The ability of excitable cells to generate rhythmic, spontaneously firing action potentials is called pacemaking. In heart, pacemaking is accomplished by rhythmic discharge of the sinoatrial node (SA node). The firing rate is determined by a specific current slowly depolarizing a membrane to the threshold level, triggering the action potential. The ionic conductance underlying the cardiac pacemaking was identified over 20 years ago and termed If (f for funny)1, 2. At the same time, a similar current was described in neurons and in retina, termed, respectively, Ih (h for hyperpolarization-activated) and Iq (q for queer)2. The unique features of If/Ih/Iq current are: (1) its activation by hyperpolarization; (2) conductance of Na+ and K+ ions (K+/Na+ ~3-4); (3) blocking by low concentration (0.1-5 mM) of extracellular Cs+; (4) enhancement by cyclic AMP (cAMP). Surprisingly, the cAMP dependence was not mediated by protein phosphorylation, but involved direct binding of the cyclic nucleotide to the channel2.
In the SA node, hyperpolarization at the termination of the cardiac action potential leads to activation of If current, which gradually depolarizes the membrane. Activation of β-adrenergic receptors by sympathetic stimulation leads to activation of Gs protein and adenylate cyclase. The synthesized cAMP binds to the channel, shifting its activation curve to more positive voltages, which results in the increase of the diastolic depolarization rate, leading to heart rate acceleration1, 2. The switch from slow-wave sleep to waking and attentiveness of the animal is correlated with cAMP-dependent changes in Ih activity in thalamic neurons. Binding of norepinephrine or serotonin to the appropriate receptors increases the intracellular cAMP concentration. Higher cAMP concentration leads to activation of Ih and facilitates the shift of these neurons from a state of rhythmic oscillation and low excitability during slow-wave sleep to a state of increased excitability and responsiveness during periods of waking, attentiveness and cognition3.
HCN family are pacemaking channels
The molecular counterpart of Ih currents was unknown until 1997. The first channel was cloned via a yeast two-hybrid system by screening for proteins interacting with N-Src4. Independently, channels underlying Ih currents were cloned by searching the EST databases for similarities with cyclic nucleotide-binding domain (CNBD) of CNG channels5.
In 1998, the common nomenclature was accepted: the new clones were designated HCN for Hyperpolarization-activated Cyclic Nucleotide-gated channels6. Structurally, HCN channels belong to the Shaker (6TM) superfamily, with 6 transmembrane domains and pore region between S5 and S6. The CNBD is located in the C-terminus region. CNBD is also present in two other groups of 6TM channels: cyclic nucleotide-gated (CNG) cation channels and EAG (KCNH) family of K+ channels. While CNG are non-selective cation channels, permeable to Ca2+, and EAG family are highly selective K+ channels, HCN channels are permeable to Na+ and K+, with a slight selectivity to the latter. In agreement with this, the P region of HCN channels contains GYG signature sequence, characteristic to most K+ channels. However, the next residue, which is D or E in most K+ channels, varies in different HCN channels2,7.
To date, four different HCN channels (HCN1-4), have been identified in mammals. When functionally expressed, they give rise to Ih-like currents, differing in their activation kinetics and cAMP modulation. Based on their similarity with other 6TM channels, one can suggest a possible tetrameric architecture for the functional unit. When heterologously expressed, HCN1 and HCN2 form functional heteromers with intermediate properties8.
The four HCN channels differ in their tissue localization. HCN1, the least sensitive to cAMP, is expressed in cerebral cortex, hippocampus, cerebellum, and facial motor nucleus11,12. In agreement with this, the cAMP dependent shift of Ih is minimal in hippocampal neurons compared to the SA node. HCN1 is the only HCN channel in retinal photoreceptors and the major isoform in dorsal root ganglia neurons11. HCN2 and HCN3 are widespread in the brain. HCN2 is most highly abundant in mamillary bodies, pontine nucleus, ventral cochlear nucleus, and nucleus of the trapezoid body, while HCN3 expression is most pronounced in supraoptic nucleus of hypothal amus1. The slowest and the most sensitive to cAMP, HCN4, is the major isoform in the SA node11,13,14. In brain, HCN4 is mostly expressed in medial habenula and in thalamus12. Ih/If current is a target of a class of agents called “specific bradycardic agents”, like UL-FS 49 (zatebradine)16, its derivative DK-AH 26815 or ZD72889. The latter has been shown to block heterologously expressed HCN110. However, the detailed data of the specificity of these agents is still unavailable. Like native Ih currents, heterologously expressed HCN channels are blocked by low concentrations of Cs+ and are unaffected by classic K+ channel blockers, Ba2+ or tetraethylammonium (TEA).
Our knowledge of HCN channels is still fragmentary, partly due to their recent discovery and lack of specific molecular tools.
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