Chromosome 3p24.1 containing the SLC4A7 gene has been identified by genome-wide association studies as one of the genomic loci that influence blood pressure. SLC4A7 encodes electroneutral Na+/HCO3– co-transporter (NBCn1) which regulates intracellular pH (pHi) in various tissues including vascular smooth muscle and endothelium, with knockout models demonstrating an altered blood pressure phenotype. We conducted a functional study of blood pressure-associated single nucleotide polymorphisms at the SLC4A7 locus in primary cultures of human vascular smooth muscle and endothelial cells.
Design and method:
Primary cultures of human umbilical vein endothelial cells and artery smooth muscle cells of different SLC4A7 genotypes were used. DNA-protein interactions were identified by formaldehyde-associated isolation of regulatory elements (FAIRE) and electrophoretic mobility shift assays (EMSA). Gene expression was quantified by allelic imbalance analyses and protein immunoblots. NBCn1 protein function was evaluated using the baseline pHi and rate of dimethyl-amiloride-insensitive, Na+/HCO3–-dependent pHi recovery following ammonium-prepulse-induced intracellular acidification. A10 cells overexpressing 326E or 326K SLC4A7 variants were used to verify primary culture results.
FAIRE identified the BP risk allele as associated with preferentially open chromatin, and EMSA identified allele-dependent DNA-protein interactions. Consistent with this, the risk allele was associated with increased NBCn1 mRNA, protein expression, availability at the cell membrane and subsequently higher Na+/HCO3–-dependent, dimethlyamiloride-insensitive net base flux and steady-state pHi compared to protective allele homozygotes. However, in the presence of Na+/H+ exchange activity, the genotypic effect of NBCn1 on net acid extrusion and steady-state pHi was detected only in vascular smooth muscle cells but not endothelial cells. There was no discernable effect of the missense polymorphism resulting in the amino acid substitution E326K.
These findings provide an insight into the molecular mechanism underlying the association of variation at the SLC4A7 locus and blood pressure, revealing an important genotypic influence on SLC4A7 expression and pHi regulation in vascular smooth muscle cells.
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