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Na+/K+ ATPase ?1 and ?3 isoforms are differentially expressed in ?- and ?-motoneurons.

ABSTRACT: The Na(+)/K(+) ATPase (NKA) is an essential membrane protein underlying the membrane potential in excitable cells. Transmembrane ion transport is performed by the catalytic ? subunits (?1-4). The predominant subunits in neurons are ?1 and ?3, which have different affinities for Na(+) and K(+), impacting on transport kinetics. The exchange rate of Na(+)/K(+) markedly influences the activity of the neurons expressing them. We have investigated the distribution and function of the main isoforms of the ? subunit expressed in the mouse spinal cord. NKA?1 immunoreactivity (IR) displayed restricted labeling, mainly confined to large ventral horn neurons and ependymal cells. NKA?3 IR was more widespread in the spinal cord, again being observed in large ventral horn neurons, but also in smaller interneurons throughout the dorsal and ventral horns. Within the ventral horn, the ?1 and ?3 isoforms were mutually exclusive, with the ?3 isoform in smaller neurons displaying markers of ?-motoneurons and ?1 in ?-motoneurons. The ?3 isoform was also observed within muscle spindle afferent neurons in dorsal root ganglia with a higher proportion at cervical versus lumbar regions. We confirmed the differential expression of ? subunits in motoneurons electrophysiologically in neonatal slices of mouse spinal cord. ?-Motoneurons were excited by bath application of low concentrations of ouabain that selectively inhibit NKA?3 while ?-motoneurons were insensitive to these low concentrations. The selective expression of NKA?3 in ?-motoneurons and muscle spindle afferents, which may affect excitability of these neurons, has implications in motor control and disease states associated with NKA?3 dysfunction.

SUBMITTER: Edwards IJ

PROVIDER: S-EPMC3722489 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Na+/K+ ATPase α1 and α3 isoforms are differentially expressed in α- and γ-motoneurons.

Edwards Ian J IJ Bruce Gareth G Lawrenson Charlotte C Howe Laura L Clapcote Steven J SJ Deuchars Susan A SA Deuchars Jim J

The Journal of neuroscience : the official journal of the Society for Neuroscience 20130601 24

The Na(+)/K(+) ATPase (NKA) is an essential membrane protein underlying the membrane potential in excitable cells. Transmembrane ion transport is performed by the catalytic α subunits (α1-4). The predominant subunits in neurons are α1 and α3, which have different affinities for Na(+) and K(+), impacting on transport kinetics. The exchange rate of Na(+)/K(+) markedly influences the activity of the neurons expressing them. We have investigated the distribution and function of the main isoforms of ...[more]

PMID: 23761886

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Project description: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.

Project description:Background and methodsMultiple Na+/K+-ATPase (NKA) α-subunit isoforms express differentially in response to salinity transfer in teleosts but we observed that the isoform nomenclature is inconsistent with the phylogenetic relationship of NKA α-genes. We cloned the catalytic NKA α-subunit isoforms in eels and medaka, analyzed the time course of their expressions in osmoregulatory tissues after transfer from freshwater (FW) to seawater (SW), and performed phylogenetic analyses to deduce an evolutionary scenario that illustrates how various duplication events have led to the current genomic arrangement of NKA α-genes in teleosts.Results and discussionFive and six α-subunits were cloned in eels and medaka respectively. In eels, the commonly-reported α1a and α1b isoforms were absent while the α1c isoform was diversified instead (α1c-1, α1c-2, α1c-3, α2, and α3 in eels). Phylogenetic estimation indicated that the α1a and α1b isoforms from salmon, tilapia, and medaka were generated by independent duplication events and thus they are paralogous isoforms. Re-examination of expression changes of known isoforms after salinity challenge revealed that the isoforms selected as predominant SW-types varied among teleost lineages. Diversification of α1 isoforms occurred by various types of gene duplication, or by alternative transcription among tandem genes to form chimeric transcripts, but there is no trend for more α1 copies in euryhaline species. Our data suggest that the isoform switching between FW (α1a predominates) and SW (α1b predominates) that occurs in salmonids is not universal in teleosts. Instead, in eels, α1c-1 was the major α-subunit upregulated gill, intestine, and kidney in SW. Localization of both NKA mRNA and protein showed consistent upregulation in gill and intestine in SW eels, but not in renal distal and collecting tubules, where low transcript expression levels were accompanied by high protein levels, suggesting a tissue-specific translational regulation that determines and fine-tunes the NKA expression. In medaka, α1b was upregulated in SW in anterior intestine while most other α-subunit isoforms were less responsive to salinity changes.ConclusionBy integrating gene expression and phylogenetic results, we propose that the major NKA α-subunits for SW acclimation were not ancestrally selected, but rather were flexibly determined in lineage-specific fashion in teleosts.

Dataset Information

Na+/K+ ATPase ?1 and ?3 isoforms are differentially expressed in ?- and ?-motoneurons.

Publications

Na+/K+ ATPase α1 and α3 isoforms are differentially expressed in α- and γ-motoneurons.

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