The role of subunit composition on prepulse facilitation of the cardiac L-type calcium channel

AbstractFacilitation of calcium current by depolarizing prepulses has been observed in many cells including cardiac muscle. The mechanism underlying prepulse facilitation is controversial with respect to the requirements of channel subunits and cAMP kinase. We found that coexpression of the cardiac α1C-a subunit with the cardiac β2a subunit significantly promotes the facilitation of IBa by strong depolarizing prepulses. The magnitude of IBa facilitation depended on the voltage potential of the prepulse and the interval duration between prepulse and test pulse. Prepulse facilitation was not affected by coexpression of AKAP79 and conditions favoring cAMP-dependent phosphorylation. Prepulse facilitation was also observed in cells expressing an α1C-a subunit which was truncated at residue 1733 removing the cAMP kinase site at Ser-1928. Facilitation was abolished by coexpression of the α2δ-1 or α2δ-3 subunit. We conclude that the expressed α1C-a β2a complex is sufficient to support prepulse facilitation. Facilitation is prevented by coexpression of the α2δ subunit

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

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 cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 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

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