Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels comprise a small subfamily of proteins within the superfamily of pore-loop cation channels. In mammals, the HCN channel family comprises four members (HCN1-4) that are expressed in heart and nervous system. The current produced by HCN channels has been known as Ih (or If or Iq). Ih has also been designated as pacemaker current, because it plays a key role in controlling rhythmic activity of cardiac pacemaker cells and spontaneously firing neurons. Extensive studies over the last decade have provided convincing evidence that Ih is also involved in a number of basic physiological processes that are not directly associated with rhythmicity. Examples for these non-pacemaking functions of Ih are the determination of the resting membrane potential, dendritic integration, synaptic transmission, and learning. In this review we summarize recent insights into the structure, function, and cellular regulation of HCN channels. We also discuss in detail the different aspects of HCN channel physiology in the heart and nervous system. To this end, evidence on the role of individual HCN channel types arising from the analysis of HCN knockout mouse models is discussed. Finally, we provide an overview of the impact of HCN channels on the pathogenesis of several diseases and discuss recent attempts to establish HCN channels as drug targets.

Pacemaker activity of spontaneously active neurons and heart cells is controlled by a depolarizing, mixed Na+/K+ current, named Ih (or I(f) in the sinoatrial node of the heart). This current is activated on hyperpolarization of the plasma membrane. In addition to depolarizing pacemaker cells, Ih is involved in determining the resting membrane potential of neurons and provides a mechanism to limit hyperpolarizing currents in these cells. Hormones and neurotransmitters that induce a rise in cyclic AMP levels increase Ih by a mechanism that is independent of protein phosphorylation, and which involves direct binding of the cyclic nucleotide to the channel that mediates Ih. Here we report the molecular cloning and functional expression of the gene encoding a hyperpolarization-activated cation channel (HAC1) that is present in brain and heart. This channel exhibits the general properties of Ih channels. We have also identified full-length sequences of two related channels, HAC2 and HAC3, that are specifically expressed in the brain, indicating the existence of a family of hyperpolarization-activated cation channels.

Hyperpolarization-activated cation (HCN) channels are believed to be involved in the generation of cardiac pacemaker depolarizations as well as in the control of neuronal excitability and plasticity. The contributions of the four individual HCN channel isoforms (HCN1±4) to these diverse functions are not known. Here we show that HCN2-de®cient mice exhibit spontaneous absence seizures. The thalamocortical relay neurons of these mice displayed a near complete loss of the HCN current, resulting in a pronounced hyperpolarizing shift of the resting membrane potential, an altered response to depolarizing inputs and an increased susceptibility for oscillations. HCN2-null mice also displayed cardiac sinus dysrhythmia, a reduction of the sinoatrial HCN current and a shift of the maximum diastolic potential to hyperpolarized values. Mice with cardiomyocytespeci®c deletion of HCN2 displayed the same dysrhythmia as mice lacking HCN2 globally, indicating that the dysrhythmia is indeed caused by sinoatrial dysfunction. Our results de®ne the physiological role of the HCN2 subunit as a major determinant of membrane resting potential that is required for regular cardiac and neuronal rhythmicity.

Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIbeta phosphorylated the IP3R and cGKIbeta, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIbeta, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIbeta in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.

A.Ludwig and X.Zong contributed equally to this work Cardiac pacemaking is produced by the slow diastolic depolarization phase of the action potential. The hyperpolarization-activated cation current (I f ) forms an important part of the pacemaker depolarization and consists of two kinetic components (fast and slow). Recently, three full-length cDNAs encoding hyperpolarization-activated and cyclic nucleotide-gated cation channels (HCN1-3) have been cloned from mouse brain. To elucidate the molecular identity of cardiac pacemaker channels, we screened a human heart cDNA library using a highly conserved neuronal HCN channel segment and identified two cDNAs encoding HCN channels. The hHCN2 cDNA codes for a protein of 889 amino acids. The HCN2 gene is localized on human chromosome 19p13.3 and contains eight exons spanning~27 kb. The second cDNA, designated hHCN4, codes for a protein of 1203 amino acids. Northern blot and PCR analyses showed that both hHCN2 and hHCN4 are expressed in heart ventricle and atrium. When expressed in HEK 293 cells, either cDNA gives rise to hyperpolarization-activated cation currents with the hallmark features of native I f . hHCN2 and hHCN4 currents differ profoundly from each other in their activation kinetics, being fast and slow, respectively. We thus conclude that hHCN2 and hHCN4 may underlie the fast and slow component of cardiac I f , respectively.