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.
Highlights
IntroductionThe Na+/K+-ATPase (sodium/potassium pump), first described in 1957 [1], is a membrane bound ion pump belonging to the family of P-type ATPases
The Na+/K+-ATPase, first described in 1957 [1], is a membrane bound ion pump belonging to the family of P-type ATPases
The a1-isoform seems to be uniformly expressed in all cells, while the a2-isoform is reported to be predominantly expressed in astrocytes and muscle, and the a3-isoform is primarily found expressed in neurons [10,11,12,13,14,15], expression in human heart is reported [16,17,18]
Summary
The Na+/K+-ATPase (sodium/potassium pump), first described in 1957 [1], is a membrane bound ion pump belonging to the family of P-type ATPases. Several members of this family catalyze active transport of cations across the cell membrane and function in maintaining the ionic gradients through hydrolysis of ATP. The Na+/K+-ATPase pumps sodium ions out of the cell and potassium ions into the cell with a stoichiometry of 3Na+ for 2K+ [2,3] It is a hetero-oligomer composed of a- and b-subunits [4] as well as in many tissues of a regulatory subunit belonging to the FXYD protein family. The a4-isoform is only found in sperm cells and is essential for male fertility [7,19]
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