The family of cyclic nucleotide-regulated channels comprises two groups: the cyclic nucleotide-gated (CNG1) channels and the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels.
Cyclic Nucleotide-Gated Channels
CNG cation channels are ion channels whose activation is mediated by the direct binding of cGMP or cAMP to the channel protein (Biel et al., 1999b; Kaupp and Seifert, 2002; Matulef and Zagotta, 2003). CNG channels are expressed in the cilia of olfactory neurones and in outer segments of rod and cone photoreceptor neurones, where they play key roles in sensory transduction. Low levels of CNG channel transcripts have also been found in a variety of other tissues, including brain, testis, kidney, and heart. CNG channels are heterotetramers composed of homologous A subunits (CNGA1–CNGA4) and B subunits (CNGB1 and CNGB3) (Bradley et al., 2001). Both types of subunits are members of the six-transmembrane segment channel superfamily. In the cytosolic C terminus, CNG channel subunits carry a cyclic nucleotide-binding domain (CNBD) that serves as activation domain. The CNBD of CNG channels reveals significant sequence similarity to the CNBDs of other cyclic nucleotide receptors (Kaupp et al., 1989). The subunit stoichiometries have been determined for the channels expressed in rod photoreceptors (3 CNGA1: 1 CNGB1a) (Weitz et al., 2002; Zheng et al., 2002; Zhong et al., 2002), cone photoreceptors (2 CNGA3: 2 CNGB3) (Peng et al., 2004), and olfactory neurons (2 CNGA2: 1 CNGA4: 1 CNGB1b) (Zheng and Zagotta, 2004). The physiological relevance of CNGA2–4 and CNGB1 subunits has been elucidated by gene deletion in mice (Brunet et al., 1996; Biel et al., 1999a; Munger et al., 2001; Huttl et al., 2005).
CNG channels pass monovalent cations, such as Na+ and K+, but do not discriminate between them. Calcium is also permeable but at the same time acts as a voltage-dependent blocker of monovalent cation permeability (Frings et al., 1995; Dzeja et al., 1999). Moreover, Ca2+ provides feedback inhibition of CNG channel activity by binding to calmodulin (Kaupp and Seifert, 2002; Matulef and Zagotta, 2003). CNG channels reveal a higher sensitivity for cGMP than for cAMP. The extent of ligand discrimination varies significantly between the individual CNG channel types. Photoreceptor channels strongly discriminate between cGMP and cAMP, whereas the olfactory channel is almost equally sensitive to both ligands.
Drugs That Act on CNG Channels
Several drugs have been reported to block CNG channels, although not with very high affinity. l-cis-diltiazem has been studied most extensively. It blocks CNG channels in a voltage-dependent manner at micromolar concentration (Haynes, 1992). The d-cis-enantiomer of diltiazem that is used therapeutically as a blocker of the L-type calcium channel is much less effective than the l-cis-enantiomer in blocking CNG channels. High-affinity binding of l-cis-diltiazem is only seen in heteromeric CNG channels containing the CNGB1 subunit (Chen et al., 1993). CNG channels are also moderately sensitive to block by some other inhibitors of the L-type calcium channel (e.g., nifedipine), the local anesthetic tetracaine, and calmodulin antagonists (Kaupp and Seifert, 2002). Interestingly, LY83583 blocks both the soluble guanylate cyclase and some CNG channels at similar concentrations (Leinders-Zufall and Zufall, 1995). H-8, which has been widely used as a nonspecific cyclic nucleotide-dependent protein kinase inhibitor, blocks CNG channels, although at significantly higher concentrations than needed to inhibit protein kinases (Wei et al., 1997).
Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels
Like CNG channels, the HCN cation channels are members of the six-transmembrane superfamily (Kaupp and Seifert, 2001; Biel et al., 2002; Robinson and Siegelbaum, 2003). In contrast to most other voltage-gated channels, HCN channels open upon hyperpolarization and close at positive potential. The cyclic nucleotides cAMP and cGMP enhance HCN channel activity by shifting the activation curve of the channels to more positive voltages. The stimulatory effect of cyclic nucleotides does not depend on protein phosphorylation but is caused by direct interaction with the HCN channel protein. The current produced by HCN channels, termed Ih, If, or Iq, is found in a variety of excitable cells, including neurones, cardiac pacemaker cells, and photoreceptors (Pape, 1996; Robinson and Siegelbaum, 2003). The best-understood function of Ih is to control heart rate and rhythm by acting as “pacemaker current” in the sinoatrial (SA) node (Stieber et al., 2004). Ih is activated during the membrane hyperpolarization following the termination of an action potential and provides an inward Na+ current that slowly depolarizes the plasma membrane. Sympathetic stimulation of SA node cells raises cAMP levels and increases Ih, thus accelerating diastolic depolarization and heart rate. Stimulation of muscarinic acetylcholine receptors slows down heart rate by the opposite action. In neurons, Ih fulfills diverse functions, including generation of pacemaker potentials (“neuronal pacemaking”), determination of resting potential, transduction of sour taste, dendritic integration, control of synaptic transmission, and plasticity (Pape, 1996; Kaupp and Seifert, 2001; Robinson and Siegelbaum, 2003).
In mammals, the HCN channel family comprises four members (HCN1–HCN4) that share approximately 60% sequence identity to each other (Gauss et al., 1998; Ludwig et al., 1998; Santoro et al., 1998; Ludwig et al., 1999). HCN channels contain six-transmembrane helices (S1–S6) and assemble in tetramers (Zagotta et al., 2003). There is evidence that HCN subunits can coassemble to form heteromers (Much et al., 2003; Robinson and Siegelbaum, 2003). The S4 segment of the channels is positively charged and serves as voltage sensor (Mannikko et al., 2002). The C terminus of HCN channels contains a CNBD that confers regulation by cyclic nucleotides (Wainger et al., 2001; Zagotta et al., 2003). When expressed in heterologous systems, all four HCN channels generate currents displaying the typical features of native Ih: 1) activation by membrane hyperpolarisation; 2) permeation of Na+ and K+ with a permeability ratio PNa/PK of approximately 0.2; 3) positive shift of voltage dependence of channel activation by direct binding of cAMP; and 4) channel block by extracellular Cs+. The HCN1–HCN4 channels mainly differ from each other with regard to their speed of activation and the extent by which they are modulated by cAMP. HCN1 is the fastest channel, followed by HCN2, HCN3, and HCN4. Unlike HCN2 and HCN4, whose activation curves are profoundly shifted by cAMP, HCN1 is only weakly affected by cAMP (Kaupp and Seifert, 2001; Biel et al., 2002; Robinson and Siegelbaum, 2003).
HCN channels are found in neurons and heart cells. In SA node cells, HCN4 represents the predominantly expressed HCN channel isoform (Ishii et al., 1999; Moosmang et al., 2001; Stieber et al., 2003). In brain, all four HCN subunits have been detected (Notomi and Shigemoto, 2004). The expression levels and regional distribution of the HCN channel mRNAs vary profoundly between the respective channel types. HCN2 is the most abundant neuronal channel and is found almost ubiquitously in the brain. By contrast, HCN1 and HCN4 are enriched in specific regions of the brain such as thalamus (HCN4) or hippocampus (HCN1). HCN3 is expressed at low density in most parts of the brain but is enriched in olfactory bulb and some hypothalamic nuclei (Notomi and Shigemoto, 2004). HCN channels have also been detected in the retina (Muller et al., 2003) and some peripheral neurones such as dorsal root ganglion neurones (Moosmang et al., 2001). The specific roles of individual HCN channel types have been defined by analysis of mouse lines deficient for HCN1 (Nolan et al., 2003), HCN2 (Ludwig et al., 2003), and HCN4 (Stieber et al., 2003).
Drugs That Act on HCN Channels
Given the key role of HCN channels in cardiac pacemaking, these channels are promising pharmacological targets for the development of drugs used in the treatment of cardiac arrhythmias and ischemic heart disease. Several blockers of native Ih channels are known. The most extensively studied blocker is ZD7288 (BoSmith et al., 1993). Low micromolar concentrations of this agent specifically block both native Ih and cloned HCN channels in a voltage-dependent manner. Three other use-dependent blockers of Ih are ivabradine (Bois et al., 1996), zatebradine (Raes et al., 1998), and cilobradine (Stieber et al., 2004). Structurally, these substances are related to verapamil, a classic L-type calcium channel blocker. These agents block Ih at concentrations comparable to ZD7288. Ivabradine is considered as a heart rate-lowering agent in the therapy of angina pectoris.
↵1 Abbreviations: CNG, cyclic nucleotide-gated; HCN, hyperpolarization-activated, cyclic nucleotide-gated; CNBD, cyclic nucleotide-binding domain; LY83583, 6-(phenyl-amino)-5,8-quinolinedione; H-8, N-2-(methyl-amino)ethyl-5-isoquinolinesulfonamide; SA, sinoatrial; ZD7288, 4-(N-ethyl-N-phenylamino-1,2-dimethyl-6-(methyl-amino) pyrimidinum chloride.
The authors serve as the Subcommittee for Cyclic Nucleotide-Regulated Channels of the Nomenclature Committee of The International Union of Pharmacology.
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