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.
Subject(s)
Brain/physiology , Heart/physiology , Ion Channels/physiology , Muscle Proteins , Sinoatrial Node/physiology , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cloning, Molecular , Cyclic Nucleotide-Gated Cation Channels , Embryo, Mammalian , Humans , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Ion Channels/chemistry , Ion Channels/genetics , Kidney , Kinetics , Membrane Potentials/physiology , Molecular Sequence Data , Muscle, Skeletal/physiology , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Organ Specificity , Potassium Channels , RNA, Messenger/analysis , RNA, Messenger/genetics , Rabbits , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Transcription, Genetic , TransfectionABSTRACT
Agrin controls the formation of the neuromuscular junction. Whether it regulates the differentiation of other types of synapses remains unclear. Therefore, we have studied the role of agrin in cultured hippocampal neurons. Synaptogenesis was severely compromised when agrin expression or function was suppressed by antisense oligonucleotides and specific antibodies. The effects of antisense oligonucleotides were found to be highly specific because they were reversed by adding recombinant agrin and could not be detected in cultures from agrin-deficient animals. Interestingly, the few synapses formed in reduced agrin conditions displayed diminished vesicular turnover, despite a normal appearance at the EM level. Thus, our results demonstrate the necessity of agrin for synaptogenesis in hippocampal neurons.
