Abstract
The pacemaker current I(f) of the sinoatrial node (SAN) is a major determinant of cardiac diastolic depolarization and plays a key role in controlling heart rate and its modulation by neurotransmitters. Substantial expression of two different mRNAs (HCN4, HCN1) of the family of pacemaker channels (HCN) is found in rabbit SAN, suggesting that the native channels may be formed by different isoforms. Here we report the cloning and heterologous expression of HCN1 from rabbit SAN and its specific localization in pacemaker myocytes. rbHCN1 is an 822-amino acid protein that, in human embryonic kidney 293 cells, displayed electrophysiological properties similar to those of I(f), suggesting that HCN1 can form a pacemaker channel. The presence of HCN1 in the SAN myocytes but not in nearby heart regions, and the electrophysiological properties of the channels formed by it, suggest that HCN1 plays a central and specific role in the formation of SAN pacemaker currents.
Highlights
The spontaneous electrical activity of mammalian heart arises from specialized myocytes of the cardiac sinoatrial node (SAN)1 Early studies of the electrical properties of SAN myocytes have revealed the existence of a hyperpolarization-activated current, named If, important in the generation and autonomic control of the diastolic (“pacemaker”) depolarization phase of the action potential [1]
It is almost identical to mHCN1 in the transmembrane regions and the cyclic nucleotide binding domain, but it has shorter cytoplasmic N and C termini
The N terminus of rbHCN1 cDNA is shorter by about 120 nucleotides, which represents a high GC-rich sequence in mouse; rbHCN1 lacks the CAG repeats corresponding to the long stretch of glutamines (poly(Q)) found in the cytoplasmic C terminus of the mouse clone (Fig. 1a)
Summary
Tissue Source—New Zealand White rabbits weighing 0.8 –1 kg were anesthetized by intramuscular injection of 4.6 mg/kg xylazine and 60 mg/kg ketamine and euthanized by cervical dislocation. Downstream of the stop codon of the uORF there was a third ATG (we refer to this ATG as M3 in Fig. 1b), which starts (in-frame with M2) a 793-amino acid ORF, which we called ⌬NrbHCN1, corresponding to a truncated form of rbHCN1, lacking the first 29 amino acids at the N terminus The sequence around this ATG showed no similarities to the Kozak consensus sequence. Radiolabeled antisense and sense cRNA probes were transcribed from the cDNA clone corresponding to the 3Ј-end of the rbHCN1 mRNA (the same probe used previously for Northern blot analysis; see above) after linearization with BamHI or KpnI (Promega), respectively, using T7 or T3 RNA polymerase (Epicentre) and 35S-labeled-UTP Both probes corresponded to the length of the insert (380 bp), and for in situ hybridization purposes they were digested to 100 nucleotides by mild alkaline hydrolysis. The images were acquired (with the same parameters for control and treated cells) by confocal microscopy (Leica TCS NT) using argon/ krypton (for rhodamine) and argon (for DAPI) lasers
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