rate, mice deficient for HCN3 displayed a profound increase in the T-wave amplitude in telemetric electrocardiographic measurements. Action potential recordings on isolated ventricular myocytes indicate that this effect was caused by an acceleration of the late repolarization phase in epicardial myocytes. Furthermore, the resting membrane potential was shifted to more hyperpolarized potentials in HCN3-deficient mice. Cardiomyocytes of HCN3-deficient mice displayed approximately 30% reduction of total I(h). At physiological ionic conditions, the HCN3-mediated current had a reversal potential of approximately -35 mV and displayed ultraslow deactivation kinetics. CONCLUSIONS: We propose that HCN3 together with other members of the HCN channel family confer a depolarizing background current that regulates ventricular resting potential and counteracts the action of hyperpolarizing potassium currents in late repolarization. In conclusion, our data indicate that HCN3 plays an important role in shaping the cardiac action potential waveform.
Subject(s)
Action Potentials/physiology , Cyclic Nucleotide-Gated Cation Channels/physiology , Ventricular Function/physiology , Animals , Cyclic Nucleotide-Gated Cation Channels/deficiency , Cyclic Nucleotide-Gated Cation Channels/genetics , Electrocardiography , Heart Rate/physiology , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Myocytes, Cardiac/physiology , Potassium Channels , Sinoatrial Node/physiologyABSTRACT
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.
