Down-regulated Expression Of The BK_Ca Channel β1 Subunit Gene In SO Cells From Hypercholesterolemic Rabbits And Cloning And Characterization Of The Rabbit BK_Ca Channel β1 Subunit Gene

The sphincter of Oddi (SO) is a smooth muscle structure with autonomic relaxation and contraction functions that encircles the end of the bile duct and plays a key role in controlling the bile flowing into the duodenum. Studies showed that cholesterol metabolic disorder is a risk factor in gallbladder cholesterol calculus formation. Gallbladder evacuation dysfunction and cholestasis are important elements for the crystallization and precipitation of supersaturated cholesterol in bile and gallstone formation. Our studies indicated that SO relaxation function was damaged in the New Zealand rabbits fed by high-cholesterol diet. SO dysfunction (SOD) was induced in hypercholesterolemic (HC) rabbits, followed by cholestasis and forming internal environment of cholesterol calculus formation. Further studies suggested that the overloading of intracellular Ca²⁺ concentration ([Ca²⁺]i) of SO cells might be one of the vital reasons for SOD in HC rabbits. And [Ca²⁺]i overloading in SO cells from HC rabbits was closely related to the function of large conductance calcium-sensitive potassium (BKCa) channels. But the mechanism is largely unknown.BKCa channels play vital role in the control of smooth muscle tone and excitability. They can be formed by two subunits, the pore-formingαsubunit and the modulatoryβ1 subunit. Their functions are highly associated with expression levels of bothαandβ1 subunits. Our previous studies indicated that the expression ofαsubunit seems no changes in SO cells from HC rabbits. Since the regulatoryβ1 subunit confers the BKCa channel higher Ca2+ sensitivity and is a key regulator in the control of BKCa channel functions, so in the first part of this study we aim at observing the expression changes ofβ1 subunit gene using RT-PCR and Western blot, and investigating the functions of BKCa channel and its effect on L-VDCC in SO cells from HC rabbits by whole cell patch clamp techniques. Furthermore, in the second part of this study we aim at cloning and characterizing the rabbit BKCa channelβ1 subunit (BK-β1) gene and its 5′-flanking region in order to analyze the structure, functional profiles and transcription regulation of this gene. The main findings of this work are as follows:1. Down-regulated expression of the BKCa channelβ1 subunit gene in SO cells from HC rabbits and its effects on the functions of the BKCa channel in SO cells from HC rabbits.Results of RT-PCR and Western blot indicated that the expression ofβ1 subunit decreased in SO cells from HC rabbits. The data of whole cell patch clamp techniques showed that the total potassium current densities, the BKCa current densities and the KV current densities of SO cells from HC rabbits decreased significantly after high-cholesterol diet; the membrane potentials of SO cells from HC rabbits were showed in the state of depolarization; the membrane capacities were decreased in SO cells from HC rabbits. Compared with the control rabbits, the SO cells of rabbits indicated an increased L-VDCC current density after high-cholesterol diet. However, neither the steady-state activation curves nor steady-state inactivation curves of SO cells showed indicated no changes in HC rabbit. We assumed that the down-regulated expression of BK-β1 gene in SO cells from HC rabbits could result in the decreased activities of BK channel and therefore triggering increased activities of L-VDCC, which probably plays a key role in SOD by HC.2. Cloning and characterization of the rabbit BK-β1 gene.The full-length cDNA of the rabbit BK-β1 gene, amplified by 3'RACE and 5'RACE from rabbit SO tissue, was 1408 bp containing a 576 bp open reading frame (ORF) encoding a peptide of 191 amino acids. The first exon contains only untranslated region (UTR); the second exon contains 24 bp of UTR and encodes amino acids 1–44; the third exon encodes amino acids 45–102 and the fourth exon encodes the remainder of the protein (aa 103–191).Semi-quantitative RT-PCR was employed to examine the expression of the BK-β1 gene in tissues of SO, aorta, liver, heart, lung, skeletal muscle, kidney, and brain from rabbits. The results indicated that the BK-β1 gene was high expressed in SO and aortal smooth muscle cells (SMC), whereas with relatively lower level expression in heart and skeletal muscle tissues and with no expression founded in tissues of liver, lung and brain. Introns of the rabbit BK-β1 gene were cloned using long and accurate PCR (LA-PCR). The length of introns in nucleotide is 3175, 1442 and 1704 bp respectively. Combined with cDNA sequence, the length of the BK-β1 genomic sequence is 7729 bp, contains four exons and three introns. All of the exon/intron junctions contain the GT/AG consensus junction sequence. Southern blot analysis indicated that the BK-β1 gene is a single copy gene in the rabbit genomic sequence.Bioinformatic analysis showed the putative topology of the rabbit BK-β1 protein with two transmembrane domains and a large extracellular loop. The BK-β1 protein contains four function domains, one PKA phosphorylation site and two N-glycosylation sites. The homology alignment and comparison showed that rabbit BK-β1 gene harbours a high homology of the nucleotide and amino acids sequence with that of human and other mammals.3. Cloning and characterization of the promoter region of the rabbit BK-β1 gene.For the first time, we isolated a 2021 bp 5'-flanking region of the rabbit BKβ1 subunit gene using Thermal Asymmetric Interlaced PCR (TAIL-PCR) and deposited it in the GenBank with accession no. DQ839485. To map the putative cis-acting elements in 5'-flanking region of the rabbit BK-β1 gene, an online software TESS was used. Analysis of the 5'-flanking region (2021 bp) did not show any canonical consensus TATA or CAAT box. However, in the proximal region of the transcription start site, several putative consensus-binding sites for transcription factors were identified. 5'-RACE assay indicated that the transcription start site of the BK-β1 gene maps to the single guanidine nucleotide (+1G), which designates the beginning of the first exon and is located at 447 bp upstream of the translation initiation codon (ATG). To assess the functional promoter activity of the isolated 5′-flanking region of the BKβ1 subunit gene, a series of 5'-deletion reporter constructs (P-2021, P-763, P-271, P-93 and P-34) were generated by inserting PCR fragments of various sizes into the pGL3-basic, a promoterless luciferase reporter vector. The promoter activities of the constructs in HEK293 cells of the rabbit were determined by luciferase reporter assays. All the constructs except P-34 (?34/+30), exhibited much higher luciferase activities than negative control of pGL-3-basic. P-763 (?763/+30) exhibited >40-fold higher luciferase activities than pGL-3-basic. Marked reduction in luciferase activities was observed in P-34 (?34/+30) compared with that of P-93 (?92/+30). The luciferase activities of P-34 is only 25% of that of P-93, suggesting that the region of ?93/+30 is the core promoter region and some essential cis-acting elements should exit within the gap region of ?93/?64.In conclusion, there are two parts in our study. In the first part, the expression of BK-β1 gene was decreased; the functions of BKCa and Kv channels were damaged and the membrane potentials were in the state of depolarization in SO cells from HC rabbits. We assumed that the down-regulated expression of BK-β1 gene in SO cells from HC rabbits could result in the decreased activities of BK channel and therefore triggering increased activities of L-VDCC, which probably plays a key role in SOD by HC. In the second part, for the first time we cloned the rabbit BK-β1 gene and its promoter region and deposited them in the GenBank with accession nos. DQ821756 (cDNA sequence) and DQ839485 (genomic sequence). The BK-β1 gene is a single copy gene and is mainly expressed in smooth muscle tissues, with the length in nucleotide 7729 bp, encoding a peptide of 191 amino acids and containing four exons and three introns, which has a high homology with that of other mammals. The promoter profiles of the rabbit BK-β1 gene indicated that no putative TATA-box exists and the transcription start site (+1G) of is located at 447 bp upstream of the translation initiation codon. In this TATA-less 5'-flanking region, the region of ?93/+30 is the core promoter region and GC-box in the gap region of ?93/?64 might play a key role in transcription initiation. Our study provides a basic knowledge of the transcriptional profiles of the rabbit BK-β1 gene.