Molecular Cloning and Characterization of Porcine Na+/K+-ATPase Isoforms α1, α2, α3 and the ATP1A3 Promoter

Abstract

Na+/K+-ATPase maintains electrochemical gradients of Na+ and K+ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na+/K+-ATPase exists in four different isoforms (α1–α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na+/K+-ATPase mutations, we have determined the porcine coding sequences of the α1–α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na+ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na+ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.

A Novel Bufalin Derivative Exhibited Stronger Apoptosis-Inducing Effect than Bufalin in A549 Lung Cancer Cells and Lower Acute Toxicity in Mice

“…Therefore, both the α1 and α2 subunits might be involved in the toxicity of BF and BF211 in mice. Interestingly, when the activity of porcine cortex Na+/K+-ATPase, which expresses the levels of the α subunits in the order α2>α3>α1 [ 35 ] is inhibited, BF211 exhibited slightly weaker effects than BF at some concentrations, such as at 0.1 μM. The weaker effects of BF211 on porcine Na+/K+-ATPase might be consistent with the weaker binding affinity of BF211 to the α2 subunit found in the Biacore assay.…”

Transcriptional regulators of Na,K-ATPase subunits

“…Aberrant DNA methylation patterns have been linked to altered gene expression in various genetic diseases and tumors (Bird, 2002 ). Higher methylated CpGs were found in the first exon of Atp1a3 , which correlated with the lower expression of this gene in pig liver (Henriksen et al, 2013 ). Manganese exposure led to sustained Atp1a3 promoter hypermethylation and downregulation of its transcript level in mice (Wang et al, 2013 ).…”

Altered Glutamate Protein Co-Expression Network Topology Linked to Spine Loss in the Auditory Cortex of Schizophrenia

“…ATP1A3 is one of three alpha subunit variants of the ATP dependent NA + /K + transporting channel expressed in the brain(69). ATP1A3 expression is neuron-specific, and is essential for returning neurons to the resting membrane potential after repeated firing(70).…”