A conserved domain in the NH2 terminus important for assembly and functional expression of pacemaker channels.
Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits. Previously, we suggested that the NH(2) termini of the mouse HCN2 isoform were important for subunit co-assembly and functional channel expression. Using an alignment strategy together with yeast two-hybrid assays, patch clamp electrophysiology, and confocal imaging, we have now identified a domain within the NH(2) terminus of the HCN2 subunit that is responsible for interactions between NH(2) termini and promoting the trafficking of functional channels to the plasma membrane. This domain is composed of 52 amino acids, is located adjacent to the putative first transmembrane segment, and is highly conserved among the mammalian HCN isoforms. This conserved domain, but not the remaining unconserved NH(2)-terminal regions of HCN2, specifically interacted with itself in yeast two-hybrid assays. Moreover, the conserved domain was important for expression of currents. Whereas relatively normal whole cell HCN2 currents were produced by channels containing only the conserved domain, further deletion of this region, leaving only a more polar and putative coiled-coil segment, eliminated HCN2 currents and resulted in proteins that localized predominantly in perinuclear compartments. Thus, we suggest that this conserved domain is the critical NH(2)-terminal determinant of subunit co-assembly and trafficking of pacemaker channels.
Tran
Neil
N
Ion Channel Laboratory, School of Kinesiology, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
Proenza
Catherine
C
Macri
Vincenzo
V
Petigara
Fiona
F
Sloan
Erin
E
Samler
Shannon
S
Accili
Eric A
EA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D017353:Gene Deletion; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007150:Immunohistochemistry; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015221:Potassium Channels; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; C578711:Hcn2 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D009124:Muscle Proteins; D015221:Potassium Channels; D020033:Protein Isoforms