Center for Integrated Protein Science CIPS-M and Zentrum für Pharmaforschung, Department Pharmazie, Pharmakologie für Naturwissenschaften, Ludwig-Maximilians-Universität München, Munich, Germany
...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 I h (or I f or I q ). I h 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 I h is also involved in a number of basic physiological processes that are not directly associated with rhythmicity. Examples for these non-pacemaking functions of I h 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.
It is highly debated how cyclic adenosine monophosphate-dependent regulation (CDR) of the major pacemaker channel HCN4 in the sinoatrial node (SAN) is involved in heart rate regulation by the ...autonomic nervous system. We addressed this question using a knockin mouse line expressing cyclic adenosine monophosphate-insensitive HCN4 channels. This mouse line displayed a complex cardiac phenotype characterized by sinus dysrhythmia, severe sinus bradycardia, sinus pauses and chronotropic incompetence. Furthermore, the absence of CDR leads to inappropriately enhanced heart rate responses of the SAN to vagal nerve activity in vivo. The mechanism underlying these symptoms can be explained by the presence of nonfiring pacemaker cells. We provide evidence that a tonic and mutual interaction process (tonic entrainment) between firing and nonfiring cells slows down the overall rhythm of the SAN. Most importantly, we show that the proportion of firing cells can be increased by CDR of HCN4 to efficiently oppose enhanced responses to vagal activity. In conclusion, we provide evidence for a novel role of CDR of HCN4 for the central pacemaker process in the sinoatrial node.
Congenital absence of cone photoreceptor function is associated with strongly impaired daylight vision and loss of color discrimination in human achromatopsia. Here, we introduce viral gene ...replacement therapy as a potential treatment for this disease in the CNGA3−/− mouse model. We show that such therapy can restore cone-specific visual processing in the central nervous system even if cone photoreceptors had been nonfunctional from birth. The restoration of cone vision was assessed at different stages along the visual pathway. Treated CNGA3−/− mice were able to generate cone photoreceptor responses and to transfer these signals to bipolar cells. In support, we found morphologically that treated cones expressed regular cyclic nucleotide-gated (CNG) channel complexes and opsins in outer segments, which previously they did not. Moreover, expression of CNGA3 normalized cyclic guanosine monophosphate (cGMP) levels in cones, delayed cone cell death and reduced the inflammatory response of Müller glia cells that is typical of retinal degenerations. Furthermore, ganglion cells from treated, but not from untreated, CNGA3−/− mice displayed cone-driven, light-evoked, spiking activity, indicating that signals generated in the outer retina are transmitted to the brain. Finally, we demonstrate that this newly acquired sensory information was translated into cone-mediated, vision-guided behavior.
Pacemaker activity of spontaneously active neurons and heart cells is controlled by a depolarizing, mixed Na+/K+ current, named Ih (or If 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.
The coassembly of homologous subunits to heteromeric complexes serves as an important mechanism in generating ion channel diversity. Here, we have studied heteromerization in the ...hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family. Using a combination of fluorescence confocal microscopy, coimmunoprecipitation, and electrophysiology we found that upon coexpression in HEK293 cells almost all dimeric combinations of HCN channel subunits give rise to the formation of stable channel complexes in the plasma membrane. We also identified HCN1/HCN2 heteromers in mouse brain indicating that heteromeric channels exist in vivo. Surprisingly, HCN2 and HCN3 did not coassemble to heteromeric channels. This finding indicates that heteromerization requires specific structural determinants that are not present in all HCN channel combinations. Using N-glycosidase F we show that native as well as recombinant HCN channels are glycosylated resulting in a 10–20-kDa shift in the molecular weight. Tunicamycin, an inhibitor of N-linked glycosylation, blocked surface membrane expression of HCN2. Similarly, a mutant HCN2 channel in which the putative N-glycosylation site in the loop between S5 and the pore helix was replaced by glutamine (HCN2N380Q) was not inserted into the plasma membrane and did not yield detectable whole-cell currents. These results indicate that N-linked glycosylation is required for cell surface trafficking of HCN channels. Cotransfection of HCN2N380Q with HCN4, but not with HCN3, rescued cell surface expression of HCN2N380Q. Immunoprecipitation revealed that this rescue was due to the formation of a HCN2N380Q/HCN4 heteromeric channel. Taken together our results indicate that subunit heteromerization and glycosylation are important determinants of the formation of native HCN channels.
Inhibition of cardiac sympathetic tone represents an important strategy for treatment of cardiovascular disease, including arrhythmia, coronary heart disease, and chronic heart failure. Activation of ...presynaptic alpha2-adrenoceptors is the most widely accepted mechanism of action of the antisympathetic drug clonidine; however, other target proteins have been postulated to contribute to the in vivo actions of clonidine.
To test whether clonidine elicits pharmacological effects independent of alpha2-adrenoceptors, we have generated mice with a targeted deletion of all 3 alpha2-adrenoceptor subtypes (alpha2ABC-/-). Alpha2ABC-/- mice were completely unresponsive to the analgesic and hypnotic effects of clonidine; however, clonidine significantly lowered heart rate in alpha2ABC-/- mice by up to 150 bpm. Clonidine-induced bradycardia in conscious alpha2ABC-/- mice was 32.3% (10 microg/kg) and 26.6% (100 microg/kg) of the effect in wild-type mice. A similar bradycardic effect of clonidine was observed in isolated spontaneously beating right atria from alpha2ABC-knockout and wild-type mice. Clonidine inhibited the native pacemaker current (I(f)) in isolated sinoatrial node pacemaker cells and the I(f)-generating hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 and HCN4 channels in transfected HEK293 cells. As a consequence of blocking I(f), clonidine reduced the slope of the diastolic depolarization and the frequency of pacemaker potentials in sinoatrial node cells from wild-type and alpha2ABC-knockout mice.
Direct inhibition of cardiac HCN pacemaker channels contributes to the bradycardic effects of clonidine gene-targeted mice in vivo, and thus, clonidine-like drugs represent novel structures for future HCN channel inhibitors.
Olfactory receptor neurons (ORNs) employ a cyclic nucleotide-gated (CNG) channel to generate a receptor current in response to an odorant-induced rise in cAMP. This channel contains three types of ...subunits, the principal CNGA2 subunit and two modulatory subunits (CNGA4 and CNGB1b). Here, we have analyzed the functional relevance of CNGB1 for olfaction by gene targeting in mice. Electro-olfactogram responses of CNGB1-deficient (CNGB1-/-) mice displayed a reduced maximal amplitude and decelerated onset and recovery kinetics compared with wild-type mice. In a behavioral test, CNGB1-/- mice exhibited a profoundly decreased olfactory performance. Electrophysiological recordings revealed that ORNs of CNGB1-/- mice weakly expressed a CNG current with decreased cAMP sensitivity, very rapid flicker-gating behavior and no fast modulation by Ca2+-calmodulin. Co-immunoprecipitation confirmed the presence of a CNGA2/CNGA4 channel in the olfactory epithelium of CNGB1-/- mice. This CNGA2/CNGA4 channel was targeted to the plasma membrane of olfactory knobs, but failed to be trafficked into olfactory cilia. Interestingly, we observed a similar trafficking defect in mice deficient for the CNGA4 subunit. In conclusion, these results demonstrate that CNGB1 has a dual function in vivo. First, it endows the olfactory CNG channel with a variety of biophysical properties tailored to the specific requirements of olfactory transduction. Second, together with the CNGA4 subunit, CNGB1 is needed for ciliary targeting of the olfactory CNG channel.
Opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated by direct binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus. ...Here, we show for the first time that in the HCN2 channel cGMP can also exert an inhibitory effect on gating via cGMP-dependent protein kinase II (cGKII)-mediated phosphorylation. Using coimmunoprecipitation and immunohistochemistry we demonstrate that cGKII and HCN2 interact and colocalize with each other upon heterologous expression as well as in native mouse brain. We identify the proximal C-terminus of HCN2 as binding region of cGKII and show that cGKII phosphorylates HCN2 at a specific serine residue (S641) in the C-terminal end of the CNBD. The cGKII shifts the voltage-dependence of HCN2 activation to 2-5 mV more negative voltages and, hence, counteracts the stimulatory effect of cGMP on gating. The inhibitory cGMP effect can be either abolished by mutation of the phosphorylation site in HCN2 or by impairing the catalytic domain of cGKII. By contrast, the inhibitory effect is preserved in a HCN2 mutant carrying a CNBD deficient for cGMP binding. Our data suggest that bidirectional regulation of HCN2 gating by cGMP contributes to cellular fine-tuning of HCN channel activity.
Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in ...brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity.
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