T., Wilhelm S., Bruening-Wright A., Maylie J., Adelman J. we reported for the first time that this C terminus of Cav1.3 translocates to the nucleus where it functions PTP1B-IN-8 as a transcriptional regulator to modulate the function of Ca2+-activated K+ channels in atrial myocytes. Nuclear translocation of the C-terminal domain name of Cav1.3 is directly regulated by intracellular Ca2+. Utilizing a null mutant mouse model, we demonstrate that ablation of Cav1.3 results in a decrease in the protein expression of myosin light chain 2, PTP1B-IN-8 which interacts and increases the membrane localization of SK2 channels. null mutant mice (null mutant mice, the possible functional coupling between Cav1.3 and SK channels was tested (17). As it turns out, Cav1.3 colocalizes with SK2 channels in atrial myocytes. Moreover, the null mutation of results in a significant decrease in siRNA-mediated gene silencing, we report for the first time that this C terminus of Cav1.3 translocates to the nucleus where it functions as a transcription factor to regulate the expression of PTP1B-IN-8 SK2 channel interacting proteins. EXPERIMENTAL PROCEDURES All animal care and procedures were approved by the University of California, Davis, Institutional Animal Care and Use Committee. Animal use was in accordance with the National Institutes of Health and institutional guidelines. Plasmid Construction pGBKT7 vector made up of the GAL4 binding domain name (BD) and pGADKT7 vector made up of the GAL4 activation domain name (AD) were used for Y2H assays (Clontech, Palo Alto, CA). A bait construct was generated in pGBKT7 vector made up of a human cardiac SK2 C-terminal fragment encoding 380C487 amino acid residues (GenBankTM accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AY258141″,”term_id”:”37955867″,”term_text”:”AY258141″AY258141) (8) and used to screen a human heart cDNA library (MATCHMAKER, Clontech) as previously described (17). The construct was generated using PCR in-frame with the GAL4 BD and subcloned into the pGBKT7 vector. Constructs for the C-terminal domains of Cav1.3 and Cav1.2 Ca2+ channels in pGBKT7 were Cav1.3-Ct, 1505C2203; and Cav1.2-Ct, 1505C2171. The numbers refer to amino acid sequences according to the rat Cav1.3 Ca2+ channel (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”D38101″,”term_id”:”736711″,”term_text”:”D38101″D38101) and rabbit cardiac Cav1.2 Ca2+ channel (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”X15539″,”term_id”:”1509″,”term_text”:”X15539″X15539), respectively. The cDNA fragments were placed in-frame with the DNA binding domain name of GAL4 using polymerase chain reaction (PCR) and subcloned into the pGBKT7 vector. All clones were sequence verified. Rat Cav1.3 (1D) Ca2+ channel in pCMV6b vector was a kind gift from Dr. S. Seino (Chiba University, Chiba, Japan) and rabbit cardiac Cav1.2 Ca2+ channel was a kind gift from Dr. Timothy Kamp (University of Wisconsin). pEYFP-N1-Cav1.3-ct was generated using the Cav1.3 cDNA fragment encoding 1505C2203 amino acid Rabbit Polyclonal to RABEP1 residues cloned in pEYFP-N1 (Clontech). The numbers refer to amino acid sequences according to the rat Cav1.3 Ca2+ channel (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”D38101″,”term_id”:”736711″,”term_text”:”D38101″D38101). Human myosin light PTP1B-IN-8 chain 2 (tag in pReceiver mammalian expression vector was purchased from GeneCopoeia (catalog number EX-T0572-M09, Rockville, MD). Antibodies The following primary antibodies were used: 1) polyclonal anti-SK2 antibodies raised in rabbits against a purified peptide corresponding to amino acid residues 542C559 of the rat SK2 (a schematic diagram of full-length Cav1.3 channel proteins with the conserved domains labeled. shows a schematic diagram of recombinant fusion proteins for the C terminus of Cav1.3 and YFP (Cav1.3-ct-YFP). Western blot analyses of atrial myocytes probed using anti-Cav1.3 antibody against the intracellular loop (and the nuclear fraction using anti-Cav1.3.
