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Regulation of Na+/K+ ATPase transport velocity by RNA editing.

Colina C, Palavicini JP, Srikumar D, Holmgren M, Rosenthal JJ - PLoS Biol. (2010)

Bottom Line: Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+)/K(+) pumps in order to maintain ion homeostasis.In this study we show that Na(+)/K(+) pump activity is tightly regulated by a novel process, RNA editing.Three codons within the squid Na(+)/K(+) ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurobiology, University of Puerto Rico-Medical Sciences Campus, San Juan, Puerto Rico.

ABSTRACT
Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+)/K(+) pumps in order to maintain ion homeostasis. In this study we show that Na(+)/K(+) pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na(+)/K(+) ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na(+) release to the extracellular solution, causing a higher turnover rate at the resting membrane potential.

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Related in: MedlinePlus

mRNAs for the squid Na+/K+ pump are edited.(A) A diagram of the intron-exon structure for the SqNaKα1 gene (the cDNA sequence is genbank EF467998). Boxes represent exons and lines introns. Arrows mark the four positions where the gene sequence contained an A and some cDNA sequences contained a G. (B) Electropherogram of the genomic sequence surrounding codon K666 compared to electropherograms of two cDNA clones, which show the first two nucleotides of the codon edited to G, resulting in the codon change K666G. (C) Positions of all four edited adenosines. Numbers refer to the editing percentage based on 50 individual cDNA clones amplified from giant axon system specific cDNA. Exon sequence is underlined. (D) Relative positions of the three codons changed by editing mapped on a cartoon of the Na+/K+ pump structure. (E) Amino acid alignment of Na+/K+ pump α subunit sequences from diverse taxa showing that the unedited amino acid at each editing site is highly conserved.
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pbio-1000540-g001: mRNAs for the squid Na+/K+ pump are edited.(A) A diagram of the intron-exon structure for the SqNaKα1 gene (the cDNA sequence is genbank EF467998). Boxes represent exons and lines introns. Arrows mark the four positions where the gene sequence contained an A and some cDNA sequences contained a G. (B) Electropherogram of the genomic sequence surrounding codon K666 compared to electropherograms of two cDNA clones, which show the first two nucleotides of the codon edited to G, resulting in the codon change K666G. (C) Positions of all four edited adenosines. Numbers refer to the editing percentage based on 50 individual cDNA clones amplified from giant axon system specific cDNA. Exon sequence is underlined. (D) Relative positions of the three codons changed by editing mapped on a cartoon of the Na+/K+ pump structure. (E) Amino acid alignment of Na+/K+ pump α subunit sequences from diverse taxa showing that the unedited amino acid at each editing site is highly conserved.

Mentions: Historically, the Na+/K+ ATPase of the squid giant axon has been one of the most actively studied native pumps. In a previous report we identified the mRNA sequences for the underlying α (EF467998) and β (EF467996) subunits [10]. Because other squid transcripts are regulated by RNA editing [17],[20], we examined whether the Na+/K+ ATPase mRNAs were as well. Sequences of 50 individual cDNA clones for the squid NaKα1 subunit, isolated from the giant axon system, showed adenosine-or-guanine variation at specific sites, a hallmark of RNA editing. To explore whether this variation was indeed due to RNA editing, we cloned the gene that encodes squid NaKα1 mRNAs (Figure 1A). The squid NaKα1 gene, which spans over 20 KB, is highly fragmented, containing 19 exons. At four positions, the gene sequence contains an A whereas some or all of the cDNA sequences contain a G (e.g., Figure 1B). Three of the sites lie at the junction with a nearby intron, as is commonly the case with other RNA editing sites (Figure 1C) [23]. Two sites lie within the same codon. Because both were guanosine in all cDNAs sequenced, the lysine at this position was always converted to glycine. To further support the idea that the A→G conversions are caused by RNA editing, we tested whether a squid editing enzyme (SqADAR2.1A (FJ478450.1); [24]) could edit these codons in vitro (Figure S1A). Using the genomic form of the full-length, mature squid NaKα1 mRNA as a substrate, recombinant SqADAR2.1A could edit all four codons. It is notable that all the information required for editing resides within the exons and that intron sequence was not required, as is commonly the case for other editing sites. Similarly, the structure that drives editing of human Kv1.1 channel mRNAs is entirely exonic [18],[25]. Interestingly, human ADAR2 (BC065545.1) can also edit codons K666G and I877V, but not R663G. Predicted folds for NaKα1 mRNA using MFOLD software show an obvious hairpin surrounding the I877V codon (Figure S1B). Using this approach, similar structures are not apparent around codons R663G and K666G. In any case, the combination of our cloning data and the in vitro editing assays verify that the A/G variation observed in Na+/K+ ATPase mRNAs is due to RNA editing.


Regulation of Na+/K+ ATPase transport velocity by RNA editing.

Colina C, Palavicini JP, Srikumar D, Holmgren M, Rosenthal JJ - PLoS Biol. (2010)

mRNAs for the squid Na+/K+ pump are edited.(A) A diagram of the intron-exon structure for the SqNaKα1 gene (the cDNA sequence is genbank EF467998). Boxes represent exons and lines introns. Arrows mark the four positions where the gene sequence contained an A and some cDNA sequences contained a G. (B) Electropherogram of the genomic sequence surrounding codon K666 compared to electropherograms of two cDNA clones, which show the first two nucleotides of the codon edited to G, resulting in the codon change K666G. (C) Positions of all four edited adenosines. Numbers refer to the editing percentage based on 50 individual cDNA clones amplified from giant axon system specific cDNA. Exon sequence is underlined. (D) Relative positions of the three codons changed by editing mapped on a cartoon of the Na+/K+ pump structure. (E) Amino acid alignment of Na+/K+ pump α subunit sequences from diverse taxa showing that the unedited amino acid at each editing site is highly conserved.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990702&req=5

pbio-1000540-g001: mRNAs for the squid Na+/K+ pump are edited.(A) A diagram of the intron-exon structure for the SqNaKα1 gene (the cDNA sequence is genbank EF467998). Boxes represent exons and lines introns. Arrows mark the four positions where the gene sequence contained an A and some cDNA sequences contained a G. (B) Electropherogram of the genomic sequence surrounding codon K666 compared to electropherograms of two cDNA clones, which show the first two nucleotides of the codon edited to G, resulting in the codon change K666G. (C) Positions of all four edited adenosines. Numbers refer to the editing percentage based on 50 individual cDNA clones amplified from giant axon system specific cDNA. Exon sequence is underlined. (D) Relative positions of the three codons changed by editing mapped on a cartoon of the Na+/K+ pump structure. (E) Amino acid alignment of Na+/K+ pump α subunit sequences from diverse taxa showing that the unedited amino acid at each editing site is highly conserved.
Mentions: Historically, the Na+/K+ ATPase of the squid giant axon has been one of the most actively studied native pumps. In a previous report we identified the mRNA sequences for the underlying α (EF467998) and β (EF467996) subunits [10]. Because other squid transcripts are regulated by RNA editing [17],[20], we examined whether the Na+/K+ ATPase mRNAs were as well. Sequences of 50 individual cDNA clones for the squid NaKα1 subunit, isolated from the giant axon system, showed adenosine-or-guanine variation at specific sites, a hallmark of RNA editing. To explore whether this variation was indeed due to RNA editing, we cloned the gene that encodes squid NaKα1 mRNAs (Figure 1A). The squid NaKα1 gene, which spans over 20 KB, is highly fragmented, containing 19 exons. At four positions, the gene sequence contains an A whereas some or all of the cDNA sequences contain a G (e.g., Figure 1B). Three of the sites lie at the junction with a nearby intron, as is commonly the case with other RNA editing sites (Figure 1C) [23]. Two sites lie within the same codon. Because both were guanosine in all cDNAs sequenced, the lysine at this position was always converted to glycine. To further support the idea that the A→G conversions are caused by RNA editing, we tested whether a squid editing enzyme (SqADAR2.1A (FJ478450.1); [24]) could edit these codons in vitro (Figure S1A). Using the genomic form of the full-length, mature squid NaKα1 mRNA as a substrate, recombinant SqADAR2.1A could edit all four codons. It is notable that all the information required for editing resides within the exons and that intron sequence was not required, as is commonly the case for other editing sites. Similarly, the structure that drives editing of human Kv1.1 channel mRNAs is entirely exonic [18],[25]. Interestingly, human ADAR2 (BC065545.1) can also edit codons K666G and I877V, but not R663G. Predicted folds for NaKα1 mRNA using MFOLD software show an obvious hairpin surrounding the I877V codon (Figure S1B). Using this approach, similar structures are not apparent around codons R663G and K666G. In any case, the combination of our cloning data and the in vitro editing assays verify that the A/G variation observed in Na+/K+ ATPase mRNAs is due to RNA editing.

Bottom Line: Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+)/K(+) pumps in order to maintain ion homeostasis.In this study we show that Na(+)/K(+) pump activity is tightly regulated by a novel process, RNA editing.Three codons within the squid Na(+)/K(+) ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurobiology, University of Puerto Rico-Medical Sciences Campus, San Juan, Puerto Rico.

ABSTRACT
Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+)/K(+) pumps in order to maintain ion homeostasis. In this study we show that Na(+)/K(+) pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na(+)/K(+) ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na(+) release to the extracellular solution, causing a higher turnover rate at the resting membrane potential.

Show MeSH
Related in: MedlinePlus