Paradigm shift: new concepts for HCN4 function in cardiac pacemaking

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are the molecular correlate of the I-f current and are critically involved in controlling neuronal excitability and the autonomous rhythm of the heart. The HCN4 isoform is the main HCN channel subtype expressed in the sinoatrial node (SAN), a tissue composed of specialized pacemaker cells responsible for generating the intrinsic heartbeat. More than 40 years ago, the I-f current was first discovered in rabbit SAN tissue. Along with this discovery, a theory was proposed that cyclic adenosine monophosphate-dependent modulation of I-f mediates heart rate regulation by the autonomic nervous system-a process called chronotropic effect. However, up to the present day, this classical theory could not be reliably validated. Recently, new concepts emerged confirming that HCN4 channels indeed play an important role in heart rate regulation. However, the cellular mechanism by which HCN4 controls heart rate turned out to be completely different than originally postulated. Here, we review the latest findings regarding the physiological role of HCN4 in the SAN. We describe a newly discovered mechanism underlying heart rate regulation by HCN4 at the tissue and single cell levels, and we discuss these observations in the context of results from previously studied HCN4 mouse models

Cyclic nucleotide-regulated channels (CNG) in GtoPdb v.2023.1

Cyclic nucleotide-gated (CNG) channels are responsible for signalling in the primary sensory cells of the vertebrate visual and olfactory systems. CNG channels are voltage-independent cation channels formed as tetramers. Each subunit has 6TM, with the pore-forming domain between TM5 and TM6. CNG channels were first found in rod photoreceptors [83, 120], where light signals through rhodopsin and transducin to stimulate phosphodiesterase and reduce intracellular cyclic GMP level. This results in a closure of CNG channels and a reduced ‘dark current’. Similar channels were found in the cilia of olfactory neurons [181] and the pineal gland [71]. The cyclic nucleotides bind to a domain in the C terminus of the subunit protein: other channels directly binding cyclic nucleotides include hyperolarisation-activated, cyclic nucleotide-gated channels (HCN), ether-a-go-go and certain plant potassium channels.The HCN channels are cation channels that are activated by hyperpolarisation at voltages negative to ~-50 mV. The cyclic nucleotides cyclic AMP and cyclic GMP directly bind to the cyclic nucleotide-binding domain of HCN channels and shift their activation curves to more positive voltages, thereby enhancing channel activity. HCN channels underlie pacemaker currents found in many excitable cells including cardiac cells and neurons [65, 192]. In native cells, these currents have a variety of names, such as Ih, Iq and If. The four known HCN channels have six transmembrane domains and form tetramers. It is believed that the channels can form heteromers with each other, as has been shown for HCN1 and HCN4 [2]. High resolution structural studies of CNG and HCN channels has provided insight into the the gating processes of these channels [139, 146, 140]. A standardised nomenclature for CNG and HCN channels has been proposed by the NC-IUPHAR Subcommittee on voltage-gated ion channels [108]

Cyclic nucleotide-regulated channels (CNG) in GtoPdb v.2021.3

Cyclic nucleotide-gated (CNG) channels are responsible for signalling in the primary sensory cells of the vertebrate visual and olfactory systems. CNG channels are voltage-independent cation channels formed as tetramers. Each subunit has 6TM, with the pore-forming domain between TM5 and TM6. CNG channels were first found in rod photoreceptors [83, 120], where light signals through rhodopsin and transducin to stimulate phosphodiesterase and reduce intracellular cyclic GMP level. This results in a closure of CNG channels and a reduced ‘dark current’. Similar channels were found in the cilia of olfactory neurons [181] and the pineal gland [71]. The cyclic nucleotides bind to a domain in the C terminus of the subunit protein: other channels directly binding cyclic nucleotides include hyperolarisation-activated, cyclic nucleotide-gated channels (HCN), ether-a-go-go and certain plant potassium channels.The HCN channels are cation channels that are activated by hyperpolarisation at voltages negative to ~-50 mV. The cyclic nucleotides cyclic AMP and cyclic GMP directly bind to the cyclic nucleotide-binding domain of HCN channels and shift their activation curves to more positive voltages, thereby enhancing channel activity. HCN channels underlie pacemaker currents found in many excitable cells including cardiac cells and neurons [64, 192]. In native cells, these currents have a variety of names, such as Ih, Iq and If. The four known HCN channels have six transmembrane domains and form tetramers. It is believed that the channels can form heteromers with each other, as has been shown for HCN1 and HCN4 [2]. High resolution structural studies of CNG and HCN channels has provided insight into the the gating processes of these channels [139, 146, 140]. A standardised nomenclature for CNG and HCN channels has been proposed by the NC-IUPHAR Subcommittee on voltage-gated ion channels [108]

Hyperpolarization-activated cyclic nucleotide-gated cation channel 3 promotes HCC development in a female-biased manner

Sex differences in hepatocellular carcinoma (HCC) development are regulated by sex and non-sex chromosomes, sex hormones, and environmental factors. We previously reported that Ncoa5(+/-) mice develop HCC in a male-biased manner. Here we show that NCOA5 expression is reduced in male patient HCCs while the expression of an NCOA5-interacting tumor suppressor, TIP30, is lower in female HCCs. Tip30 heterozygous deletion does not change HCC incidence in Ncoa5(+/-) male mice but dramatically increases HCC incidence in Ncoa5(+/-) female mice, accompanied by hepatic hyperpolarization-activated cyclic nucleotide-gated cation channel 3 (HCN3) overexpression. HCN3 overexpression cooperates with MYC to promote mouse HCC development, whereas Hcn3 knockout preferentially hinders HCC development in female mice. Furthermore, HCN3 amplification and overexpression occur in human HCCs and correlate with a poorer prognosis of patients in a female-biased manner. Our results suggest that TIP30 and NCOA5 protect against female liver oncogenesis and that HCN3 is a female-biased HCC driver

cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells

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. The involvement of cAMP-dependent regulation of HCN4 in the chronotropic heart rate response is a matter of debate. Here the authors use a knockin mouse model expressing cAMP-insensitive HCN4 channels to discover an inhibitory nonfiring cell pool in the sinoatrial node and a tonic and mutual interaction between firing and nonfiring pacemaker cells that is controlled by cAMP-dependent regulation of HCN4, with implications in chronotropic heart rate responses

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy

Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications

KCNMA1 Encoded Cardiac BK Channels Afford Protection against Ischemia-Reperfusion Injury

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+-and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioninglike effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and reoxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP),but differed upon IP. While the area of infarction comprised 28 +/- 3% of the area at risk in wild-type, it was increased to 58 +/- 5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations

The two-pore channel TPCN2 mediates NAADP-dependent Ca2+-release from lysosomal stores

Second messenger-induced Ca2+-release from intracellular stores plays a key role in a multitude of physiological processes. In addition to 1,4,5-inositol trisphosphate (IP3), Ca2+, and cyclic ADP ribose (cADPR) that trigger Ca2+-release from the endoplasmatic reticulum (ER), nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a cellular metabolite that mediates Ca2+-release from lysosomal stores. While NAADP-induced Ca2+-release has been found in many tissues and cell types, the molecular identity of the channel(s) conferring this release remained elusive so far. Here, we show that TPCN2, a novel member of the two-pore cation channel family, displays the basic properties of native NAADP-dependent Ca2+-release channels. TPCN2 transcripts are widely expressed in the body and encode a lysosomal protein forming homomers. TPCN2 mediates intracellular Ca2+-release after activation with low-nanomolar concentrations of NAADP while it is desensitized by micromolar concentrations of this second messenger and is insensitive to the NAADP analog nicotinamide adenine dinucleotide phosphate (NADP). Furthermore, TPCN2-mediated Ca2+-release is almost completely abolished when the capacity of lysosomes for storing Ca2+ is pharmacologically blocked. By contrast, TPCN2-specific Ca2+-release is unaffected by emptying ER-based Ca2+ stores. In conclusion, these findings indicate that TPCN2 is a major component of the long-sought lysosomal NAADP-dependent Ca2+-release channel

Disturbed Processing of Contextual Information in HCN3 Channel Deficient Mice

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 rhythmand 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