Tissue-specific Versus Isoform-specific Differences in Cation Activation Kinetics of the Na,K-ATPase

280

246

Three isoforms (␣1, ␣2, and ␣3) of the catalytic (␣) subunit of the plasma membrane (PM) Na ؉ pump have been identified in the tissues of birds and mammals. These isoforms differ in their affinities for ions and for the Na ؉ pump inhibitor, ouabain. In the rat, ␣1 has an unusually low affinity for ouabain. The PM of most rat cells contains both low (␣1) and high (␣2 or ␣3) ouabain affinity isoforms, but precise localization of specific isoforms, and their functional significance, are unknown. We employed high resolution immunocytochemical techniques to localize ␣ subunit isoforms in primary cultured rat astrocytes, neurons, and arterial myocytes. Isoform ␣1 was ubiquitously distributed over the surfaces of these cells. In contrast, high ouabain affinity isoforms (␣2 in astrocytes, ␣3 in neurons and myocytes) were confined to a reticular distribution within the PM that paralleled underlying endoplasmic or sarcoplasmic reticulum. This distribution is identical to that of the PM Na͞Ca exchanger. This raises the possibility that ␣1 may regulate bulk cytosolic Na ؉ , whereas ␣2 and ␣3 may regulate Na ؉ and, indirectly, Ca 2؉ in a restricted cytosolic space between the PM and reticulum. The high ouabain affinity Na ؉ pumps may thereby modulate reticulum Ca 2؉ content and Ca 2؉ signaling.

Section: Resultsmentioning

confidence: 80%

mentioning

confidence: 99%

Na ⁺ pump low and high ouabain affinity α subunit isoforms are differently distributed in cells

Juhaszova

Blaustein

1997

280

246

“…The Na pump's functional properties are known to vary depending on the particular subunit combination of its isoforms, as well as the cellular͞tissue environment in which these combinations are located (2)(3)(4)(5)(6). In addition, there are also variations in the pump's isoform composition that differ in their tissue͞ species distribution and in their developmental stage (2)(3)(4).…”

mentioning

confidence: 99%

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

Hoffman

Wickrema

Potapova

et al. 2002

This study is aimed at identifying the Na pump isoform composition of human erythroid precursor cells and mature human erythrocytes. We used purified and synchronously growing human erythroid progenitor cells cultured for 7-14 days. RNA was extracted from the progenitor cells on different days and analyzed by RT-PCR. The results showed that only the ␣1, ␣3, ␤2, and ␤3 subunit isoforms and the ␥ modulator were present. Northern analysis of the erythroid progenitor cells again showed that ␤2 but not ␤1 or ␣2 isoforms were present. The erythroid cells display a unique ␤ subunit expression profile (called ␤-profiling) in that they contain the message for the ␤2 isoform but not ␤1, whereas leukocytes and platelets are known to have the message for the ␤1 but not for the ␤2 isoform. This finding is taken to indicate that our preparations are essentially purely erythroid and free from white cell contamination. Western analysis of these cultured progenitor cells confirmed the presence of ␣1, ␣3, (no ␣2), ␤2, ␤3, and ␥ together now with clear evidence that ␤1 protein was also present at all stages. Western analysis of the Na pump from mature human erythrocyte ghosts, purified by ouabain column chromatography, has also shown that ␣1, ␣3, ␤1, ␤2, ␤3, and ␥ are present. Thus, the Na pump isoform composition of human erythroid precursor cells and mature erythrocytes contains the ␣1 and ␣3 isoforms of the ␣ subunit, the ␤1, ␤2, and ␤3 isoforms of the ␤ subunit, and the ␥ modulator.

“…If the abundance of reticulocyte mRNA is a measure of the abundance of protein in the mature red cell (most of the copies of Na,K-ATPase contain the ␣1 isoform of the ␣ subunit, and few contain the ␣3 isoform), then the complexation of ␣1 with either ␤2 or ␤3 should govern most of the kinetics, implying that complexes containing ␣3 should have a minor influence. Most studies link the catalytic activity overwhelmingly to the ␣ subunit, and studies of different combinations of ␣ and ␤ subunits (9)(10)(11)(12) have not yet shown any dramatic differences in their kinetic characteristics. However, in none of the aforementioned studies was uncoupled sodium efflux investigated.…”

Section: Discussionmentioning

confidence: 99%

“…␤3 was recently found in human placenta and various rat tissues (6). Although ␣ is the catalytically important subunit (7,8), kinetic studies of combinations of ␣1, ␣2, and ␣3 with ␤1 and ␤2 (9)(10)(11)(12) indicated that the kinetic parameters of the Na,K-ATPase can be influenced by different combinations of isoforms of the ␣ and ␤ subunits. However, uncoupled sodium efflux was not investigated in any of these studies.…”

mentioning

confidence: 99%

Na,K-ATPase subunit isoforms in human reticulocytes: Evidence from reverse transcription–PCR for the presence of α1, α3, β2, β3, and γ

Stengelin

Hoffman²

1997

The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium eff lux mode between the human red blood cell Na,KATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoformspecific primers were used. The ␣1 and ␣3 isoforms of the ␣ subunit, and the ␤2 and ␤3 isoforms of the ␤ subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found ␤2 but not ␤1 in reticulocyte single-stranded cDNA, and ␤1 but not ␤2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that ␣1, ␣2, and ␣3 as well as all three ␤ isoforms were present. The extent to which the kinetic properties of uncoupled sodium eff lux might depend on different isoform combinations is not yet known.