Dataset Information

Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels.

ABSTRACT: We have isolated a novel cDNA, that appears to represent a new class of ion channels, by using the yeast two-hybrid system and the SH3 domain of the neural form of Src (N-src) as a bait. The encoded polypeptide, BCNG-1, is distantly related to cyclic nucleotide-gated channels and the voltage-gated channels, Eag and H-erg. BCNG-1 is expressed exclusively in the brain, as a glycosylated protein of approximately 132 kDa. Immunohistochemical analysis indicates that BCNG-1 is preferentially expressed in specific subsets of neurons in the neocortex, hippocampus, and cerebellum, in particular pyramidal neurons and basket cells. Within individual neurons, the BCNG-1 protein is localized to either the dendrites or the axon terminals depending on the cell type. Southern blot analysis shows that several other BCNG-related sequences are present in the mouse genome, indicating the emergence of an entire subfamily of ion channel coding genes. These findings suggest the existence of a new type of ion channel, which is potentially able to modulate membrane excitability in the brain and could respond to regulation by cyclic nucleotides.

SUBMITTER: Santoro B

PROVIDER: S-EPMC25120 | biostudies-literature | 1997 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels.

Santoro B B Grant S G SG Bartsch D D Kandel E R ER

Proceedings of the National Academy of Sciences of the United States of America 19971201 26

We have isolated a novel cDNA, that appears to represent a new class of ion channels, by using the yeast two-hybrid system and the SH3 domain of the neural form of Src (N-src) as a bait. The encoded polypeptide, BCNG-1, is distantly related to cyclic nucleotide-gated channels and the voltage-gated channels, Eag and H-erg. BCNG-1 is expressed exclusively in the brain, as a glycosylated protein of approximately 132 kDa. Immunohistochemical analysis indicates that BCNG-1 is preferentially expressed ...[more]

PMID: 9405696

Similar Datasets

Project description:Human ether-á-go-go (eag)-related gene (hERG) potassium channels play a critical role in cardiac repolarization and are characterized by unusually slow closing (deactivation) kinetics. The N-terminal "eag" domain and a C-terminal C-linker/cyclic nucleotide-binding homology domain (CNBHD) are required for regulation of slow deactivation. The region between the S4 and S5 transmembrane domains (S4-S5 linker) is also implicated in this process, but the mechanism for regulation of slow deactivation is unclear. Here, using an eag domain-deleted channel (hERG ?eag) fused to Citrine fluorescent protein, we found that most channels bearing individual alanine mutations in the S4-S5 linker were directly regulated by recombinant eag domains fused to a cyan fluorescent protein (N-eag-CFP) and had robust Förster resonance energy transfer (FRET). Additionally, a channel bearing a group of eight alanine residues in the S4-S5 linker was not measurably regulated by N-eag-CFP domains, but robust FRET was measured. These findings demonstrate that the eag domain associated with all of the S4-S5 linker mutant channels. In contrast, channels that also lacked the CNBHD (hERG ?eag ?CNBHD-Citrine) were not measurably regulated by N-eag-CFP nor was FRET detected, suggesting that the C-linker/CNBHD was required for eag domains to directly associate with the channel. In a FRET hybridization assay, N-eag-CFP had robust FRET with a C-linker/CNBHD-Citrine, suggesting a direct and specific interaction between the eag domain and the C-linker/CNBHD. Lastly, coexpression of a hERG subunit lacking the CNBHD and the distal C-terminal region (hERG ?pCT-Citrine) with hERG ?eag-CFP subunits had FRET and partial restoration of slow deactivation. Collectively, these findings reveal that the C-linker/CNBHD, but not the S4-S5 linker, was necessary for the eag domain to associate with the channel, that the eag domain and the C-linker/CNBHD were sufficient for a direct interaction, and that an intersubunit interaction between the eag domain and the C-linker/CNBHD regulated slow deactivation in hERG channels at the plasma membrane.

Project description:Analysis of transcriptome in moss Physcomitrella patens CNGCb null mutant at 25 and 34 degrees C for 30 minutes. Results provide insight into role of CNGCb in acquired thermotolerance induced by non-lethal heat treatment. Typically at dawn of a hot summer day, land plants need precise molecular thermometers to sense harmless increments in the ambient temperature to timely develop a heat-shock response (HSR) and accumulate protective heat shock proteins (Hsps), in anticipation of upcoming harmful temperatures at mid-day. Here, we found that the CNGCb gene from Physcomitrella patens and its Arabidopsis ortholog CNGC2, encode for a component of cyclic nucleotide gated Ca2+ channels acting as the primary thermosensors of land plant cells. Disruption of CNGCb or CNGC2 produced a hyper-thermosensitive phenotype, giving rise to a HSR and acquired thermotolerance at significantly milder heat-priming treatments than in wild type plants. In an aequorin-expressing moss, CNGCb loss-of-function caused altered Ca2+ signaling and a sustained Ca2+ influx. Patch clamp recordings on moss protoplasts showed the presence of three distinct thermo-responsive Ca2+-channels in wild type cells. Deletion of CNGCb led to a total absence of one, and it increased the open probability of the remaining two thermo-responsive Ca2+ channels. Thus, both in Arabidopsis and moss, CNGC2 and CNGCb are expected to form with other related CNGCs, heteromeric Ca2+ channels in the plasma membrane that respond to mild increments in the ambient temperature by triggering an optimal HSR, leading to the onset of plant acquired thermotolerance. The WT moss tissues were heat-shocked for a half an hour at 34°C and 38°C and CNGCb at 25and 34°C followed by liquid nitrogen freezing. Total RNA was isolated using RNeasy Mini Kit (QIAGEN, Hilden, Germany) and two biological replicate samples for each treatment, were extracted. An Agilent-certified microarray service lab (MOgene, LC, St. Louis, MO, USA) was used to verify the integrity of the RNA and perform the microarray experiments. Two biological replicates were performed.

Dataset Information

Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels.

Publications

Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets